Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria

Background: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens. Methodology/Principal Findings: We compared the identification efficiency of MALDI-TOF MS with that of PhoenixH, APIH and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7 % of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63 % of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn’s kappa values: 0.9 to 0.93). Conclusions/Significance: MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria

Effectiveness of strategies to increase the validity of findings from association studies: size vs. replication

<p>Abstract</p> <p>Background</p> <p>The capacity of multiple comparisons to produce false positive findings in genetic association studies is abundantly clear. To address this issue, the concept of false positive report probability (FPRP) measures "the probability of no true association between a genetic variant and disease given a statistically significant finding". This concept involves the notion of prior probability of an association between a genetic variant and a disease, making it difficult to achieve acceptable levels for the FPRP when the prior probability is low. Increasing the sample size is of limited efficiency to improve the situation.</p> <p>Methods</p> <p>To further clarify this problem, the concept of true report probability (TRP) is introduced by analogy to the positive predictive value (PPV) of diagnostic testing. The approach is extended to consider the effects of replication studies. The formula for the TRP after k replication studies is mathematically derived and shown to be only dependent on prior probability, alpha, power, and number of replication studies.</p> <p>Results</p> <p>Case-control association studies are used to illustrate the TRP concept for replication strategies. Based on power considerations, a relationship is derived between TRP after k replication studies and sample size of each individual study. That relationship enables study designers optimization of study plans. Further, it is demonstrated that replication is efficient in increasing the TRP even in the case of low prior probability of an association and without requiring very large sample sizes for each individual study.</p> <p>Conclusions</p> <p>True report probability is a comprehensive and straightforward concept for assessing the validity of positive statistical testing results in association studies. By its extension to replication strategies it can be demonstrated in a transparent manner that replication is highly effective in distinguishing spurious from true associations. Based on the generalized TRP method for replication designs, optimal research strategy and sample size planning become possible.</p

Global Analysis of Circulating Immune Cells by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Background: MALDI-TOF mass spectrometry is currently used in microbiological diagnosis to characterize bacterial populations. Our aim was to determine whether this technique could be applied to intact eukaryotic cells, and in particular, to cells involved in the immune response. Methodology/Principal Findings: A comparison of frozen monocytes, T lymphocytes and polymorphonuclear leukocytes revealed specific peak profiles. We also found that twenty cell types had specific profiles, permitting the establishment of a cell database. The circulating immune cells, namely monocytes, T lymphocytes and polymorphonuclear cells, were distinct from tissue immune cells such as monocyte-derived macrophages and dendritic cells. In addition, MALDI-TOF mass spectrometry was valuable to easily identify the signatures of monocytes and T lymphocytes in peripheral mononuclear cells. Conclusions/Significance: This method was rapid and easy to perform, and unlike flow cytometry, it did not require any additional components such as specific antibodies. The MALDI-TOF mass spectrometry approach could be extended t

Visualization and Curve-Parameter Estimation Strategies for Efficient Exploration of Phenotype Microarray Kinetics

The Phenotype MicroArray (OmniLog® PM) system is able to simultaneously capture a large number of phenotypes by recording an organism's respiration over time on distinct substrates. This technique targets the object of natural selection itself, the phenotype, whereas previously addressed '-omics' techniques merely study components that finally contribute to it. The recording of respiration over time, however, adds a longitudinal dimension to the data. To optimally exploit this information, it must be extracted from the shapes of the recorded curves and displayed in analogy to conventional growth curves.The free software environment R was explored for both visualizing and fitting of PM respiration curves. Approaches using either a model fit (and commonly applied growth models) or a smoothing spline were evaluated. Their reliability in inferring curve parameters and confidence intervals was compared to the native OmniLog® PM analysis software. We consider the post-processing of the estimated parameters, the optimal classification of curve shapes and the detection of significant differences between them, as well as practically relevant questions such as detecting the impact of cultivation times and the minimum required number of experimental repeats.We provide a comprehensive framework for data visualization and parameter estimation according to user choices. A flexible graphical representation strategy for displaying the results is proposed, including 95% confidence intervals for the estimated parameters. The spline approach is less prone to irregular curve shapes than fitting any of the considered models or using the native PM software for calculating both point estimates and confidence intervals. These can serve as a starting point for the automated post-processing of PM data, providing much more information than the strict dichotomization into positive and negative reactions. Our results form the basis for a freely available R package for the analysis of PM data

