Periplaneta americana Arginine Kinase as a Major Cockroach Allergen among Thai Patients with Major Cockroach Allergies

Periplaneta americana is the predominant cockroach (CR) species and a major source of indoor allergens in Thailand. Nevertheless, data on the nature and molecular characteristics of its allergenic components are rare. We conducted this study to identify and characterize the P. americana allergenic protein. A random heptapeptide phage display library and monoclonal antibody (MAb) specific to a the P. americana component previously shown to be an allergenic molecule were used to identify the MAb-bound mimotope and its phylogenic distribution. Two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, peptide mass fingerprinting, and BLAST search were used to identify the P. americana protein containing the MAb-specific epitope. We studied the allergenicity of the native protein using sera of CR-allergic Thai patients in immunoassays. The mimotope peptide that bound to the MAb specific to P. americana was LTPCRNK. The peptide has an 83–100% identity with proteins of Anopheles gambiae, notch homolog scalloped wings of Lucilia cuprina, delta protein of Apis mellifera; neu5Ac synthase and tyrosine phosphatase of Drosophila melanogaster, and a putative protein of Drosophila pseudoobscura. This finding implies that the mimotope-containing molecule of P. americana is a pan-insect protein. The MAb-bound protein of P. americana was shown to be arginine kinase that reacted to IgE in the sera of all of the CR-allergic Thai patients by immunoblotting, implying its high allergenicity. In conclusion, our results revealed that P. americana arginine kinase is a pan-insect protein and a major CR allergen for CR-allergic Thai patients

Identification of VPA1327 (vopT) as a Novel Genetic Marker for Detecting Pathogenic Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative bacterium that is ubiquitous in marine and estuarine environments. The pathogenic strains can cause foodborne gastroenteritis especially when raw or undercooked seafood contaminated with pathogenic V. parahaemolyticus are consumed. This study aims to obtain a novel species-specific genetic marker using comparative genomics and to investigate their prevalence in both clinical and seafood isolates of V. parahaemolyticus. VPA1327 (vopT) was identified as a unique gene of this organism, but it was reported present in only 33.3% of V. parahaemolyticus (n=72 isolates). Interestingly, vopT was found exclusively in clinical isolates. The combination PCR analysis was used to determine the presence of three genes (tdh, trh, and vopT) in 36 clinical isolates. Four genotypes were classified namely, tdh+trh-vopT + (24 isolates, 66.7%), tdh+trh+vopT - (2 isolates, 5.6%), tdh-trh+vopT - (2 isolates, 5.6%) and tdh-trh-vopT- (8 isolates, 22.2%). Furthermore, all vopT+ isolates were tdh+ isolates, and none of them coexist with trh+ isolates. This study has provided strong evidence to support vopT as an additional virulence marker for the detection of V. parahaemolyticus with the pathogenic potential to cause human disease

Effect of rhodomyrtone on iNOS expression in THP-1 monocytes.

<p>THP-1 cells were treated with rhodomyrtone at concentrations from 0.195 to 1.56 µg/ml for 24 h. Cells were collected and RNA was extracted for the determination of iNOS mRNA levels using real-time PCR (A). Gene expression was calculated relative to β-actin mRNA and the untreated monocytes and expressed as log₂ fold change (ΔΔCt). Data are the mean ± SEM from three independent experiments and two technical replications. * p<0.05, compared with the untreated controls. # p<0.05, compared with the monocytes treated with LPS. THP-1 monocytes were stimulated with heat-killed MRSA at 10⁶, 10⁷, 10⁸, and 10⁹ cfu/ml, followed by rhodomyrtone (0.39 or 1.56 µg/ml) for 24 h. Cells were collected and RNA was extracted for the determination of iNOS mRNA levels by real-time PCR (B). * p<0.05, compared to no rhodomyrtone controls. The supernatant was collected and used to measure NO production using the Griess assay (C). Data are present the mean ± SEM from three independent experiments and two technical replicates. * p<0.05, compared with untreated monocytes.</p

Effect of rhodomyrtone on the expression of TLR2 and CD14 by THP-1 monocytes stimulated with MRSA.

