iTRAQ experimental design for plasma biomarker discovery

There is considerable interest in using mass spectrometry for biomarker discovery in human blood plasma. We investigated aspects of experimental design for large studies that require analysis of multiple sample sets using iTRAQ reagents for sample multiplexing and quantitation. Immunodepleted plasma samples from healthy volunteers were compared to immunodepleted plasma from patients with osteoarthritis in eight separate iTRAQ experiments. Our analyses utilizing ProteinPilot software for peptide identification and quantitation showed that the methodology afforded excellent reproducibility from run to run for determining protein level ratios (coefficient of variation 11.7%), in spite of considerable quantitative variances observed between different peptides for a given protein. Peptides with high variances were associated with lower intensity iTRAQ reporter ions, while immunodepletion prior to sample analysis had a negligible affect on quantitative variance. We examined the influence of different reference samples, such as pooled samples or individual samples on calculating quantitative ratios. Our findings are discussed in the context of optimizing iTRAQ experimental design for robust plasma-based biomarker discovery.     

Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.     

Induction of sister-chromatid exchanges in Chinese hamster V79 cells by exposure to the photochemical reaction products of toluene plus NO2 in the gas phase

A study was made on the induction of sister-chromatid exchanges (SCE) in cultured Chinese hamster V79 cells exposed to the photochemical reaction products of toluene plus NO2 in the gas phase. The photochemical reaction products of toluene plus NO2 were obtained by photochemical reaction of a toluene--NO2/dry air system in a photochemical smog chamber and then exposed to cultured cells for 2 h using a system for in vitro gas exposure. SCEs were induced at all concentrations of the photochemical reaction products employed in the present study, and the highest SCE frequency observed for the highest concentration tested for each component was 3.6 times higher than that of the control. Cytogenotoxicity which was evaluated with induced-SCEs of the photochemical reaction products of toluene plus NO2 was much the same as that of the previously reported photochemical reaction products of propylene plus NO2 (Shiraishi and Bandow, 1985), but was considerably stronger than that of typical gaseous air pollutants such as NO2 alone and O3 alone.     

Inactivation of microbes and macromolecules by atmospheric-pressure plasma jets

Plasma is ionized gas, which is found in various forms in nature and can also be generated artificially. A variety of cold atmospheric-pressure plasmas are currently being investigated for their clinical utility, and first studies reporting on the treatment of patients showed that plasma treatment may support the wound healing process. One of the benefits of plasma treatment is the effective inactivation of bacteria including tenacious pathogens such as Pseudomonas aeruginosa or multiresistant Staphylococcus aureus (MRSA). Neither the molecular mechanisms promoting wound healing nor those underlying bacterial inactivation are fully understood yet. The review has a focus on plasma jets, a particular type of cold atmospheric-pressure plasma sources featuring an indirect treatment whereby the treated substrates do not come into contact with the plasma directly but are exposed to the plasma-emitted reactive species and photons. Such plasma jets are being employed as tools in basic research regarding the effects of plasmas on biological samples. This review provides a brief overview on the recent clinical investigations into the benefits of cold atmospheric-pressure plasmas. It then describes our current understanding of the mechanisms leading to bacterial inactivation and inactivation of biomacromolecules gained by employing plasma jets.     

Proteomic signatures in antibiotic research

Antibiotics disturb the physiological homeostasis of bacterial cells by interfering with essential cellular functions or structures. The bacterial proteome adjusts quickly in response to antibiotic challenge. This physiological response is specifically tailored to overcome the inflicted damage and, thus, closely linked to the antibiotic target and mechanism of action. In a way, the proteome mirrors the antibiotic insult. This connection can be exploited to guide the development of novel antibiotics. By using structurally different antibiotics, which cause the same physiological disturbance, proteomic signatures diagnostic of the mechanism of action can be defined. These proteomic signatures inform about mechanism-related differential protein expression as well as about protein modifications. This review recapitulates how antibiotic proteomic signatures are established and highlights areas of antibiotic research benefiting most from proteomic signatures. Antibacterial research programs designed to structurally advance existing antibiotic classes profit from rapid in vivo mechanism of action confirmation. What is more, a comprehensive reference compendium of antibiotic proteomic signatures allows rapid mechanism of action identification of those structurally novel compounds that inhibit known targets. Finally, novel proteomic response profiles indicate unprecedented mechanisms. Here, the proteome profile provides evidence on the nature of the antibiotic-caused physiological disturbance leading to testable hypotheses on the mechanism of action.     

Feldspar Tunneling by Fungi along Natural Productivity Gradients

Recently, it was hypothesized that ectomycorrhizas hyphae are involved in mineral tunneling. We evaluated the role of ectomycorrhizas in mineral weathering and the ecosystem influx of basic cations by correlating mineral tunnel density to ectomycorrhizas density in two forest productivity gradients. The gradients, two gentle slopes in northern Sweden, are the result of groundwater movement and are characterized by reduced productivity upslope due to lower nitrogen availability. As expected, ectomycorrhizas density in the O horizon was higher upslope, where nitrogen availability was lower and where the vegetation was dominated by ectomycorrhizas plant species. We consistently found that tunnel formation in mineral grains was more intense in nutrient-poor sites, indicating a higher contribution of fungi to ecosystem influx of potassium and calcium. ectomycorrhizas density was positively correlated with feldspar tunnel density in the upper 2 cm of the E horizon. This suggests that ectomycorrhizas are involved in mineral tunneling. We discuss the possible involvement of ericoid mycorrhizas and saprotrophic fungi in feldspar tunneling and the role of the weathering status of mineral grains as additional factors controlling mineral funneling.     

Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies--COPD biomarker discovery study

2-D gel electrophoresis has been used for more than three decades to study the protein complement of organisms, tissues, and cells. Three issues are holding back large-scale proteomics studies: low-throughput, high technical variation, and study designs lacking statistical power. We identified image analysis as the central factor connecting these three issues. By developing an improved image analysis workflow we shortened project timelines, decreased technical variation, and thus enabled large-scale proteomics studies that are statistically powered. Rather than detecting protein spots on each gel image and matching spots across gel images, the improved workflow is based on aligning images first, then creating a consensus spot pattern and finally propagating the consensus spot pattern to all gel images for quantitation. This results in a data table without gaps. As an example we show here a study aimed at discovering circulating biomarkers for chronic obstructive pulmonary disease (COPD). Eight candidate biomarkers were identified by comparing plasma from 24 smokers with COPD and 24 smokers without COPD. Among the candidates are proteins such as plasma retinal-binding protein (RETB) and fibrinogen that had previously been linked to the disease and are frequently monitored in COPD patients, as well as other proteins such as apolipoprotein E (ApoE), inter-alpha-trypsininhibitor heavy chain H4 (ITIH4), and glutathione peroxidase.