Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning

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Not AvailableArunachal Pradesh, the largest mountainous state of India, is situated in the northeastern part of the Himalayan region and characterized by high annual rainfall, forest vegetation and diversity in soils. Information on the soils of the state is essential for scientific land use planning and sustainable production. A soil resource inventory and subsequent database creation for thematic mapping using a Geographical Information System (GIS) is presented in this paper. Physiographically, Arunachal Pradesh can be divided into four distinct zones: snow-capped mountains (5500 m amsl); lower Himalayan ranges (3500 m amsl); the sub-Himalayan Siwalik hills (700 m amsl); and the eastern Assam plains. Soils occurring in these physiographic zones are lnceptisols (37 percent), Entisols (35 per- cent), Ultisols (14 percent) and Alfisols (0.5 percent). The remaining soils can be classed as miscellaneous. Soil resource inventory studies show that the soils of the warm perhumid eastern Himalayan ecosystem, with a ‘thermic’ temperature regime, are lnceptisols and Entisols; and that they are highly acidic in nature. Soils of the warm perhumid Siwalik hill ecosystem, with a ‘hyperthermic’ temperature regime, are also Entisols and lnceptisols with a high to moderate acidic condition. The dominant soils of the northeastern Purvachal hill ecosystem, with ‘hyperthermic’ and ‘thermic’ temperature regimes, are Ultisols and Inceptisols. lnceptisols and Entisols are the dominant soils in the hot and humid plain ecosystem. Steeply sloping landform and high rainfall are mainly responsible for a high erosion hazard in the state. The soil erosion map indicates that very severe (20 percent of TGA) to severe (25 percent of TGA) soil erosion takes place in the warm per-humid zone, whereas, moderate erosion takes place in the Siwalik hills and hot, humid plain areas. This is evident from the soil depth class distribution of Arunachal Pradesh, which shows that shallow soils cover 20 percent of the TGA of the state. Most of the the state is covered by hills and agri- cultural practices are limited to valley regions, However, the soils of other physiographic zones (lower altitudinal, moderately hilly terrain) provide scope for plantations, such as orange, banana and tea plantations.Not Availabl

Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX(3)CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX(3)CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX(3)CL1, CX(3)CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo

Effects of Educational Intervention among Reproductive Age Group Women on Safe Abortion

Introduction: Many reproductive aged women needlessly die due to unsafe abortion even when they seek help to terminate their unwanted pregnancy. These deaths could have been prevented had they been aware that safe abortion service was available to them. The study aimed at finding out the effectiveness of the education intervention in improving knowledge among reproductive age group women regarding the safe abortion. Methods: An experimental intervention was carried out on safe abortion education among the reproductive age group women. The impact of pre- and post- intervention was evaluated by using a set of structured questionnaire in local language. The obtained data was analyzed by using the Excel and Statistical Package for the Social Sciences, version 12.0 for windows and result was interpreted. Results: The post intervention finding revealed a significantly higher (p= 0.001) mean on knowledge among participants about safe abortion compared to pre-observational test. The mean difference between the pre-test and post-test was 64.1% (Pre-test 11.18 ±12.88 Post-test 75.28 ±9.56). The research hypothesis was accepted with p value paired t-test at <0.001 (0.05). The result supports that the educational intervention was effective in increasing safe abortion awareness among reproductive aged group women. Conclusion: The safe abortion educational intervention program was instrumental to improve reproductive age women’s knowledge considerably about safe abortion service.  Keywords: educational intervention; knowledge; safe abortion; women.  

Cytotoxicity of Dinitrotoluenes (2,4-DNT, 2,6-DNT ) to MCF-7 and MRC-5 Cells

DNTs are considered possibly carcinogenic to humans (Group 2B) because there is inadequate evidence in humans for carcinogenicity though there is sufficient evidence in experimental animals. In this study, MCF-7 (breast) and MRC-5 (lung) cells were exposed to a serial dilution of 2,4 and 2,6 DNTs (control, 1-500 ppm) in 96 well tissue culture plates. After various time intervals (24, 48, 72 and 96 hrs) the plates were washed, and 100μl fluorescein diacetate solution (10 μg/ml in PBS) was added column wise to each well, and incubated at 37°C for 30 - 60 min before reading the fluorescence with a spectrofluorometer at excitation and emission wavelengths of 485 and 538 nm respectively. Spectrofluorometeric readings were converted to percentages of cell survival. Regression analysis was conducted to determine the relationship between cell survival and exposed concentration. Linear equations derived from the regression analysis were used to calculate the LC50 values. Results indicated that 2,6 DNT was more toxic to breast cells; LC50 values were 445 and 292 ppm at 24 and 48 hours respectively compared to 2,4 DNT showing LC50 values of 570 and 407 ppm at 24 and 48 hours, respectively. No significant differences in toxicity existed between the two chemicals with regard to lung cells. Contrary to the above observation, 2,4 DNT was more toxic to breast cells; LC50 values were 407 and 238 ppm at 24 and 48 hours respectively compared to lung cells showing LC50 values of 527 and 402 ppm at 24 and 48 hours respectively. No significant difference existed for 2,6 DNT between the two cell lines. Lungs cells were more resistant to the two chemicals