Bacterial biocatalysts play a key role in our transition to a bio-based, post-petroleum economy. However, the discovery of new biocatalysts is currently limited by our ability to analyze genomic information and our capacity of functionally screening for desired activities. Here, we present a simple workflow that combines functional metaproteomics and metagenomics, which facilitates the unmediated and direct discovery of biocatalysts in environmental samples. To identify the entirety of lipolytic ...[more]
Project description:Chelocardin is an atypical tetracycline and its mechanism of action is discussed controversially in literature. Therefore, we analyzed the bacterial response of Bacillus subtilis to chelocardin and as a control for classical tetracyclines to tetracycline by proteomic profiling. The induced marker proteins mirror the damage in the cell due the antibiotic stress. Proteomic profiling was carried out by 2D-PAGE and spot identification by nUPLC-ESI-MS/MS. The proteomic profile of chelocardin demonstrates a dual mechanism of action with protein biosynthesis inhibition and membrane damage.
Project description:The diazotrophic bacterium Rhodobacter capsulatus is able to synthesize two nitrogenases, a molybdenum-dependent and an alternative Mo-free iron-only nitrogenase, enabling growth with molecular dinitrogen (N2) as sole nitrogen source. The Mo response of the wild type and a mutant lacking the high-affinity molybdate transporter, ModABC, were analyzed by proteome profiling.
Project description:The diazotrophic bacterium Rhodobacter capsulatus synthesizes a molybdenum nitrogenase and an alternative iron-only nitrogenase, enabling growth with molecular dinitrogen as sole nitrogen source. Regulation of nitrogen fixation was analyzed by proteome profiling of wild-type and mutant strains lacking the transcriptional regulators NifA, AnfA, and MopAB.
Project description:We use HDX-MS to characterise the binding interface between the tick evasin P672 and the chemokine CCL8, in order to assist in the development of peptides capable of mimicking P672 anti-inflammatory activity.
Project description:Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s - known as SAPKs (Stress/ABA-activated Protein Kinases). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2-box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2-box and ABA-box. Surprisingly, mutations in the SnRK2-box did not drastically affect sugarcane SnRK2 activity, in contrast to previous observations for the homologous proteins in Arabidopsis. Also, we found that the ABA-box might have a role in SnRK2 activation in the absence of PP2C phosphatase. Taken together, our results demonstrate that both C-terminal domains of sugarcane SnRK2 proteins play a role in protein activation and activity.
Project description:Based on UPLC-Q-TOF-MS metabolomics technology, urine samples of 1072 subjects from 9 centers, including normal control, phlegm and blood stasis (PBS) syndrome and Qi and Yin deficiency (QYD) syndrome, and other syndromes of CHD, were conducted to find biomarkers. Among them, the discovery set (n = 125) and the test set (n = 337) were used to identify and validate biomarkers, and the validation set (n = 610) was used for the application and evaluation of the support vector machine (SVM) prediction model.
Project description:We designed siRNAs against pyk-reg-90 and pyk-reg-10 using the Dharmacon algorithm (Dharmacon siDESIGN http://www.dharmacon.com/sidesign/). Each of four highest-ranking siRNA sequences for pyk-reg-90 and pyk-reg-10 respectively was tested in our and pyk-reg-90, we used a pool of different siRNAs that included the two most effective siRNAs. The cellsperformance was assessed at 24-hr intervals until 96 hrs posttransfection by qRT-PCR. For both pyk-reg-10resuspended in 1X siRNA buffer (Dharmacon, LaFayette CO, USA) to a stock concentration of 50 ?M. Theexperiments. These siRNAs were were transfected with the corresponding siRNA pool at the final concentration of 100nM and 200nM by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol for further analysis. As control, we used a pool of non-targetting siRNAs (Dharmacon).
Project description:We selected and characterized doxorubicin/5FU resistant HCT-8/R clones from sensitive parental cells, analyzing several parameters: cell cycle phase distribution, apoptotic activity, expression, distribution and functionality of the P-gp efflux pump, ultrastructural features, invasiveness and transcriptomic profiles.
Project description:Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a representative gel was selected, on which a total of 136 CBB-stained spots were numbered and subjected to in-gel digestion and label-free quantitative LC-MSE (MS/MS in data-independent acquisition mode). The analysis provided the assignment of 1-25 (average eight) nonredundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. The quantity data from the LC-MSE analysis were further analyzed to reconstruct the quantity distribution on the 2D gel for each of the assigned proteins (a native protein map). This work provides not only a reference library containing 199 native protein maps for rat plasma proteome, but also useful information for subsequent analysis for protein-protein interactions.
Project description:Salt stress is a rising threat to agriculture system. The accumulation of salts near the plant roots hampers the normal uptake of water causing osmotic stress and ionic toxicity to the plant. Thompson Seedless is a popular table grape variety of Vitis vinifera L., which is sensitive to salt stress when grown on its own roots; grafting it onto a wild rootstock such as 110 Richtor (110R) makes it tolerant to salt stress. In the present study, shotgun-proteomics approach was used for the investigation of salt stress induced molecular response of own rooted and 110R grafted Thompson Seedless grapevines. A salt stress experiment was conducted on sixteen month old potted grapevines. The grapevines were treated with 150mM NaCl solution for seven days and the control vines were irrigated with tap water. The young leaf samples were collected from control and treated vines at three time-points viz. 6 hours, 48 hours and 7 days of salt stress. The stress responsive proteins identified through statistical analysis revealed a distinct response to salinity in both the vines.