Development and characterization of a cell line WAF from freshwater shark Wallago attu

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.     

A multi-step phosphorelay two-component system impacts on tolerance against dehydration stress in common wheat

Wheat is an important staple crop, and its productivity is severely constrained by drought stress (DS). An understanding of the molecular basis of drought tolerance is necessary for genetic improvement of wheat for tolerance to DS. The two-component system (TCS) serves as a common sensor-regulator coupling mechanism implicated in the regulation of diverse biological processes (including response to DS) not only in prokaryotes, but also in higher plants. In the latter, TCS generally consists of two signalling elements, a histidine kinase (HK) and a response regulator (RR) associated with an intermediate element called histidine phosphotransferase (HPT). Keeping in view the possible utility of TCS in developing water use efficient (WUE) wheat cultivars, we identified and characterized 62 wheat genes encoding TCS elements in a silico study; these included 7 HKs, 45 RRs along with 10 HPTs. Twelve of the 62 genes showed relatively higher alterations in the expression under drought. The quantitative RT-PCR (qRT-PCR)-based expression analysis of these 12 TCS genes was carried out in wheat seedlings of a drought sensitive (HD2967) and a tolerant (Dharwar Dry) cultivar subjected to either dehydration stress or cytokinin treatment. The expression of these 12 genes under dehydration stress differed in sensitive and tolerant genotypes, even though for individual genes, both showed either up-regulation or down-regulation. In response to the treatment of cytokinin, the expression of type-A RR genes was higher in the tolerant genotype, relative to that in the sensitive genotype, the situation being reverse for the type-B RRs. These results have been discussed in the context of the role of TCS elements in drought tolerance in wheat.     

Regulation of lipid metabolism by Dicer revealed through SILAC mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.     

Detection, isolation and confirmation of Crimean-Congo hemorrhagic fever virus in human, ticks and animals in Ahmadabad, India, 2010-2011

Background

In January 2011, human cases with hemorrhagic manifestations in the hospital staff were reported from a tertiary care hospital in Ahmadabad, India. This paper reports a detailed epidemiological investigation of nosocomial outbreak from the affected area of Ahmadabad, Gujarat, India.

Principal Findings

Samples from 3 suspected cases, 83 contacts, Hyalomma ticks and livestock were screened for Crimean-Congo hemorrhagic fever (CCHF) virus by qRT-PCR of which samples of two medical professionals (case C and E) and the husband of the index case (case D) were positive for CCHFV. The sensitivity and specificity of indigenous developed IgM ELISA to screen CCHFV specific antibodies in human serum was 75.0% and 97.5% respectively as compared to commercial kit. About 17.0% domestic animals from Kolat, Ahmadabad were positive for IgG antibodies while only two cattle and a goat showed positivity by qRT-PCR. Surprisingly, 43.0% domestic animals (Buffalo, cattle, sheep and goat) showed IgG antibodies in the adjoining village Jivanpara but only one of the buffalo was positive for CCHFV. The Hyalomma anatolicum anatolicum ticks were positive in PCR and virus isolation. CCHFV was isolated from the blood sample of case C, E in Vero E-6 cells and Swiss albino mice. In partial nucleocapsid gene phylogeny from CCHFV positive human samples of the years 2010 and 2011, livestock and ticks showed this virus was similar to Tajikistan (strain TAJ/{"type":"entrez-nucleotide","attrs":{"text":"H08966","term_id":"873788","term_text":"H08966"}}H08966), which belongs in the Asian/middle east genetic lineage IV.

Conclusions

The likely source of CCHFV was identified as virus infected Hyalomma ticks and livestock at the rural village residence of the primary case (case A). In addition, retrospective sample analysis revealed the existence of CCHFV in Gujarat and Rajasthan states before this outbreak. An indigenous developed IgM ELISA kit will be of great use for screening this virus in India.     

Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog??s (MS) basal medium containing 8.9???M benzyladenine (BA) produced the most suitable explant material. Six?days of explant pre-culture and 5?min inoculation with Agrobacterium culture in MS medium, containing 8.9???M BA, 9.3???M kinetin, and 0.4?mg?l^?1 thiamine at pH?5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20?mg?l^?1 in the selection medium for the initial 10?d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3?%. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5?% of the independent transgenic lines from self-fertilized T₁ progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.     

An ethnobotanical survey of medicinal plants used in Terai forest of western Nepal

ABSTRACT: BACKGROUND: Nepal Himalayas have been known as a rich source for valuable medicinal plants since Vedic periods. Present work is the documentation of indigenous knowledge on plant utilization as natural remedy by the inhabitants of terai forest in Western Nepal. METHODS: Study was conducted during 2010-2011 following standard ethnobotanical methods. Data about medicinal uses of plants were collected by questionnaire, personal interview and group discussion with pre identified informants. Voucher specimens were collected with the help of informants, processed into herbarium following standard methods, identified with the help of pertinent floras and taxonomic experts, and submitted in Department of Botany, Butwal Multiple Campus, Tribhuvan University, Nepal for future references. RESULTS: During the present study 66 medicinal plant species belonging to 37 families and 60 genera has been documented. These plants were used to treat various diseases and ailments grouped under 11 disease categories, with the highest number of species (41) being used for gastro-intestinal disorders, followed by dermatological disorders (34). In the study area the informants' consensus about usages of medicinal plants ranges from 0.93 to 0.97 with an average value of 0.94. Herbs (53 %) were the primary source of medicine, followed by trees (23 %). Curcuma longa (84 %) and Azadirachta indica (76 %) are the most frequently and popularly used medicinal plant species in the study area. Acacia catechu, Bacopa monnieri, Bombax ceiba, Drymaria diandra, Rauvolfia serpentina, and Tribulus terrestris are threatened species which needs to be conserved for future use. CONCLUSIONS: The high degree of consensus among the informants suggests that current use and knowledge are still strong, and thus the preservation of today's knowledge shows good foresight in acting before much has been lost. The connections between plant use and conservation are also important ones, especially as the authors note that neither the local inhabitants nor the government is addressing the potential loss of valuable species in this region.     

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.