Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris

The hemagglutinin (HA) gene of novel Swine Origin Influenza A/California/04/2009 (H1N1) was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA-synthetic gene having ?? secretory tag under the control of AOX1 promoter was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having single and multiple copy integrants of the expression cassettes were screened for the expression of full length HA protein in the culture supernatant. In order to completely exploit the expression potential of the P. pastoris expression system, a systematic investigation on the influence of gene copy number on the expression of the recombinant protein was made. A panel of Pichia clones carrying increasing copies of the heterologous gene was selected based on Geneticin resistance and SYBR green-based quantitative real-time PCR approach. Using these strategies, recombinant Pichia transformants carrying up to a maximum of four to six copies of the transgene were identified. After optimising the expression conditions for shaker flask culture, the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the expression level of H1N1HA recombinant protein. Our findings clearly suggest that the gene dosage effect play a vital role in high level expression of the pandemic Influenza HA protein in yeast system.     

A Correlation of the Platelet Count with D-Dimer Levels as an Indicator for Component Therapy in Children with Dengue Hemorrhagic Fever

Dengue Fever (DF) may evolve into two life threatening forms—Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). DHF is associated with increased vascular permeability and plasma leakage causing thrombocytopenia and loss of clotting factors into the third space and may result in bleeding initially due to thrombocytopenia and later due to disseminated intravascular coagulation (DIC), often as a terminal event. Prompt recognition and treatment of minor bleeds in DF children with incipient DIC with component therapy may be associated with improved survival while failure to do so is usually catastrophic. A sensitive marker for early DIC is the presence of D-dimer (DD) in the blood. To determine the correlation between the severity of thrombocytopenia and early DIC in children with DHF. The impact of additional factors like age and shock will also be evaluated. Case control prospective study of 60 DHF sero -positive children (1–15 years) with thrombocytopenia. After clinical evaluation they were divided into two equal groups based on the degree of thrombocytopenia (more than/less than 30,000/mm³). PT/APTT and DD levels were estimated in all children of both groups and statistical correlation was done. There was no significant difference in the DD levels between the two groups. However, children in either group, presenting with clinical features of shock and thrombocytopenia had significantly higher DD levels. Empirical component therapy in children with DHF based purely on their low platelet counts may not be justified. However, in DHF children with thrombocytopenia and features of shock, aggressive component therapy may prevent subsequent bleeding and may be justified.     

Impact Assessment on the Aquatic Ecosystem in the Vicinity of an Operating Nuclear Power Plant at the Kalpakkam Coastal Site

An inherent problem in electricity generation is that much of the heat produced cannot be converted into electricity and is dissipated in nearby heat sinks, such as lakes, rivers and oceans. In India, such heat rejection to water bodies is governed by legislation from ministry of environment and forests and central and state pollution control boards. New standards for thermal discharge issued by MoE&F in 1998 seem to be very stringent. But in the absence of relevant data for Indian sites appear to be somewhat justified. In order to make a realistic assessment of the impact of thermal discharge on aquatic ecosystems, the Department of Atomic Energy initiated the first major thermal ecological study in the country at two of its operating nuclear power plants, one at a coastal site in Kalpakkam and the other on the banks of river Kali at Kaiga. The 5 years duration co-ordinated research project involved researchers from eight different universities and national laboratories and experts from NIO, NIOT, CECRI, BARC, NPCIL and MoE&F. In view of the fact that the most of the forthcoming nuclear power plants in the country are coastally located, this article discusses the findings of Thermal Ecological Study at Kalpakkam. A major achievement of the study was to map the precise boundaries of the small mixing zone, the region of confluence of the heated water with the recipient water body, wherein the thermal plume effected a ∆T of only 3–5 °C due to rapid inundation and mixing with turbulent sea water. This caused a limited impact on the growth and distribution of the relatively sedentary benthic flora and fauna, though none of the motile forms, including fishes, were affected. Beyond the mixing zone, the life appeared to be as near normal as possible. No change in any physico-chemical parameter was noticed, except for a small expected decrease in dissolved oxygen at the discharge point. At Kalpakkam, position of the mixing zone shifts periodically due to formation of a natural sand bar by sea currents. This allows restoration of parameters, especially temperature to more conducive values and results in recolonization of the previously impacted sites. An ecologically encouraging finding of the study was that the impact was restricted in space and reversible in time. The data generated are expected to aid regulatory agencies in formulation of more appropriate stipulations for thermal discharge and also guide power industry to design eco-friendly technologies in future.

     

Molecular identification of man-eating carnivores from scat samples

We describe a simple, rapid molecular method for identifying man-eating carnivores. We designed mitochondrial control region based human-specific primers. These selectively amplify human DNA. We sequenced PCR amplicons and the presence of human remains was confirmed in two tiger scats collected in conflicting sites. PCR-based human-specific amplification with DNA extracted from scats is a confirmatory test for the presence of human remains. The protocol is useful in identifying man-eating carnivores.