Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Traditional Chinese medicine Bak Foong Pills alters uterine quiescence – Possible role in alleviation of dysmenorrhoeal symptoms

Since contractility of the uterus appears to be the major source of pain during dysmenorrhoea, alleviation of the contractions is believed to be a possible treatment strategy. Bak Foong Pills, a traditional Chinese formulation for use in gynaecological disorders, has long been thought as effective in the treatment of dysmenorrhoeal symptoms. The present study thus aims to investigate whether ethanol extract of Bak Foong Pills (BFP-Ex) or its constituent herbs may have direct effects on alleviating dysmenorrhoeal symptoms by altering uterine tone. This was investigated using isolated uterine preparations and intracellular messenger analysis of adenylate cyclase, via [³H]-adenine assay, and calcium, with fluorometry imaging, in myometrial cultures. BFP-Ex can stimulate uterine relaxation following oxytocin-induced contractions ex-vivo. Attempted inhibition of BFP-Ex's relaxatory response with a nitric oxide inhibitor and adenylate cyclase inhibitor, however, had no significant effect, suggesting that most of BFP-Ex's relaxatory response was not due to increases in NO or cAMP. Further studies on tetramethylpyrazine (TMP), a major active ingredient of BFP-Ex, indicated that TMP could modulate intracellular calcium levels in favour of uteri relaxation. The ability of Bak Foong Pills to alleviate menstrual pain may be due to direct regulation of uterine tone.     

The quest for the function of simple epithelial keratins

Simple epithelial keratins K8 and K18 are components of the intracellular cytoskeleton in the cells of the single-layered sheet tissues inside the body. As members of the intermediate filament family of proteins, their function has been a matter for debate since they were first discovered. Whilst there is an indisputable case for a structural cell-reinforcing function for keratins in the mutilayered squamous epithelia of external barrier tissues, some very different stress-protective features now seem to be emerging for the simple epithelial keratins. Even the emerging evidence of pathological mutations in K8/K18 looks very different from mutations in stratified epithelial keratins. K8/K18-like keratins were probably the first to evolve and, whilst stratified epithelial (keratinocyte) keratins have diversified into a large group of keratins highly specialised for providing mechanical stability, the simple epithelial keratins have retained early features that may protect the internal epithelia from a broader range of stresses, including osmotic stress and chemical toxicity.     

Solvent accessibility in native and isolated domain environments: general features and implications to interface predictability

A non-redundant database of 4536 structural domains, comprising more than 790,000 residues, has been used for the calculation of their solvent accessibility in the native protein environment and then in the isolated domain environment. Nearly 140,000 (18%) residues showed a change in accessible surface area in the above two conditions. General features of this change under these two circumstances have been pointed out. Propensities of these interfacing amino acid residues have been calculated and their variation for different secondary structure types has been analyzed. Actual amount of surface area lost by different secondary structures is higher in the case of helix and strands compared to coil and other conformations. Overall change in surface area in hydrophobic and uncharged residues is higher than that in charged residues. An attempt has been made to know the predictability of interface residues from sequence environments. This analysis and prediction results have significant implications towards determining interacting residues in proteins and for the prediction of protein-protein, protein-ligand, protein-DNA and similar interactions.     

Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis - a multicenter trial

The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs.     

Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.     

High environmental temperature and low pH stress alter the gill phenotypic plasticity of Hoven's carp Leptobarbus hoevenii

Climate warming and low pH environment are known to negatively impact all levels of aquatic organism from cellular to organism and population levels. For ammonotelic freshwater species, any abiotic factor fluctuation will cause disturbance to the fish, specifically at the gills which act as a multifunctional organ to support all biological processes. Therefore, this study was designed to investigate the effect of temperature (28 vs. 32°C) and pH (7.0 vs. 5.0) stress on the gill plasticity of Hoven's carp after 20 days of continuous exposure. The results demonstrated that high temperature and low pH caused severe changes on the primary and secondary lamellae as well as the cells within lamellae. An increasing trend of the proportion available for gas exchange was noticed at high temperature in both pH exposures, which resulted from a reduction of the primary lamellae width with elongated and thinner secondary lamellae compared to fishes at ambient temperature. Following exposure to high temperature and acidic pH, Hoven's carp experienced gill modifications including aneurysm, oedema, hypertrophy, curling of secondary lamellae, epithelial lifting, hyperplasia and lamellae fusion. These modifications are indicators of the coping mechanism of Hoven's carp to the changing environment in order to survive.     

Interactive effect of nitrogen fertilizer and plant density on photosynthetic and agronomical traits of cotton at different growth stages

Individual effects of application of nitrogen (N) and plant densities (PD) were reported in various studies; however an interactive effect of N and PD in cotton was not studied. To explore the benefits of interactive effects of N fertilizer and PD to increase the quality of cotton. This study was carried out in randomized complete block design (RCBD) with split plot arrangement. In split plot arrangement, main plot was consisted of N application rate and in sub plots different PD were done. There were two nitrogen levels; low N level (F1) 120 kg ha⁻¹ and high N level (F2) 180 kg ha⁻¹ and three planting densities; 8 plants m⁻² as low density (LD), 10 plants m⁻² as medium density (MD) and 12 plants m⁻² as high density (HD). In this study we observed the interactive effect of N application levels and PD on cotton photosynthetic and agronomic traits of various stages of development. Results showed that cotton growth and N contents was varied among treatments on different development stages. Plant biomass production, photosynthetic rate (Pn), intercellular CO₂ (C_i), water use efficiency (WUE) and N contents were unaffected at the seedling stage by N application rate and PD, however, the highest Pn, C_i and N contents was at squaring stage followed by blooming stage. Higher seed cotton yield and lint yield were obtained F1 with HD, and F2 with MD yielded the highest N contents and cotton yield among treatments. We found that the squaring stage was more critical, followed by the blooming stage when considering N rate and PD.     

Genotyping of Toxoplasma gondii Isolates from Wild Boars in Peninsular Malaysia

Toxoplasma gondii is a parasitic protozoan that infects nearly one-third of the world population. The present study was done to isolate and genotype T. gondii from wild boar from forests of Pahang, Malaysia. A total of 30 wild boars'' blood, heads and hearts were obtained for this study and 30 (100.0%) were found to be seropositive when assayed with modified agglutination test (MAT≥6). The positive samples were inoculated into mice and T. gondii was only isolated from samples that had strong seropositivity (MAT≥1∶24).The isolates were subjected to PCR-RFLP analysis and all the Peninsular Malaysia isolates of T. gondii are of clonal type I.