<p>THP-1 monocytes were stimulated with heat-killed MRSA at 10⁶, 10⁷, 10⁸ or 10⁹ cfu/ml, followed by treatment rhodomyrtone (0.39 or 1.56 µg/ml) for 6 h. Cells were collected and RNA was assayed for the amount of mRNA for TLR2 (A) and CD14 (B) using real-time PCR. Data are the mean ± SEM from three separate experiments and two technical replicates. * p<0.05, ** p<0.01, *** p<0.001, compared with no rhodomyrtone controls. ‡ p<0.01, compared with 0.39 µg/ml rhodomyrtone treatment alone. # p<0.001, compared with untreated monocytes. Median fluorescent intensity (MFI) of CD14 (C) expressed on the cell surface of THP-1 cells untreated or treated with PMA or rhodomyrtone (0.39 or 1.56 µg/ml) for 6 h. *** p<0.001, compared with the untreated monocytes.</p

Phagocytic activity of THP-1 monocytes determined by flow cytometry.

<p>THP-1 monocytes were treated with or without 1.56 µg/ml rhodomyrtone, after which they were incubated with CFSE-labeled killed MRSA at ratios of 1∶100 and 1∶1,000 (monocytes:bacteria) for 30 or 60 min. Experimental data are shown in the top panel and summarized in the bottom panel, where data are the mean ± SD percent phagocytic activity of monocytes, determined from the mean fluorescence intensity compared to control monocytes.</p

Primers for real-time PCR.

<p>Primers for real-time PCR.</p

Isolation and Characterization of scFv Antibody against Internal Ribosomal Entry Site of Enterovirus A71

Enterovirus A71 (EV-A71) is one of the causative agents of hand-foot-mouth disease, which can be associated with neurocomplications of the central nervous system. A limited understanding of the virus’s biology and pathogenesis has led to the unavailability of effective anti-viral treatments. The EV-A71 RNA genome carries type I internal ribosomal entry site (IRES) at 5′ UTR that plays an essential role in the viral genomic translation. However, the detailed mechanism of IRES-mediated translation has not been elucidated. In this study, sequence analysis revealed that the domains IV, V, and VI of EV-A71 IRES contained the structurally conserved regions. The selected region was transcribed in vitro and labeled with biotin to use as an antigen for selecting the single-chain variable fragment (scFv) antibody from the naïve phage display library. The so-obtained scFv, namely, scFv #16-3, binds specifically to EV-A71 IRES. The molecular docking showed that the interaction between scFv #16-3 and EV-A71 IRES was mediated by the preferences of amino acid residues, including serine, tyrosine, glycine, lysine, and arginine on the antigen-binding sites contacted the nucleotides on the IRES domains IV and V. The so-produced scFv has the potential to develop as a structural biology tool to study the biology of the EV-A71 RNA genome

Effect of rhodomyrtone on the bactericidal activity of phagocytes.

<p>THP-1 monocytes (A) and PMA-activated THP-1 monocytes (B) were incubated with MRSA at the multiplicity of infection of 1 for 30 min. Cells were treated with rhodomyrtone at 0.0975 (0.25MIC), 0.39 (1MIC), or 1.56 (4MIC) µg/ml for the times indicated. Negative-control cells were treated with the vehicle (0.0078% DMSO) alone. Data are the mean ± SD of 3 independent experiments. ** p<0.01, compared with DMSO controls.</p

Pro-inflammatory cytokine expression by THP-1 monocytes in response to MRSA stimulation, followed by rhodomyrtone treatment.

<p>The monocytes were incubated with heat-killed MRSA at 10⁶, 10⁷, 10⁸, and 10⁹ cfu/ml, followed by rhodomyrtone treatment (0.39 and 1.56 µg/ml) for 6 h. Monocytes were collected and RNA was isolated for the measurement of mRNA levels of IL-1β (A), TNF-α (B), and IL-6 (C) by real-time PCR. Gene expression was calculated relative to β-actin gene and expressed as log₂ fold change. The data are the mean ± SEM from three independent experiments and two technical replicates. p values were calculated with reference to no rhodomyrtone values at corresponding doses. * p<0.05, ** p<0.01, *** p<0.001. # p<0.001, compared with the unstimulated monocytes.</p