Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates

ABSTRACT: BACKGROUND: Chikungunya (CHIK) is currently endemic in South and Central India and exist as co-infections with dengue in Northern India. In 2010, New Delhi witnessed an outbreak of CHIK in the months October-December. This was the first incidence of a dominant CHIK outbreak in Delhi and prompted us to characterize the Delhi virus strains. We have also investigated the evolution of CHIK spread in India. FINDINGS: Clinical samples were subjected to RT-PCR to detect CHIK viral RNA. The PCR amplified products were sequenced and the resulting sequences were genetically analyzed. Phylogenetic analysis based on partial sequences of the structural proteins E1 and E2 revealed that the viruses in the latest outbreak exhibited ECSA lineage. Two novel mutations, E1 K211E and E2 V264A were observed in all Delhi isolates. In addition, CHIKV sequences from eight states in India were analyzed along with Delhi sequences to map the genetic diversity of CHIKV within the country. Estimates of average evolutionary divergence within states showed varying divergence among the sequences both within the states and between the states. We identified distinct molecular signatures of the different genotypes of CHIKV revealing emergence of a new signature in the New Delhi clade. Statistical analyses and construction of evolutionary path of the virus within the country revealed gradual spread of one specific strain all over the country. CONCLUSION: This study has identified unique mutations in the E1 and E2 genes and has revealed the presence of ancestral CHIKV population with maximum diversity circulating in Maharashtra. The study has further revealed the trend of CHIK spread in India since its first report in 1963 and its subsequent reappearance in 2005.     

The genetic structure of taro: a comparison of RAPD and isozyme markers

Germplasm characterization and evolutionary process in viable populations are important links between the conservation and utilization of plant genetic resources. Here, an investigation is made, based on molecular and biochemical techniques for assessing and exploiting the genetic variability in germplasm characterization of taro, which would be useful in plant breeding and ex situ conservation of taro plant genetic resources. Geographical differentiation and phylogenetic relationships of Indian taro, Colocasia esculenta (L.) Schott, were analyzed by random amplified polymorphic DNA (RAPD) and isozyme of seven enzyme systems with specific reference to the Muktakeshi accession, which has been to be proved resistant to taro leaf blight caused by P. colocasiae. The significant differentiations in Indian taro cultivars were clearly demonstrated by RAPD and isozyme analysis. RAPD markers showed higher values for genetic differentiation among taro cultivars and lower coefficient of variation than those obtained from isozymes. Genetic differentiation was evident in the taro accessions collected from different regions of India. It appears that when taro cultivation was introduced to a new area, only a small fraction of genetic variability in heterogeneous taro populations was transferred, possibly causing random differentiation among locally adapted taro populations. The selected primers will be useful for future genetic analysis and provide taro breeders with a genetic basis for selection of parents for crop improvement. Polymorphic markers identified in the DNA fingerprinting study will be useful for screening a segregating population, which is being generated in our laboratory aimed at developing a taro genetic linkage map.     

Cholinergic location of delta-opioid receptors in canine atria and SA node

Delta-opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and <8% were adrenergic. DORs colocalized 75-85% with synapsin in tissue slices from both atria and SA node. The colocalization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Colocalization between DOR and VAChT was 75-85% regardless of the source. Overlap between DOR and TH was uniformly low, ranging from 8% to 17%. Western blots with synaptosomal extracts confirmed two DOR-positive bands at molecular masses corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes, largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals support the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.     

Healthcare infrastructure, contraceptive use and infant mortality in Uttar Pradesh, India

This paper analyzes data on approximately 30,000 women from a survey in Uttar Pradesh in 1995 together with the data from surveys of public and private providers of healthcare and family planning services. A framework was developed for analyzing the effects of quality of services on utilization, and for understanding the gradual evolution of the healthcare infrastructure. The empirical results from logistic regressions for use of female sterilization and IUD showed significant effects of quality of services in government and private hospitals, and of socioeconomic variables such as education, caste, and an index of household possessions. Secondly, models for infant mortality of children born in the preceding 3-year period showed significant effects of socioeconomic variables, quality of healthcare services and birth spacing. Lastly, analysis of data at a more aggregated (Primary Sampling Unit) level indicated differential effects of economic development on the quality of services available in the public and private facilities.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists.

