Systematic analysis of the regulation of type three secreted effectors in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

Background  

The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions.     

Markers of Endogenous Desaturase Activity and Risk of Coronary Heart Disease in the CAREMA Cohort Study

Background

Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20∶5n-3) and DHA (C22∶6n-3), are known to prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20∶4n-6), however, remain unclear. δ-5 and δ-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20∶4n-6 to C20∶3n-6 and C18∶3n-6 to C18∶2n-6 ratios are markers of endogenous δ-5 and δ-6 desaturase activities, but have never been studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as well as genotypes of FADS1 rs174547 and CHD incidence.

Methods

We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors.

Results

The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20∶5n-3 and C22∶6n-3 and increased δ-5 and δ-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline δ-5 desaturase activity was associated with reduced CHD risk (P for trend = 0.02), especially among those carrying the high desaturase activity genotype (AA): HR (95% CI) = 0.35 (0.15–0.81) for comparing the extreme quintiles. High plasma DHA levels were also associated with reduced CHD risk.

Conclusion

In this prospective cohort study, we observed a reduced CHD risk with an increased C20∶4n-6 to C20∶3n-6 ratio, suggesting that δ-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other independent studies.     

Detection and estimation of aflatoxins using both chemical and biological techniques

Production of aflatoxins on both natural (rice and corn) and semisynthetic (YES) media was conducted using an identified toxin-producing strain ofAspergillus flavus. TheA flavus strain was able to produce 4 types of aflatoxins, namely B₁, B₂, G₁, and G₂ on rice, corn, and YES media. Quantitative data showed that the concentrations of aflatoxins B₁ and G₁ produced were 52, 40.3, and 39.6; and 64.7, 45.0, and 58.0jug for 50g of rice, corn, and YES media, respectively. In comparison, the yielded amounts of aflatoxins B₂ and G₂ were much lower: 11.5, 17.9, and 17.5; and 28.S, 40.3, and 39.5 μg for 50 g of rice, corn, and YES media, respectively. A bioassay was conducted using the following 5 standard bacterial strains:Bacillus megaterium. Bacillus subtilis, Streptococcus faecal is, Staphylococcus epidermidis, andParacoccus denitrificans as well as a field strain of Candida albicans. All strains exceptP denitrificans showed varied degrees of inhibition when applied with crude aflatoxins at 5 to 40μg/mL. The minimum concentration of crude aflatoxins needed to inhibitP denitrificans was 10μg/mL. Moreover,Candida albicans was not inhibited at any concentration of aflatoxins applied in this work. Both undiluted and diluted (1/10, 1/100, and 1/1000) bacterial broth cultures showed a direct relationship between the diameter of inhibition zones and the concentrations of crude aflatoxins. Mean diameters of (7.0–20.5), (5–14), (4.5–13.0), (3.0–12.0), and (1.5–11.0) mm were observed when various concentrations of aflatoxins were applied usingB megaterium, S epidermidis, S faecal is, B subtilis, andP denitrificans, respectively. Field trials were applied to testify the validity of our data. A 1/100 dilution was prepared from each strain of 4 different species to estimate aflatoxins in samples of contaminated corn. Both chemical and biological assays were carried out at the same time. Data revealed that the most sensitive organism inhibited by as low as 7.5μg aflatoxins/mL wasB megaterium giving an inhibition zone of 10.5 mm, followed byS epidermidis with an inhibition zone of 7.5mm. In relation, the other 2 organisms were less sensitive to crude aflatoxins. Similarly, the biological assay was applied to detect aflatoxins in some samples of wheat, corn, peanut, rice, and poultry rations. Of the 14 wheat and 10 corn samples, only 4 wheat and 2 corn samples were found to be positive. The same results were obtained using TLC analysis.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Meta‐analysis on blood transcriptomic studies identifies consistently coexpressed protein–protein interaction modules as robust markers of human aging

