Comparative analysis of midgut bacterial communities in three aedine mosquito species from dengue-endemic and non-endemic areas of Rajasthan, India.

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue-endemic and non-endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.-related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue-endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real-time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.

Microbial community structure at different fermentation stages of kutajarista, a herbal formulation.

Kutajarista is an Ayurvedic fermented herbal formulation prescribed for gastrointestinal disorders. This herbal formulation undergoes a gradual fermentative process and takes around 2 months for production. In this study, microbial composition at initial stages of fermentation of Kutajarista was assessed by culture independent 16S rRNA gene clone library approach. Physicochemical changes were also compared at these stages of fermentation. High performance liquid chromatography-mass spectrometry analysis showed that Gallic acid, Ellagic acid, and its derivatives were the major chemical constituents recovered in this process. At 0 day of fermentation, Lactobacillus sp., Acinetobacter sp., Alcaligenes sp., and Methylobacterium sp. were recovered, but were not detected at 8 day of fermentation. Initially, microbial diversity increased after 8 days of fermentation with 11 operational taxonomic units (OTUs), which further decreased to 3 OTUs at 30 day of fermentation. Aeromonas sp., Pseudomonas sp., and Klebsiella sp. dominated till 30 day of fermentation. Predominance of γ- Proteobacteria and presence of gallolyl derivatives at the saturation stage of fermentation implies tannin degrading potential of these microbes. This is the first study to highlight the microbial role in an Ayurvedic herbal product fermentation.

DNA barcoding of nymphalid butterflies (Nymphalidae: Lepidoptera) from Western Ghats of India.

We have checked the utility of DNA barcoding for species identification of nymphalid butterflies from Western Ghats of India by using 650 bp sequence of mitochondrial gene cytochrome c oxidase subunit I. Distinct DNA barcoding gap (i.e. difference between intraspecies and interspecies nucleotide divergence), exists between species studied here. When our sequences were compared with the sequences of the conspecifics submitted from different geographic regions, nine cases of deep intraspecies nucleotide divergences were observed. In spite of this, NJ (Neighbour Joining) clustering analysis successfully discriminated all species. Observed cases of deep intraspecies nucleotide divergences certainly warrant further study.

Development and characterization of novel cell lines from Etroplus suratensis and their applications in virology, toxicology and gene expression.

Four novel cell lines from tissues of eye, gill, kidney and brain of Etroplus suratensis were developed and characterized. The cell lines of eye, gill, kidney and brain were sub-cultured for 245, 185, 170 and 90 passages, respectively, since 2008. These cell lines showed predominantly epithelial-like cells. Effects of temperature and foetal bovine serum concentration on the growth of these cell lines were examined and optimum growth was found at the temperature of 28° C with 20% foetal bovine serum. All the four cell lines were successfully cryopreserved and revived at different passage levels. Cell-cycle analysis of these cell lines was carried out by fluorescence-activated cell sorting. Polymerase chain reaction (PCR) products obtained from the cells and tissues of E. suratensis with primers specific to the conserved region of 16S ribosomal RNA and cytochrome oxidase I genes of E. suratensis revealed the origin of cell lines from E. suratensis. Antibodies raised against the tissues and cells of eye, kidney and gill were highly cross reacted to their specific tissue and cells of E. suratensis. Chromosomal analysis revealed that E. suratensis cells have a normal diploid karyotype with 2n = 48. The cells of these cell lines were successfully transfected with pEGFP vector DNA. The eye (IEE), gill (IEG) and kidney (IEK) cell lines were found to be susceptible to nodavirus but resistant to infectious pancreatic necrosis virus (IPNV). The cells of gill, kidney and eye were applied to test the cytotoxicity of tannery effluents.

Antibacterial activities of multi drug resistant Myroides odoratimimus bacteria isolated from adult flesh flies (Diptera: sarcophagidae) are independent of metallo beta-lactamase gene.

