Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein.

Dengue virus (DENV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADE) of infection. Phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DENV infection. This study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection.

Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand.

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.

Helicobacter pylori cagA, vacA and iceA genotypes in northern Thai patients with gastric disease.

Helicobacterpylori, a common infectious bacterium, has been linked to chronic gastritis, peptic ulcer and gastric cancer. Gastric biopsy specimens were obtained from 58 northern Thai patients with gastritis, 28 with gastric ulcer, 45 with duodenal ulcer and 4 with gastric cancer. cagA, vacA s1 and iceA gene was found in 88, 98, and 89% of the specimens, respectively. For vacA, the frequency of subtype s1a, s1c and combined sla and s1c was 40, 16, and 41%, respectively. The frequency of subtype s1a/m1 and s1a/s1c/m1 was 27 and 20%, respectively. Fifty-three patients (39%) were infected with multiple vacA genotypes but there was no association with clinical outcome. cagA positive and mixed vacA s1a and s1c strains were found in significantly more cases of duodenal ulcer than gastritis (p < 0.05). For iceA, subtype iceA1 reached a frequency of 60%, whereas subtype iceA2 was only 24%.

Use of different PCR primers and gastric biopsy tissue from CLO test for the detection of Helicobacter pylori.

Four different DNA loci were assessed for the detection of H. pylori by PCR on gastric biopsy specimens. PCR, with a primer specific 860 bp DNA fragment, was the most sensitive, with a detection limit of 0.02 pg H. pylori DNA, corresponding to approximately 10 organisms. Nested-PCR of the 860-bp DNA fragment was 10-fold more sensitive than single-step PCR. The sensitivity and specificity of the four PCR methods, in comparison to the results obtained from histology and the urease test, are as follows: 80.7% and 76% for the hpaA gene; 100% and 76% for the 16S rRNA gene; 84.6% and 80.0% for the 860-bp DNA fragment; 61.5% and 84.0% for the ureC (glmM) gene, respectively. The sensitivity of nested-PCR for the 860-bp DNA fragment was 100%. This nested-PCR gave positive results for eight specimens which were negative by conventional methods. PCR can be performed on gastric biopsy specimens obtained from the CLO test.