Characterization of the effect of LPS on dendritic cell subset discrimination in spleen

The Gram‐negative bacterial endotoxin lipopolysaccharide (LPS) is a potent inflammatory mediator and a leading cause of bacterial sepsis. While LPS is known to activate antigen‐presenting cells, here we find that LPS down‐regulates expression of CD11c and CD11b on splenic dendritic cell subsets, thus confounding the ability to identify these subsets following treatment. This has implications with regard to tracking the response to LPS in terms of the cell subsets involved, and should be considered whenever such studies are undertaken.     

A rapid and sensitive HPLC-MS method for the detection of plasma and cellular rifampicin

Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC-MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze-thaw cycles, but were significantly affected by heat-inactivation (58 degrees C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8h period.     

Activity of crude and fractionated extracts by lactic acid bacteria (LAB) isolated from local dairy,meat, and fermented products against Staphylococcus aureus

This study aimed to evaluate anti-staphylococcal properties of crude and fractionated extracts of lactic acid bacteria (LAB) isolated from local meat, dairy, and fermented products. A total of 36 LAB isolates were obtained and identified via 16S rDNA sequencing. Cell-free supernatant (CFS) of all isolates exhibiting a statistically significant inhibition against Staphylococcus aureus (ρ < 0.05), with six LAB isolates exhibiting a more prevalent inhibition. The inhibition effects of cell wall and intracellular extracts from the six prevalent isolates were evaluated. Lactobacillus plantarum USM8613 was the most prominent isolate with both CFS and cell wall extract exhibiting the most prevalent inhibition against S. aureus. Scanning electron micrographs showed alteration of S. aureus membrane morphology upon CFS treatment, suggesting an anti-staphylococcal effect via membrane destruction. Confocal laser scanning micrographs showed inhibition against biofilm formations by S. aureus in porcine skins upon CFS treatment. The CFS from L. plantarum USM8613 was separated into protein, lipid, and polysaccharide fractions for evaluation of anti-staphylococcal activity and chemical characterization. All fractions inhibited growth of S. aureus (ρ < 0.05), with protein fractions exhibiting stronger inhibition effect. Data from our present study showed that extracts from LAB could be applied as biopreservatives in the food industries and/or as an antimicrobial agent against bacterial infections for cosmeceutical and pharmaceutical uses.     

A Variable Sampling Interval Synthetic Xbar Chart for the Process Mean

The usual practice of using a control chart to monitor a process is to take samples from the process with fixed sampling interval (FSI). In this paper, a synthetic X¯ control chart with the variable sampling interval (VSI) feature is proposed for monitoring changes in the process mean. The VSI synthetic X¯ chart integrates the VSI X¯ chart and the VSI conforming run length (CRL) chart. The proposed VSI synthetic X¯ chart is evaluated using the average time to signal (ATS) criterion. The optimal charting parameters of the proposed chart are obtained by minimizing the out-of-control ATS for a desired shift. Comparisons between the VSI synthetic X¯ chart and the existing X¯, synthetic X¯, VSI X¯ and EWMA X¯ charts, in terms of ATS, are made. The ATS results show that the VSI synthetic X¯ chart outperforms the other X¯ type charts for detecting moderate and large shifts. An illustrative example is also presented to explain the application of the VSI synthetic X¯ chart.     

Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.     

Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27

Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili–mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili–related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.Thermus thermophilus HB27 is a Gram-negative, rod-shaped, and extremely thermophilic eubacterium isolated from a geothermal area (1). This organism grows at temperatures up to 85 °C and has an optimal growth temperature of 70 °C. The thermostable enzymes obtained from members of the genus Thermus are of considerable interest because of their potential in research, biotechnological, and industrial applications (2, 3). In addition, T. thermophilus HB27 is a suitable laboratory model for genetic manipulation, as it is easily cultured under laboratory conditions and has a natural transformation system that is much more efficient than those of other Thermus spp. (4). Intriguingly, thermophiles are also found in biofilms, enclosed within a matrix consisting of extracellular polymeric substances, in various natural and artificial thermal environments (5, 6). Bacteria form biofilms in order to adapt and survive in harsh environments (7, 8). Over the past few decades, biofilm formation has been a major focus of microbial research and, as such, has been studied in relationship to bacterial pathogenesis, immunology, biofouling, microbial technology, and industrial applications (7, 9–12).Members of the genus Thermus, like many other thermophiles, have evolved two main mechanisms for thermoadaption. One is biofilm formation, which confers protection against environmental stresses such as high temperature and the presence of antibiotics (8). In previous studies, a novel exopolysaccharide, TA-1, was isolated from a T. aquaticus YT-1 biofilm, and both its primary structure and its immunological activity were determined (13). In addition, we showed that the overexpression of uridine diphosphate (UDP)-galactose-4′-epimerase (GalE), which catalyzes the reversible interconversion of UDP-galactose and UDP-glucose, in T. thermophilus HB27 increases biofilm production because of the enzyme''s involvement in an important step of exopolysaccharide (EPS)¹ biosynthesis (14). The other mechanism that enables Thermus to thrive in extreme habitats is natural transformation (i.e. the ability to take up free DNA). In hot environments, natural transformation allows the horizontal exchange of genetic information between extremophiles, including of genes that promote thermoadaptation (15–17). Recent studies showed that the type IV pili (T4P) on the cell surface of T. thermophilus HB27 not only are required for natural transformation (18, 19), but also mediate adhesion and twitching motility (20). Also, together with the degree of EPS production, the presence of T4P on the bacterial cell surface contributes to the regulation of biofilm formation (21). However, despite extensive research on the physiological, biochemical, and genetic traits of thermophiles, the mechanisms underlying these functions and their role in thermal adaptation have not been fully elucidated (16, 22–24).Advances in the field of phosphoproteomics have come from high-resolution mass spectrometry and prokaryotic genome sequencing, which have confirmed the phosphorylation of many bacterial proteins on serine/threonine and tyrosine residues (25, 26). In surveys of phosphorylation-related functions, bacterial serine, threonine, and tyrosine phosphoproteins have been shown to regulate many physiological and adaptation processes, such as central carbon catabolism, the heat shock response, osmolarity, starvation, EPS synthesis, virulence, and sporulation (25–27). These observations have been followed by more detailed, species-specific phosphoproteomics investigations, including in Bacillus subtilis (28), Escherichia coli (29), Lactococcus lactis (30), Halobacterium salinarum (31), Klebsiella pneumonia (32), Pseudomonas spp. (33), Rhodopseudomonas palustris (34), and T. thermophilus HB8 (35). In this study, the role played by the global phosphorylation network of the thermophile T. thermophilus HB27 in the physiological processes that mediate the stress responses and thermotolerance of this bacterium was examined. Specifically, we used strong cation exchange (SCX) chromatography and titanium dioxide (TiO₂) (28–30) enrichment to characterize the phosphoproteomic map of T. thermophilus HB27. Genetic manipulation of this strain indicated that phosphorylation of the PilF protein, which contains an ATP-binding motif (TTC1622/pilF) and drives T4P formation, is involved in both EPS production and piliation, thereby influencing the biofilm formation during thermophilic adaptation.     

Attenuation of fibroblast growth factor signaling by poly-N-acetyllactosamine type glycans

Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-β-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells.     

Integrating NOE and RDC using sum-of-squares relaxation for protein structure determination

We revisit the problem of protein structure determination from geometrical restraints from NMR, using convex optimization. It is well-known that the NP-hard distance geometry problem of determining atomic positions from pairwise distance restraints can be relaxed into a convex semidefinite program (SDP). However, often the NOE distance restraints are too imprecise and sparse for accurate structure determination. Residual dipolar coupling (RDC) measurements provide additional geometric information on the angles between atom-pair directions and axes of the principal-axis-frame. The optimization problem involving RDC is highly non-convex and requires a good initialization even within the simulated annealing framework. In this paper, we model the protein backbone as an articulated structure composed of rigid units. Determining the rotation of each rigid unit gives the full protein structure. We propose solving the non-convex optimization problems using the sum-of-squares (SOS) hierarchy, a hierarchy of convex relaxations with increasing complexity and approximation power. Unlike classical global optimization approaches, SOS optimization returns a certificate of optimality if the global optimum is found. Based on the SOS method, we proposed two algorithms—RDC-SOS and RDC–NOE-SOS, that have polynomial time complexity in the number of amino-acid residues and run efficiently on a standard desktop. In many instances, the proposed methods exactly recover the solution to the original non-convex optimization problem. To the best of our knowledge this is the first time SOS relaxation is introduced to solve non-convex optimization problems in structural biology. We further introduce a statistical tool, the Cramér–Rao bound (CRB), to provide an information theoretic bound on the highest resolution one can hope to achieve when determining protein structure from noisy measurements using any unbiased estimator. Our simulation results show that when the RDC measurements are corrupted by Gaussian noise of realistic variance, both SOS based algorithms attain the CRB. We successfully apply our method in a divide-and-conquer fashion to determine the structure of ubiquitin from experimental NOE and RDC measurements obtained in two alignment media, achieving more accurate and faster reconstructions compared to the current state of the art.     

Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.