FLESH FLIES (DIPTERA: Sarcophagidae) are well known cause of myiasis and their gut bacteria have never been studied for antimicrobial activity against bacteria. Antimicrobial studies of Myroides spp. are restricted to nosocomial strains. A Gram-negative bacterium, Myroides sp., was isolated from the gut of adult flesh flies (Sarcophaga sp.) and submitted to evaluation of nutritional parameters using Biolog GN, 16S rRNA gene sequencing, susceptibility to various antimicrobials by disc diffusion method and detection of metallo ß-lactamase genes (TUS/MUS). The antagonistic effects were tested on Gram-negative and Gram-positive bacteria isolated from human clinical specimens, environmental samples and insect mid gut. Bacterial species included were Aeromonas hydrophila, A. culicicola, Morganella morganii subsp. sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp., Serratia sp., Kestersia sp., Ignatzschineria sp., Bacillus sp. The Myroides sp. strain was resistant to penicillin-G, erythromycin, streptomycin, amikacin, kanamycin, gentamycin, ampicillin, trimethoprim and tobramycin. These strain showed antibacterial action against all bacterial strains except W. confusa, Ignatzschineria sp., A. hydrophila and M. morganii subsp. sibonii. The multidrug resistance of the strain was similar to the resistance of clinical isolates, inhibiting growth of bacteria from clinical, environmental and insect gut samples. The metallo ß-lactamase (TUS/MUS) genes were absent, and resistance due to these genes was ruled out, indicating involvement of other secretion machinery.

Onychomycosis due to Achaetomium strumarium.

Onychomycosis is the most common infection of the toe-nails or finger-nails and it may be caused by a large variety of fungal species. Achaetomium species which belong to the phylum Ascomycota (Family Chaetomiaceae), are usually soil saprophytes or endophytic fungi which have been rarely reported as human or animal pathogens. Here, we report a case of onychomycosis caused by Achaetomium strumarium in a healthy person who showed involvement of all fingers of both hands with yellowish brown discoloration. The causative agent isolated was identified as Achaetomium species by morphology, colony morphometry and growth at high temperature and as A. strumarium from DNA sequence of ITS region. Onychomycosis from this case responded satisfactorily with per os (P. O.; oral) and topical application of Terbinafine.

Keratitis by a rare pathogen Colletotrichum gloeosporioides: A case report.

Colletotrichum species have been reported infrequently as the cause of keratitis or subcutaneous lesions. The patient we describe developed keratitis after ocular trauma. The sample from the corneal scrapings grew Colletotrichum gloeosporioides as identified from morphological characters and DNA sequence of the 'Internal Transcribed Spacer' (ITS) region. The patient underwent topical application of amphotericin-B followed by itraconazole and natamycin treatment. Simultaneous oral voriconazole regimen leads to complete regression of corneal ulcer. This report highlights the fact that early and accurate identification and therapy can resolve keratitis caused by rare pathogen C. gloeosporioides.

Uptake of clinical yeast isolates by human epithelial cell line.

OBJECTIVE: The occurrence of yeast infections in humans has increased, with the species belonging to genus Candida still being the most common cause of infection. Nevertheless, infections caused by less common yeasts have been widely reported in recent years. The main objective of this study was to assess the potential of these less common saprophytic yeasts to invade the host cell, which is essential for causing systemic infections. MATERIAL AND METHODS: Various yeast isolates were identified by DNA sequence information of PCR amplified ITS region. The purported saprophytic yeasts were characterized for internalization by mammalian cells in vitro, by staining the F-actin. CONCLUSION: The identification of different yeast isolates from various patients revealed that 70% of the isolates belonged to the genus Candida, while remaining 30% of the isolates were yeasts not belonging to genus Candida. These non-Candida clinical isolates, either in yeast or hyphal forms, were efficiently internalized by human epithelial cells. The internalization was marked by a process of actin polymerization surrounding the invading yeast. Such uptake by epithelial cells signifies traversal of cell barrier by yeast cells during infection in vivo.

Cloning of two hexokinase isoenzyme sequences from Drosophila melanogaster.

