Detection of Brugia malayi in laboratory and wild-caught Mansonioides mosquitoes (Diptera: Culicidae) using Hha I PCR assay

An Hha 1 based polymerase chain reaction (PCR) assay developed for the detection of Brugia malayi, the causative agent of Brugian lymphatic filariasis, was evaluated for its sensitivity in the laboratory and for its usefulness in measuring changes in transmission of the disease in the field. Laboratory studies showed that the new assay was highly sensitive in comparison with the standard dissection and microscopy technique. The assay can detect as little as 4 pg of parasite DNA or a single microfilaria in pools of up to 100 mosquitoes. The optimum pool size for convenience was found to be 50 mosquitoes per pool. The efficacy of PCR assay was evaluated in filariasis control programmes in operation in endemic areas of Kerala State, South India. The infection rates obtained by the Hha I PCR assay and the conventional dissection and microscopy technique were 1.2% and 1.7% respectively in operational areas and 8.3% and 4.4% respectively, in check areas, which were not significantly different (P < 0.05). Thus, the Hha I PCR assay was found to be as sensitive as the conventional technique and hence it can be used as a new epidemiological tool for assessing parasite infection in field-collected mosquitoes.     

Nanoscale organization of hedgehog is essential for long-range signaling

Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: An organochlorine pesticide

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL⁻¹ to 1000 ng mL⁻¹ to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL⁻¹ with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.     

Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells

Lack of effective treatment options for the management of hormone refractory prostate cancer (PCA) reinforce the great need to develop novel compounds that act singly or in combination. 2-Methoxyestradiol (2-ME₂) is an endogenous estrogenic metabolite that has been reported to work as an antiproliferative agent in various tumor models including prostate. Recently conducted clinical trial in hormone refractory prostate cancer (HRPC) patients concluded that 2-ME₂ was safe and well tolerated. However this study identified bioavailability of 2-ME₂ as a limiting factor. Here we report the ability of a combination of 2-ME₂ and eugenol (4-allyl-2-methoxyphenol) as an approach for enhancing anticancerous activities in prostate cancer cells. Combining 2-ME₂ with eugenol (i) inhibited growth of prostate cancer cells and induced apoptosis at lower concentrations than either single agent alone; (ii) analysis of the data using combination index (CI) showed CI values of 0.4 indicating strong synergistic interaction; (iii) increased population of cells G₂/M phase by 4.5-fold (p = 0.01); (iv) significantly reduced expression of antiapoptotic protein Bcl-2 and enhanced expression of proapoptotic protein Bax. Combination induced apoptosis was not affected in PC-3 cells that over-express or lack Bcl-2 but was associated with loss of mitochondrial membrane potential. Since 2-ME₂ was well tolerated in phase II trail in patients with HRPC; and eugenol is consumed by humans in the form of spices, the combination of 2-ME₂ with eugenol may offer a new clinically relevant treatment regimen. Combining these agents may allow ameliorating any adverse effects of either 2-ME₂ or eugenol alone by reducing their individual concentrations should these two agents be developed for human use.     

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC₅₀ dose of 1 μg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13–C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.     

Characterization of an immortalized oviduct cell line from the cynomolgus monkey (Macaca fascicularis)

To establish reproductive biological techniques in mammals, it is important to understand the growth environment of the embryo. Oviduct epithelial cells are in close proximity to the embryo during pre-implantation development. We, therefore, established an immortalized oviduct epithelial cell line from the cynomolgus monkey, evaluated the usefulness of these cells as feeder cells for embryo culture, and investigated the gene expression of several growth factors and cytokines in the cells. The immortalized cells were positive for the anti-cytokeratin antibody, as determined by immunocytochemistry, indicating that they are epithelial. They also expressed oviductin, which is specific to oviduct epithelial cells, glyceraldehyde-3-phosphate dehydrogenase (control), leukemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor 1, transforming growth factor beta-2, and interleukin 4. Mouse embryo development was improved when the immortalized cells were used as feeder cells. This cell line is also useful for studying the factors secreted by oviduct epithelial cells.     

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods

Aims: The study describes the development of simple and rapid DNA extraction method in combination with loop‐mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. Methods and Results: In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X‐100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. Conclusions: The template DNA extraction method developed in this study for food samples is simple, rapid and cost‐effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. Significance and Impact of the Study: The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.