<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> as a live vehicle for oral immunization against chicken anemia virus

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens.     

Differential suppression of menstrual fluid prostaglandin F2a, prostaglandin E2, 6-keto prostaglandin F1a and thromboxane B2 by suprofen in women with primary dysmenorrhea

Eleven women with primary dysmenorrhea completed a randomized, double-blind, placebo-controlled, three-way cross-over study comparing 200 and 400mg suprofen. Menstrual fluid volume did not change. Mean+/-S.E.M. menstrual fluid PGF2a was significantly suppressed from 18.9+/-1.9 microg (placebo) to 10.9+/-1.7 and 9.3+/-2.1 microg with 200 and 400 mg suprofen, respectively (p=<0.005). PGE2 dropped from 7.8+/-0.9 to 4.6+/-0.8 and 4.6+/-1.1 microg (p=<0.05) and TxB2 from 17.5+/-4.3 to 7.5+/-2.9 and 3.6+/-1.3 microg (p=<0.01), respectively. 6-Keto PGF1a was significantly suppressed (2.7+/-0.4 to 1.9+/-0.5 microg, p=<0.025) with only 400 mg suprofen. Six subjects rated placebo poor and five fair to very good. In contrast, nine rated suprofen excellent to fair while two rated poor. Thus, suprofen was clinically effective but the differential suppression of prostanoids favors 200mg which spares 6-keto PGF1a.     

Comparative proteomic analysis of different developmental stages of the edible mushroom Termitomyces heimii

Background

Termitomyces heimii is a basidiomycete fungus that has a symbiotic relationship with termites, and it is an edible mushroom with a unique flavour and texture. T. heimii is also one of the most difficult mushrooms to cultivate throughout the world. Little is known about the growth and development of these mushrooms, and the available information is insufficient or poor. The purpose of this study was to provide a base of knowledge regarding the biological processes involved in the development of T. heimii. The proteomic method of 2 dimensional difference gel electrophoresis 2D-DIGE was used to determine and examine the protein profiles of each developmental stage (mycelium, primordium and fruiting body). Total proteins were extracted by TCA-acetone precipitation.

Results

A total of 271 protein spots were detected by electrophoresis covering pH 3–10 and 10–250 kDa. Selected protein spots were subjected to mass spectrometric analyses with matrix-assisted laser desorption/ionisation (MALDI TOF/TOF). Nineteen protein spots were identified based on peptide mass fingerprinting by matching peptide fragments to the NCBI non-redundant database using MASCOT software. The 19 protein spots were categorised into four major groups through KEGG pathway analysis, as follows: carbohydrate metabolism, energy metabolism, amino acid metabolism and response to environmental stress.

Conclusions

The results from our study show that there is a clear correlation between the changes in protein expression that occur during different developmental stages. Enzymes related to cell wall synthesis were most highly expressed during fruiting body formation compared to the mycelium and primordial stages. Moreover, enzymes involved in cell wall component degradation were up-regulated in the earlier stages of mushroom development.     

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

Background

Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.

Methods and Findings

CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates.

Conclusions

The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.     

Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium

This study was designed to investigate the ability of Clostridium saccharoperbutylacetonicum N1-4 to produce butanol in a limited nutrient medium using mixtures of glucose and butyric acid as substrates. Specific combinations of glucose and butyric acid were found to influence the enhancement and retardation of butanol production as well as the reduction and modulation of the number of bacterial cells. Increasing the butyric acid concentration leads to the inhibition of bacterial growth, whereas the presence of (0?C5?g/L) butyric acid and (0?C10?g/L) glucose enhances the butanol production. The combination of 5?g/L butyric acid with 5 and 10?g/L of glucose was found to be the most suitable, but the use of glucose at concentrations greater than 10?g/L shifted the optimal butyric acid concentrations to 10 and 15?g/L for maximum butanol production signifying the requirement of a specific combination of glucose and butyric acid for enhanced butanol production in the fermentation process. C. saccharoperbutylacetonicum N1-4 demonstrated the ability to produce butanol in the absence of glucose, but no acetone or ethanol was produced under these conditions, reflecting the nature of the pathways involved in the production of butanol using only butyric acid. Ten grams per litre of butyric acid was found able to produce 13?g/L of butanol in the presence of 20?g/L of glucose, and 0.7?g/L butanol was produced in the absence of glucose. This study indicates the importance of the glucose to butyric acid ratio to the enhancement of butanol production.     

Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters

ABSTRACT: Enterovirus 71 (EV71) causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP11-100) protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP11-100 candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP11-100 candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP11-100 recombinant protein as a candidate vaccine in an alternative animal model system.     

In Vitro Antifungal Susceptibility of <Emphasis Type="Italic">Neoscytalidium dimidiatum</Emphasis> Clinical Isolates from Malaysia

Neoscytalidium dimidiatum is an opportunistic fungus causing cutaneous infections mostly, which are difficult to treat due to antifungal resistance. In Malaysia, N. dimidiatum is associated with skin and nail infections, especially in the elderly. These infections may be mistaken for dermatophyte infections due to similar clinical appearance. In this study, Neoscytalidium isolates from cutaneous specimens, identified using morphological and molecular methods (28 Neoscytalidium dimidiatum and 1 Neoscytalidium sp.), were evaluated for susceptibility towards antifungal agents using the CLSI broth microdilution (M38-A2) and Etest methods. Amphotericin B, voriconazole, miconazole and clotrimazole showed high in vitro activity against all isolates with MIC ranging from 0.0313 to 1 µg/mL. Susceptibility towards fluconazole and itraconazole was noted in up to 10% of isolates, while ketoconazole was inactive against all isolates. Clinical breakpoints for antifungal drugs are not yet available for most filamentous fungi, including Neoscytalidium species. However, the results indicate that clinical isolates of N. dimidiatum in Malaysia were sensitive towards miconazole, clotrimazole, voriconazole and amphotericin B, in vitro.     

Gelam Honey Attenuates Carrageenan-Induced Rat Paw Inflammation via NF-κB Pathway

The activation of nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of a number of inflammatory diseases. In this study, we investigated the anti-inflammatory mechanism of Gelam honey in inflammation induced rats via NF-κB signalling pathway. Rats paw edema was induced by subplantar injection of 1% carrageenan into the right hind paw. Rats were pre-treated with Gelam honey at different doses (1 or 2 g/kg, p.o.) and NSAID Indomethacin (10 mg/kg, p.o.), in two time points (1 and 7 days). Our results showed that Gelam honey at both concentrations suppressed the gene expressions of NF-κB (p65 & p50) and IκBα in inflamed rats paw tissues. In addition, Gelam honey inhibited the nuclear translocation and activation of NF-κB and decreased the cytosolic degradation of IκBα dose dependently in inflamed rats paw tissues. The immunohistochemical expressions of pro-inflammatory mediators COX-2 and TNF-α were also decreased in inflamed rats paw tissues when treated with Gelam honey. The results of our findings suggest that Gelam honey exhibits its inhibitory effects by attenuating NF-κB translocation to the nucleus and inhibiting IκBα degradation, with subsequent decrease of inflammatory mediators COX-2 and TNF-α.     

Isolation of trinucleotide microsatellite markers for Mystus nemurus

Twelve single-locus trinucleotide microsatellite markers were developed to characterize the Asian river catfish, Mystus nemurus, an important food fish in Southeast Asia. They were obtained by using a rapid method, namely, the 5′ anchored PCR enrichment protocol. The specific primers were designed to flank the repeat sequences and these were subsequently used to characterize 90 unrelated fish from Malaysia. The number of alleles per locus ranged from 2 (MnVj2-281) to 12 (MnBp8-4-43b) while the levels of heterozygosity ranged from 0.0444 (MnVj2-1-19) to 0.7458 (MnVj2-291). The text was submitted by the authors in English.