Prednisolone-Induced Predisposition to Femoral Head Separation and the Accompanying Plasma Protein Changes in Chickens

Femoral head separation (FHS) is an idiopathic bone problem that causes lameness and production losses in commercial poultry. In a model of prednisolone-induced susceptibility to FHS, the changes in plasma proteins and peptides were analyzed to find possible biomarkers. Plasma samples from control and FHS-susceptible birds were depleted of their high abundance proteins by acetonitrile precipitation and were then subjected to cation exchange and reverse-phase (RP) fractionations. Analysis with matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) showed several differentially expressed peptides, two of which were isolated by RP-HPLC and identified as the fragments of apolipoprotein A-I. The acetonitrile fractionated plasma proteins were subjected to reduction/alkylation and trypsin digestion followed by liquid chromatography and tandem mass spectrometry, which showed the absence of protocadherin 15, vascular endothelial growth factor-C, and certain transcription and ubiquitin-mediated proteolytic factors in FHS-prone birds. It appears that prednisolone-induced dyslipidemia, vascular, and tissue adhesion problems may be consequential to FHS. Validity of these biomarkers in our model and the natural disease must be verified in future using traditional approaches. Biomarker Insights Lameness because of femoral head separation (FHS) is a production and welfare problem in the poultry industry. Selection against FHS requires identification of the birds with subclinical disease with biomarkers from a source such as blood. Prednisolone can induce femoral head problems and predisposition to FHS. Using this experimental model, we analyzed the plasma peptides and proteins from normal and FHS-prone chickens by mass spectrometry to identify differentially expressed peptides and proteins. We found two peptides, both derived from apolipoprotein A-I, quantitatively elevated and two proteins, protocadherin 15 and VEGF-C, that were conspicuously absent in FHS-susceptible birds

Updip rupture of the 2004 Sumatra earthquake extended by thick indurated sediments

During subduction, weak, unlithified sediments are scraped off the down-going plate and accumulate near the subduction trench axis. The weak nature of the sediments usually impedes the propagation of fault rupture during an earthquake. However, measurements of slip during the 2004 Sumatra–Andaman Mw 9.2 earthquake show that fault rupture propagatedupdip, extending unusually close to the subduction trench, in the southern part of the rupture area. Here we present seismic reflection images of the southern part of the 2004 Sumatra–Andaman earthquake rupture area. We show that sedimentary strata, greater than 4?km in thickness, form coherent blocks that have been thrust onto the continental margin during subduction. The blocks form a 130-km-wide plateau overlying the seismogenic zone and the plate boundary megathrust lies near to the base of the sediments. The sediments consist of the Nicobar and Bengal Fan turbidites and exhibit strong internal cohesion. We suggest that dewatering and lithification of the sediments during burial made them unusually competent and strong, thus enabling rupture during the 2004 earthquake to propagate beneath the plateau, close to the Sunda Trench. Extending fault rupture so close to the trench, and thus further seaward, may have enhanced the tsunami hazard by displacing a greater thickness of water

Phylogenetic classification and identification of bacteria by mass spectrometry

Bacteria are a convenient source of intrinsic marker proteins, which can be detected efficiently by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The patterns of protein masses observed can be used for accurate classification and identification of bacteria. Key to the reliability of the method is a robust and standardized procedure for sample preparations, including bacterial culturing, chemical treatment for bacterial cell wall disruption and for protein extraction, and mass spectrometry analysis. The protocol is an excellent alternative to classical microbiological classification and identification procedures, requiring minimal sample preparation efforts and costs. Without cell culturing, the protocol takes in general <1 h