Novel functional polymorphism on PADI-4 gene and its association with arthritis onset

BackgroundCitrullinated proteins formed by peptidyl arginine deiminases (PADIs) deimination of arginine residues in proteins are of particular interest in arthritis pathogenesis. Polymorphisms on the PADI-4 gene lead to the malfunctioning of PADIs leading to the onset of arthritis.ObjectiveThe present study was conducted to determine the polymorphisms on the PADI-4 gene and their association with rheumatoid arthritis (RA) as well as Osteoarthritis (OA).MethodologyTo achieve the above-mentioned objective a case-control study was conducted. Blood samples were collected from RA, OA, and control subjects. DNA was extracted from each blood sample by modified organic method and was quantified as well as qualified by DNA gel electrophoresis and Nanodrop. Patients were tested for rs874881, rs11203366, rs11203367, rs2240336, rs2240337, rs2240339, rs1748033 and rs2240340 polymorphic sites by amplifying targeted regions through PCR with site-specific primers. Genotyping was performed by Restriction Fragment Length Polymorphism and direct sequencing method. Mutations were identified by analyzing sequences on BioEdit software. Allelic, genetic, and multiple site analysis were performed by SHEsis and PLINK software. Change in the amino acid sequence was identified by MEGA 6.0 software.ResultsPolymorphisms were identified on all targeted polymorphic sites except rs2240337 in both RA and OA individuals. In addition, two novel mutations were also identified in exon 4 identified i-e SCV000804840: c.218T > C and SCV000807675: c.241G > T. All the SNPs except rs11203366 were found to be significantly associated with RA at an allelic level whereas all SNP’s have been significant risk factors in the onset of OA. At genotypic level rs874881, rs11203366, rs2240339, SCV000804840 and SCV000807675 were significantly associated to RA development whereas rs874881, rs11203366, rs11203367, rs2240339, SCV000804840 and SCV000807675 were genetic risk factors in OA onset. Haplotype analysis indicated that GACCACGCC and GACCACGCT were highly significant in disease development. Polymorphisms identified altered the functioning of PADIs by altering their amino acid sequence.ConclusionIn conclusion, it was found that PADI-4 gene polymorphism was not only involved in the onset of RA but was also found to be a significant risk factor in OA onset.     

Humoral immune response to filarial antigens in chyluria

Humoral immune parameters like total immunoglobulins and specific antibody levels in serum were studied in filarial chyluria patients. Mean serum IgG was significantly reduced in this group compared to normal controls, while IgA and IgM levels remained comparable to controls. Anti-filarial antibody titre as measured by enzyme-linked immunosorbent assay also was significantly reduced. However, the total and specific IgE antibody titre was similar to that of controls. Specific IgE contents of the patients’ sera could be related to their microfilaraemic status.     

INSECTICIDE RESISTANCE IN THE GROUND SPIDER,Pardosa sumatrana (THORELL, 1890; ARANEAE: LYCOSIDAE)

Elevated levels of insecticides detoxifying enzymes, such as esterases, glutathione S‐transferases (GSTs), and cytochrome P‐450 monooxygenases, act in the resistance mechanisms in insects. In the present study, levels of these enzymes in the insecticide‐resistant ground spider Pardosa sumatrana (Thorell, 1890) were compared with a susceptible population (control) of the same species. Standard protocols were used for biochemical estimation of enzymes. The results showed significantly higher levels of nonspecific esterases and monooxygenases in resistant spiders compared to controls. The activity of GSTs was lower in the resistant spiders. Elevated levels of nonspecific esterases and monooxygenases suggest their role in metabolic resistance in P. sumatrana. The reduced levels of total protein contents revealed its possible consumption to meet energy demands.     

