Schematic presentation of the <i>phaCAB</i> operon of <i>R. eutropha</i> PHB^-4.

<p>Primers <i>phaCAB_fw</i> and <i>phaCAB_rv</i> were applied to generate the 4,860-bp DNA fragment comprising the <i>phaCAB</i> operon. The PCR fragment was subcloned into vector pJET1.2/blunt, and subsequently fragments of three independent hybrid plasmids were sequenced. The arrow marks the observed G320A mutation in <i>phaC</i> causing a stop codon obviously leading to a truncated and non functional PHA synthase (PhaC) in strain PHB^-4.</p

Impact of the Core Components of the Phosphoenolpyruvate-Carbohydrate Phosphotransferase System, HPr and EI, on Differential Protein Expression in <i>Ralstonia eutropha</i> H16

In <i>Ralstonia eutropha</i> H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly­(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/EI deletion mutants H16Δ<i>ptsH</i>, H16Δ<i>ptsI</i>, and H16Δ<i>ptsHI</i> when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner–Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16Δ<i>ptsHI</i>. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16Δ<i>ptsHI</i>, and to gain insight into the molecular response of <i>R. eutropha</i> to the deletion of HPr/EI

Quantification of differentially expressed proteins in <i>R. eutropha</i> H16 and mutant <i>R. eutropha</i> PHB^-4 as based on image fusion of 2D PAGE gels (Fig. 2).

<p>Spot quantities are given as % volume (representing the relative portion of an individual spot of the total protein present on the respective average fusion image). To facilitate comparision of spot quantities, panel A presents spots with relative quantities close to 1, while spots with higher quantities are shown in panel B. Quantification was done with Delta 2D software. Suffixes (a, b, and others) indicate isoforms of the same protein species present in the gels.</p

Changes in the proteomes of the PHA-negative mutant <i>R. eutropha</i> PHB^-4 compared to the wild type <i>R. eutropha</i> H16.

<p>Cells were cultivated in MSM medium under conditions promoting PHB synthesis. Extracted proteins were focused using pH 5 to 8 nonlinear strips. Dual channel images were generated by employing the Delta2D software. (A) Cells of the exponential growth phase. Wild type H16, blue spots; mutant PHB^-4, orange spots. (B) Cells of the stationary growth phase. Wild type H16, blue spots; mutant PHB^-4, orange spots. Arrows and numbers mark proteins with significantly different levels of expression in comparison to those of wild type H16 and mutant PHB^-4. Detailed information about the detected proteins is compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095907#pone-0095907-t002" target="_blank">Table 2</a> and Fig. 3.</p

Central metabolism of <i>R. eutropha</i> H16 and mutant PHB^-4 with regard to the results of proteome analyses.

<p>The numbers in the scheme indicate the following involved enzymes: 1, glucokinase; 2, phosphogluconate dehydratase; 3, phospho-2-keto-3-desoxygluconate aldolase; 4, glyceraldehyde-3-phosphate dehydrogenase; 5, phosphoglycerate dehydrogenase; 6, phosphoglyceromutase; 7, enolase; 8, pyruvate kinase; 9, pyruvate dehydrogenase/decarboxylase (E1 of PDHC); 10, dihydrolipoamide acetyltransferase (E1 of PDHC); 11, dihydrolipoamide dehydrogenase (E3 of PDHC); 12, acetoin dehydrogenase enzyme system; 13, acetyl-CoA acetyltransferase; 14, acetoacetyl-CoA reductase; 15, PHB synthase; 16, 3-oxoacid-CoA transferase; 17, 3-hydroxybutyrate dehydrogenase; 18, citrate synthase; 19, aconitase; 20, isocitrate dehydrogenase; 21, 2-oxoacid dehydrogenase multienzyme complex; 22, succinyl-CoA synthetase; 23, succinate dehydrogenase; 24, fumarase; 25, malate dehydrogenase; 26, citrate lyase.</p

Differentially expressed proteins obtained by proteome analysis.

<p>Differentially expressed proteins obtained by proteome analysis.</p

Additional file 1: of Comparative evaluation of patients’ and physicians’ satisfaction with interferon beta-1b therapy

Full data set used for congruence analysis. Satisfaction with interferon beta-1b therapy as evaluated by patients and their physicians after 6 months of treatment and at the 24 months/study end visit. For a subgroup of patients, records of an electronic patient diary allowed to assess adherence to the injection schedule in the first 6 months and in the last 12 months of the study. Moreover, the table provides the information, for which patients drug-related adverse events were reported. (XLSX 30 kb

Picking Vanished Proteins from the Void: How to Collect and Ship/Share Extremely Dilute Proteins in a Reproducible and Highly Efficient Manner

Successful proteome analyses of highly dilute samples are strongly dependent on optimized workflows considering especially sample preparation prior to highly sensitive mass spectrometric analysis. Various methods are available for enrichment of proteome samples, each characterized by specific advantages and disadvantages limiting their general application as a method of choice. Here we suggest an optimized universal protocol ensuring reproducibility and effective enrichment of dilute samples by commercial affinity beads. By comparably assessing the performance of the new protocol with selected standard enrichment techniques, we show the seamless application of the enrichment in common mass spectrometry based proteomic workflows. Further, novel applications are suggested including a facile storage and shipping of desiccated, trapped proteome samples at ambient temperatures and usage of the affinity beads for gel-free proteomic approaches

Visualization of the inferred dynamic gene regulatory network for the responder group

Each gene is represented by a node, and gene regulatory interactions are shown by directed edges. Solid lines, activating effects; dashed lines, inhibitory effects. The hypothesized network was reconstructed from quantitative real-time RT-PCR data by the modified LASSO method.<p><b>Copyright information:</b></p><p>Taken from "Molecular discrimination of responders and nonresponders to anti-TNFalpha therapy in rheumatoid arthritis by etanercept"</p><p>http://arthritis-research.com/content/10/3/R50</p><p>Arthritis Research & Therapy 2008;10(3):R50-R50.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2483439.</p><p></p

Time-Resolved Analysis of Cytosolic and Surface-Associated Proteins of <i>Staphylococcus aureus</i> HG001 under Planktonic and Biofilm Conditions

Staphylococcal biofilms are associated with persistent infections due to their capacity to protect bacteria against the host’s immune system and antibiotics. Cell-surface-associated proteins are of great importance during biofilm formation. In the present study, an optimized biotinylation approach for quantitative GeLC–MS-based analysis of the staphylococcal cell-surface proteome was applied and the cytoplasmic protein fraction was analyzed to elucidate proteomic differences between colony biofilms and planktonic cells. The experimental setup enabled a time-resolved monitoring of the proteome under both culture conditions and the comparison of biofilm cells to planktonic cells at several time points. This allowed discrimination of differences attributed to delayed growth phases from responses provoked by biofilm conditions. Biofilm cells expressed CcpA-dependent catabolic proteins earlier than planktonic cells and strongly accumulated proteins that belong to the SigB stress regulon. The amount of the cell-surface protein and virulence gene regulator Rot decreased within biofilms and MgrA-dependent regulations appeared more pronounced. Biofilm cells simultaneously up-regulated activators (e.g., SarZ) as well as repressors (e.g., SarX) of RNAIII. A decreased amount of high-affinity iron uptake systems and an increased amount of the iron-storage protein FtnA possibly indicated a lower demand of iron in biofilms