A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C-terminal hexapeptide sequence Tyr-Phe-Ser-Pro-Arg-Leu-NH2 of PBAN1-33NH2; whereas library II (D-Phe library) was based on the sequence of the PBAN lead linear antagonist Arg-Tyr-Phe-d-Phe-Pro-Arg-Leu-NH2. In both libraries the Pro residue was replaced by the BBC building unit Nalpha-(omega-aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N-terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl3I for selective deprotection of the Boc group from the building unit prior to on-resin amino-end to backbone-nitrogen (AE-BN) cyclization, during solid-phase synthesis with Fmoc chemistry.     

Status and distribution of anthropogenic pressure in the buffer zone of Nanda Devi Biosphere Reserve in western Himalaya, India

Nanda Devi Biosphere Reserve (NDBR) (2236.74 km²), extending over three civil districts viz, Chamoli, Pithoragarh and Almora of the state of Uttar Pradesh, is an important protected area of the western Himalaya owing to its rich biological and cultural diversity. The human (n = 1856) and livestock population (n = 7785) of 19 settlements in the buffer zone as well as seasonal grazing by 15 000–20 000 migratory sheep and goats were the major sources of anthropogenic pressure in the reserve. The intensity of wood-cutting and lopping of a total of 35 sampled woody species was assessed in the buffer zone as parameters of anthropogenic pressure. Anthropogenic pressure showed restricted distribution due to the sparse location of human settlements and seasonal use of biomass resources by majority of the inhabitants, who live a trans-humane lifestyle. Intensive wood-cutting was limited to within 2 km radius of human settlements while tree lopping was mainly done by the migratory shepherds around timber-line (3600–3800 m). Of the total cut trees, the proportion of 20–60 cm girth class cut trees was recorded more. The regeneration percentage of tree species was higher in Chamoli and Pithoragarh sectors as compared to the Almora sector of the buffer zone. Fuelwood plantation in the degraded lands around villages through peoples participation, introduction of alternate fuel devices and employment opportunities have been suggested to minimize the dependence of local people on biomass resources and ensure the biodiversity conservation in the NDBR.     

Discovery and parallel synthesis of a new class of cathepsin K inhibitors.

Peptidomimetic aminomethyl ketones have been identified as a new class of cathepsin K inhibitors. Traditional and high-speed parallel synthesis techniques were applied to investigate this series. Structure-activity relationships were established, and certain analogues were characterized with IC(50) values in the range 200-500 nM.     

Vascular anastomoses in twin placentae and their recognition.

166 placentae from multiple pregnancies (including three triplets and 17 monochorral) have been studied by injection corrosion technique, using cellulose acetate buterite in acetone, under continuous pressure within physiological limits. It has been shown that vascular anastomoses in twin placentae are usually multiple. The direct type is frequently found in monochorial twin placentae and is mostly arterial or venous in nature. The indirect type is observed more commonly in dichorial twin placentae and is mostly arteriovenous in nature. Both types are almost universally found in monochorial monoamniotic placentae. The presence of direct anastomotic channels has a highly significant association with the incidence of marginal vessels and an exaggeration of parameters of stress to an abnormal quantum--both singly and in combinations of three or more-, and is independent of the pattern of branching. In the ontogenetic time-sequence perspective, direct anastomoses, marginal vessels and parameters of stress in a twin placenta can be regarded as different phases of the sequelae of an altered vascular relationship caused by disturbances in the vicinity of the 12th week of gestation. It has been further shown that direct vascular anastomoses are a greater risk to the twins than indirect ones. The importance of early and timely recognition of such channels, by an examination of twin placentae for the presence of marginal vessels, is stressed.