Computational design of Phe-Tyr dipeptide and preparation,characterization, cytotoxicity studies of Phe-Tyr dipeptide loaded PLGA nanoparticles for the treatment of hypertension

Phe-Tyr dipeptide which was investigated in Wakame food with greatest ACE-inhibitory activity is used as a pharmaceutical drug for the treatment of hypertension, cardiovascular diseases, and diabetic nephropathy. To improve the bioavailability of Phe-Tyr, a delivery system based on poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with Phe-Tyr (Phe-Tyr-PLGA NPs) for treating hypertension and cardiovascular diseases was prepared in this study. In the experiments, poly(lactic-co-glycolic acid) (PLGA) and Phe-Tyr dipeptide-loaded PLGA nanoparticles were prepared using the double emulsion (w/o/w) method. The characterizations of the nanoparticles were performed with a UV–vis spectrometer, the Zeta-sizer system, and FTIR spectrometer. The optimum size of the Phe-Tyr dipeptide-loaded PLGA nanoparticle was obtained with a 213.8 nm average particle size, and a 0.061 polydispersity index, ?19.5 mV zeta potential, 34% of loaded and 90.09% of encapsulation efficiency. From TEM analysis, it was clearly seen that the dipeptide loaded nanoparticles had the spherical and non-aggregated morphology and Phe-Tyr dipeptide loaded-PLGA nanoparticles were obtained successfully. Cell toxicity of nanoparticles at different concentrations was assayed with XTT methods on L929 fibroblast cells. This study determined that the nanoparticles have low toxicity at lower concentration and toxicity augmented with increasing concentration of dipeptide. To analyze the effect of solvents on structure of Phe-Tyr, Molecular dynamics simulation was performed with GROMACS program and molecular orbital calculations were carried out to obtain structural and electronic properties of dipeptide. Moreover, molecular docking calculations were also employed to model and predict protein–drug interactions.     

Impact of Laboratory Test Use Strategies in a Turkish Hospital

ObjectivesEliminating unnecessary laboratory tests is a good way to reduce costs while maintain patient safety. The aim of this study was to define and process strategies to rationalize laboratory use in Ankara Numune Training and Research Hospital (ANH) and calculate potential savings in costs.MethodsA collaborative plan was defined by hospital managers; joint meetings with ANHTA and laboratory professors were set; the joint committee invited relevant staff for input, and a laboratory efficiency committee was created. Literature was reviewed systematically to identify strategies used to improve laboratory efficiency. Strategies that would be applicable in local settings were identified for implementation, processed, and the impact on clinical use and costs assessed for 12 months.ResultsLaboratory use in ANH differed enormously among clinics. Major use was identified in internal medicine. The mean number of tests per patient was 15.8. Unnecessary testing for chloride, folic acid, free prostate specific antigen, hepatitis and HIV testing were observed. Test panel use was pinpointed as the main cause of overuse of the laboratory and the Hospital Information System test ordering page was reorganized. A significant decrease (between 12.6–85.0%) was observed for the tests that were taken to an alternative page on the computer screen. The one year study saving was equivalent to 371,183 US dollars.ConclusionHospital-based committees including laboratory professionals and clinicians can define hospital based problems and led to a standardized approach to test use that can help clinicians reduce laboratory costs through appropriate use of laboratory tests.     

Evaluation of the kinetics and mechanism of action of anti‐integration host factor‐mediated disruption of bacterial biofilms

The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice‐like network wherein a member of the DNABII family of DNA‐binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using non‐typeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF‐specific and mediated by binding of transiently dissociated IHF by anti‐IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA‐binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.     

The Genome Sequence of the Cyanobacterium Oscillatoria sp. PCC 6506 Reveals Several Gene Clusters Responsible for the Biosynthesis of Toxins and Secondary Metabolites