Hexokinase coding DM1 and DM2 sequences were obtained from genomic DNA of a Drosophila melanogaster cell line by PCR amplification strategy. Both the sequences were found to encode an enzyme with a molecular weight of 50,000 Da. Amino acid sequence alignment of DM1 and DM2 shows approximately 45% homology with yeast and human hexokinases. The sequences also indicated the presence of conserved amino acid residues and motifs that are present in mammalian hexokinases and are involved in the binding of different substrates. Southern blot analysis suggests that the D. melanogaster genome contain a single copy of DM1 and DM2 sequences. Northern analysis indicates DM1 is expressed as more than one transcript in adult as well as in the D.Mel2 cell line. DM2 is expressed as a single transcript in adult flies. Expression levels for DM1 and DM2 encoded message were found to be similar in different stages of development as seen by RT-PCR. The biotechnological significance of these sequences in metabolic engineering of cells is discussed.

Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species.

Mosquitoes are vectors for the transmission of many human pathogens that include viruses, nematodes and protozoa. For the understanding of their vectorial capacity, identification of disease carrying and refractory strains is essential. Recently, molecular taxonomic techniques have been utilized for this purpose. Sequence analysis of the mitochondrial 16S rRNA gene has been used for molecular taxonomy in many insects. In this paper, we have analysed a 450 bp hypervariable region of the mitochondrial 16S rRNA gene in three major genera of mosquitoes, Aedes, Anopheles and Culex. The sequence was found to be unusually A+T rich and in substitutions the rate of transversions was higher than the transition rate. A phylogenetic tree was constructed with these sequences. An interesting feature of the sequences was a stretch of Ts that distinguished between Ae-des and Culex on the one hand, and Anopheles on the other. This is the first report of mitochondrial rRNA sequences from these medically important genera of mosquitoes.

Effect of cryopreservation on lipid peroxidation in chick cornea.

A mechanism suggested to cause injury to the preserved organs in vitro is the generation of oxygen free radicals either during preservation or after transplantation due to reperfusion. Methods to suppress generation of oxygen free radicals may lead to improved methods of organ preservation. In this study, increase in the levels of lipid peroxidation in chick cornea after cryopreservation is reported. Addition of fetal bovine serum (FBS) in cryopreservation medium was found to prevent lipid peroxidation. Addition of FBS was also found to be protective towards corneal viability during cryopreservation.

Functional analysis of Drosophila melanogaster hexokinase Hex-A locus: multiple Initiator-like elements enhance DPE containing promoter activity.

Flight muscle Hexokinase-A (HEX-A) is the most conserved and essential hexokinase isoenzyme among Drosophila species. In this study, the Hex-A locus, encoding the HEX-A isoenzyme, has been analysed for the elements regulating its expression. By sequencing the 5' ends of Hex-A cDNA amplified by 5' RACE, we identified a transcription start site that overlapped the Initiator and downstream promoter elements. A 214 bp sequence, encompassing transcription start sites and promoter elements, was required for minimal promoter activity. DNA sequence to the 5' end of the minimal promoter element did not demonstrate any promoter activity; however, its inclusion with the basal promoter element enhanced the promoter activity. Oligonucleotide competition and site-directed mutagenesis identified the Initiator-like sequences, TCAWT, present in this region that were responsible for enhancing the promoter activity. The Hex-A locus is expressed as a single protein in Drosophila cell line, whereas in pupae, larvae and adult flies, it is expressed as two distinct types.

Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate.

Oxidation of NADH by rat brain microsomes was stimulated severalfold on addition of vanadate. During the reaction, vanadate was reduced, oxygen was consumed, and H2O2 was generated with a stoichiometry of 1:1 for NADH/O2, as in the case of other membranes. Extra oxygen was found to be consumed over that needed for H2O2 generation specifically when brain microsomes were used. This appears to be due to the peroxidation of lipids known to be accompanied by a large consumption of oxygen. Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate has been demonstrated. This activity was obtained specifically with the polymeric form of vanadate and with NADH, and was inhibited by the divalent cations Cu2+, Mn2+, and Ca2+, by dihydroxyphenolic compounds, and by hemin in a concentration-dependent fashion. In the presence of a small concentration of vanadate, addition of an increasing concentration of Fe2+ gave increasing lipid peroxidation. After undergoing lipid peroxidation in the presence of NADH and vanadate, the binding of quinuclidinyl benzylate, a muscarinic antagonist, to brain membranes was decreased.