The bodily decline that occurs with advancing age strongly impacts on the prospects for future health and life expectancy. Despite the profound role of age in disease etiology, knowledge about the molecular mechanisms driving the process of aging in humans is limited. Here, we used an integrative network-based approach for combining multiple large-scale expression studies in blood (2539 individuals) with protein–protein Interaction (PPI) data for the detection of consistently coexpressed PPI modules that may reflect key processes that change throughout the course of normative aging. Module detection followed by a meta-analysis on chronological age identified fifteen consistently coexpressed PPI modules associated with chronological age, including a highly significant module (P = 3.5 × 10⁻³⁸) enriched for ‘T-cell activation’ marking age-associated shifts in lymphocyte blood cell counts (R² = 0.603; P = 1.9 × 10⁻¹⁰). Adjusting the analysis in the compendium for the ‘T-cell activation’ module showed five consistently coexpressed PPI modules that robustly associated with chronological age and included modules enriched for ‘Translational elongation’, ‘Cytolysis’ and ‘DNA metabolic process’. In an independent study of 3535 individuals, four of five modules consistently associated with chronological age, underpinning the robustness of the approach. We found three of five modules to be significantly enriched with aging-related genes, as defined by the GenAge database, and association with prospective survival at high ages for one of the modules including ASF1A. The hereby-detected age-associated and consistently coexpressed PPI modules therefore may provide a molecular basis for future research into mechanisms underlying human aging.     

Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

The distribution and quantitation of 125I-alpha-bungarotoxin (alpha-BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetanic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, alpha-BTX was bound to approximately 90% of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of alpha-BTX relative to this membrane was determined to be 46,000 +/- 27% sites/mum2.     

Identification of messenger and long noncoding RNAs associated with gallbladder cancer via gene expression profile analysis

The present study aimed to investigate the long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in the progression of gallbladder cancer and explore the potential physiopathologic mechanisms of gallbladder cancer in terms of competing endogenous RNAs (ceRNAs). The original lncRNA and mRNA expression profile data (nine gallbladder cancer tissues samples and nine normal gallbladder samples) in GSE76633 was downloaded from the Gene Expression Omnibus database. Differentially expressed mRNAs and lncRNAs between gallbladder cancer tissue and normal control were selected and the pathways in which they are involved were analyzed using bioinformatics analyses. MicroRNAs (miRNAs) were also predicted based on the differentially expressed mRNAs. Finally, the co-expression relation between lncRNA and mRNA was analyzed and the ceRNA network was constructed by combining the lncRNA-miRNA, miRNA-mRNA, and lncRNA-mRNA pairs. Overall, 373 significantly differentially expressed mRNAs and 47 lncRNAs were identified between cancer and normal tissue samples. The upregulated genes were significantly enriched in the extracellular matrix (ECM)-receptor interaction pathway, while the downregulated genes were involved in the complement and coagulation cascades. Altogether, 128 co-expression relations between lncRNA and mRNA were obtained. In addition, 196 miRNA-mRNA regulatory relations and 145 miRNA-lncRNA relation pairs were predicted. Finally, the lncRNA-miRNA-gene ceRNA network was constructed by combining the three types of relation pairs, such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6. mRNAs and lncRNAs may be involved in gallbladder cancer progression via ECM-receptor interaction pathways and the complement and coagulation cascades. Moreover, ceRNAs such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6 can also be implicated in the pathogenesis of gallbladder cancer.     

Systematic Testing of Literature Reported Genetic Variation Associated with Coronary Restenosis: Results of the GENDER Study

Background

Coronary restenosis after percutaneous coronary intervention still remains a significant problem, despite all medical advances. Unraveling the mechanisms leading to restenosis development remains challenging. Many studies have identified genetic markers associated with restenosis, but consistent replication of the reported markers is scarce. The aim of the current study was to analyze the joined effect of previously in literature reported candidate genes for restenosis in the GENetic DEterminants of Restenosis (GENDER) databank.

Methodology/Principal Findings

Candidate genes were selected using a MEDLINE search including the terms ‘genetic polymorphism’ and ‘coronary restenosis’. The final set included 36 genes. Subsequently, all single nucleotide polymorphisms (SNPs) in the genomic region of these genes were analyzed in GENDER using set-based analysis in PLINK. The GENDER databank contains genotypic data of 2,571,586 SNPs of 295 cases with restenosis and 571 matched controls. The set, including all 36 literature reported genes, was, indeed, significantly associated with restenosis, p = 0.024 in the GENDER study. Subsequent analyses of the individual genes demonstrated that the observed association of the complete set was determined by 6 of the 36 genes.

Conclusion

Despite overt inconsistencies in literature, with regard to individual candidate gene studies, this is the first study demonstrating that the joint effect of all these genes together, indeed, is associated with restenosis.     

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.