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites.The presence in the environment of cyanobacteria producing toxins represents a risk for human and animal health (5). It is thus important to develop methodologies to detect these toxic microorganisms in water reservoirs, and so far, PCR-based amplification of genes responsible for the biosynthesis of these toxins is the best method (1). We have been interested in the study of cyanobacteria producing the neurotoxins anatoxin-a and homoanatoxin-a (2, 17, 18). Our goals were first to identify the genes responsible for the biosynthesis of these toxins and to deepen our knowledge of this interesting group of bacteria. For that, we chose Oscillatoria sp. PCC 6506 (4, 6, 14), a filamentous benthic cyanobacterium, as our model microorganism and initiated the sequencing of its genome.Whole-genome shotgun sequencing of Oscillatoria sp. PCC 6506 was performed using the 454 technology (GATC, Konstanz, Germany), giving 677,304 reads, with a total number of 164 megabases, representing an estimated coverage of 24. Assembly yielded 419 contigs consisting of 6,729,249 bases with a G+C content of 43.40%. Automatic annotation of this genome using the MaGe pipeline system (16) predicted 6,007 coding sequences (CDS), 61 fragments of CDS, and 84 genes coding for RNA (4 rRNA genes, 70 tRNA genes, and 10 small noncoding RNAs [ncRNAs]).Oscillatoria sp. PCC 6506 is an aerobic photosynthetic and diazotrophic cyanobacterium that shows transient and dispersed gas vesicles (4, 6). We found 37 genes coding for proteins involved in photosynthesis and the nif gene cluster, responsible for nitrogen fixation. However, we could not identify any gene coding for a hydrogenase (15), as for Synechococcus sp. {"type":"entrez-nucleotide","attrs":{"text":"BG043511","term_id":"12489990","term_text":"BG043511"}}BG043511 (8). Four genes were annotated as gas vesicle protein (gvp) genes (13).Oscillatoria sp. PCC 6506 produces anatoxin-a and homoanatoxin-a, and we recently identified the ana cluster of genes, responsible for the biosynthesis of these alkaloids (3, 10, 11). We also identified the cluster of genes (cyr) responsible for the biosynthesis of cylindrospermopsin (9, 12). Cyanobacteria are known to produce a wide range of secondary metabolites (7), and 3.7% of the total annotated CDS in this genome were dedicated to secondary metabolism. We identified, beside the ana and cyr clusters, four other clusters of genes containing polyketide synthase and nonribosomal peptide synthase coding genes, likely involved in the biosynthesis of secondary metabolites.We found 51 transposase genes in the Oscillatoria sp. PCC 6506 genome, some close to the biosynthetic gene clusters identified, suggesting horizontal gene transfer events.In conclusion, we present here the sequence of the first genome of an Oscillatoria cyanobacterium strain, a very common genus found in the environment, and the first genome of a neurotoxin-producing cyanobacterium.     

Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (?Ψ_m) in living cells and tissues at low values (100-140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ?Ψ_m values (180-220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ?Ψ_m from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ?Ψ_m is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ?Ψ_m was found. From the data we conclude that high matrix ATP/ADP ratios keep ?Ψ_m at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ?Ψ_m-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ?Ψ_m according to the Mitchell Theory.     

Infection with Helicobacter pylori Is Associated with Protection against Tuberculosis

Background

Helicobacter pylori, a lifelong and typically asymptomatic infection of the stomach, profoundly alters gastric immune responses, and may benefit the host in protection against other pathogens. We explored the hypothesis that H. pylori contributes to the control of infection with Mycobacterium tuberculosis.

Methodology/Principal Findings

We first examined M. tuberculosis-specific IFN-γ and H. pylori antibody responses in 339 healthy Northern Californians undergoing routine tuberculin skin testing. Of 97 subjects (29%) meeting criteria for latent tuberculosis (TB) infection (LTBI), 45 (46%) were H. pylori seropositive. Subjects with LTBI who were H. pylori-seropositive had 1.5-fold higher TB antigen-induced IFN-γ responses (p = 0.04, ANOVA), and a more Th-1 like cytokine profile in peripheral blood mononuclear cells, compared to those who were H. pylori seronegative. To explore an association between H. pylori infection and clinical outcome of TB exposure, we evaluated H. pylori seroprevalence in baseline samples from two high risk TB case-contact cohorts, and from cynomolgus macaques experimentally challenged with M. tuberculosis. Compared to 513 household contacts who did not progress to active disease during a median 24 months follow-up, 120 prevalent TB cases were significantly less likely to be H. pylori infected (AOR: 0.55, 95% CI 0.0.36–0.83, p = 0.005), though seroprevalence was not significantly different from non-progressors in 37 incident TB cases (AOR: 1.35 [95% CI 0.63–2.9] p = 0.44). Cynomolgus macaques with natural H. pylori infection were significantly less likely to progress to TB 6 to 8 months after M. tuberculosis challenge (RR: 0.31 [95% CI 0.12–0.80], p = 0.04).

Conclusions/Significance

H. pylori infection may induce bystander effects that modify the risk of active TB in humans and non-human primates. That immunity to TB may be enhanced by exposure to other microbial agents may have important implications for vaccine development and disease control.