Vanadate-stimulated NADH oxidation requires polymeric vanadate, phosphate and superoxide.

NADH oxidation, catalyzed by the microsomal enzyme system is stimulated on addition of polymeric vanadate. Maximum stimulation by polymeric vanadate was obtained in the presence of phosphate buffer. The small stimulation obtained by metavanadate (500 microM) increased on acidification followed by neutralization, or on adding a trace amount of polymeric vanadate (1 microM).

Vanadate-stimulated NADH oxidation by xanthine oxidase: an intrinsic property.

Vanadate-dependent oxidation of NADH by xanthine oxidase does not require the presence of xanthine and therefore is not due to cooxidation. Addition of NADH or xanthine had no effect on the oxidation of the other substrate. Oxidation of NADH was high at acid pH and oxidation of xanthine was high at alkaline pH. The specific activity was relatively very high with NADH. Concentration-dependent oxidation of NADH Concentration-dependent oxidation of NADH was obtained in the presence of the polymeric form of vanadate, but not orthovanadate or metavanadate. Both NADH and NADPH were oxidized, as in the nonenzymatic system. Oxidation of NADH, but not xanthine, was inhibited by KCN, ascorbate, MnCl2, cytochrome c, mannitol, Tris, epinephrine, norepinephrine, and triiodothyronine. Oxidation of NADH was accompanied by uptake of oxygen and generation of H2O2 with a stoichiometry of 1:1:1 for NADH:O2:H2O2. A 240-nm-absorbing species was formed during the reaction which was different from H2O2 or superoxide. A mechanism of NADH oxidation is suggested wherein Vv and O2 receive one electron each successively from NADH followed by VIV giving the second electron to superoxide and reducing it to H2O2.

NADH-dependent polyvanadate reduction by microsomes.

NADH-dependent reduction of polyvanadate was observed by using rat liver microsomes as the enzyme source. The reduced vanadate form obtained was blue in color with a broad absorption maximum in the red region around 650 nm. Microsomes and phosphate anions were found to be essential for polyvanadate reduction. The rate and the extent of formation of blue color compound was dependent on the amount of vanadate present. Cytochrome b5 was found to be involved in this SOD-insensitive reaction. The rate of disappearance of the blue-colored compound was dependent on concentration of NADH and was found to be sensitive to SOD. Catalase and Mn2+, which inhibit oxygen consumption accompanying NADH oxidation, increased both the rate and extent of the blue color compound formed. The results suggest that vanadate acts as an electron acceptor.

Reduction of vanadate by a microsomal redox system.

The reduction of vanadate catalyzed by rat liver microsomes is demonstrated. This reaction is SOD-insensitive. It is specific for NADH and polyvanadate and is not obtained with metavanadate and NADPH.

Generation of H2O2 in brain mitochondria.

Generation of H2O2 by rat brain mitochondria using succinate and glycerol-1-phosphate as substrates has been demonstrated. Earlier workers were unable to detect this activity in sucrose-Tris buffer. We found that this was due to a lag in the expression of activity in sucrose medium. Using phosphate buffer (50 mM), good rates are now obtained. Generation of H2O2 by rat brain mitochondria required the presence of antimycin A and was dependent on the substrates succinate and glycerol-1-phosphate. Low rates were obtained with NAD+-linked substrates and none with choline, glutamate, and NADH. The Km and Vmax values for H2O2 generation were considerably lower than the corresponding values for the respective dehydrogenase activity, measured by dye reduction. Oxygen-radical scavengers inhibited H2O2 generation, suggesting oxygen radical involvement. Depletion of ubiquinone from mitochondria resulted in loss of H2O2 generation. Reconstitution of such depleted particles with ubiquinone restored the capacity to generate H2O2 in a concentration-dependent manner. Levels of H2O2 production were found to be maximal in cerebellum. Brain mitochondria from rabbit, hamster, mouse, and guinea pig also have the capacity to generate H2O2 on oxidation of glycerol-1-phosphate.