Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models.

Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection ar

Quantification of sulfatides and lysosulfatides in tissues and body fluids by liquid chromatography-tandem mass spectrometry

Sulfatides are found in brain as components of myelin, oligodendrocytes, and neurons but are also present in various visceral tissues. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A, leading to severe white matter disease due to the accumulation of sulfatides and lysosulfatides. To study the physiological role of sulfatides, accessible and sensitive quantitative methods are required. We developed a sensitive LC/MS/MS method to quantify total sulfatide and lysosulfatide content as well as individual molecular species in urine and plasma from MLD patients and plasma and tissues from an MLD mouse model. Our results demonstrate that the method can quantify a wide range of sulfatide concentrations and can be used to quantify total sulfatide content and levels of individual molecular species of sulfatides in tissues, cells, and body fluids. Even though plasma sulfatides and lysosulfatides would seem attractive candidate biomarkers that could possibly correlate with the severity of MLD and be of use to monitor the effects of therapeutic intervention, our results indicate that it is unlikely that the determination of these storage products in plasma will be useful in this respec

Quantitation of newly synthesized proteins by pulse labeling with azidohomoalanine

Measuring protein synthesis and degradation rates on a proteomic scale is an important step toward modeling the kinetics in complicated cellular response networks. A gel-free method, able to quantify changes in the formation of new proteins on a 15 min timescale, compatible with mass spectrometry is described. The methionine analogue, azidohomoalanine (azhal), is used to label newly formed proteins during a short pulse-labeling period following an environmental switch in Escherichia coli. Following digestion a selective reaction against azhal-containing peptides is applied to enrich these peptides by diagonal chromatography. This technique enables quantitation of hundreds of newly synthesized proteins and provides insight into immediate changes in newly synthesized proteins on a proteomic scale after an environmental perturbatio

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatus pulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment

The Hfq regulon of Neisseria meningitidis

The conserved RNA-binding protein, Hfq, has multiple regulatory roles within the prokaryotic cell, including promoting stable duplex formation between small RNAs and mRNAs, and thus hfq deletion mutants have pleiotropic phenotypes. Previous proteome and transcriptome studies of Neisseria meningitidis have generated limited insight into differential gene expression due to Hfq loss. In this study, reversed-phase liquid chromatography combined with data-independent alternate scanning mass spectrometry (LC-MSE) was utilized for rapid high-resolution quantitative proteomic analysis to further elucidate the differentially expressed proteome of a meningococcal hfq deletion mutant. Whole-cell lysates of N. meningitidis serogroup B H44/76 wild-type (wt) and H44/76 Delta hfq (Delta hfq) grown in liquid growth medium were subjected to tryptic digestion. The resulting peptide mixtures were separated by liquid chromatography (LC) prior to analysis by mass spectrometry (MSE). Differential expression was analyzed by Student's t-test with control for false discovery rate (FDR). Reliable quantitation of relative expression comparing wt and Delta hfq was achieved with 506 proteins (20%). Upon FDR control at q <= 0.05, 48 up- and 59 downregulated proteins were identified. From these, 81 were identified as novel Hfq-regulated candidates, while 15 proteins were previously found by SDS/PAGE/MS and 24 with microarray analyses. Thus, using LC-MSE we have expanded the repertoire of Hfq-regulated proteins. In conjunction with previous studies, a comprehensive network of Hfq-regulated proteins was constructed and differentially expressed proteins were found to be involved in a large variety of cellular processes. The results and comparisons with other gram-negative model systems, suggest still unidentified sRNA analogs in N. meningitidi