Identification and characterization of cysteine proteinases of Trypanosoma evansi

Trypanosoma evansi is a causative agent of `surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

A Method for Evaluation of Product Lifecycle Alternatives under Uncertainty

In each stage of product development, we need to take decisions, by evaluating multiple product alternatives based on multiple criteria. Classical evaluation methods like weighted objectives method assumes certainty about information available during product development. However, designers often must evaluate under uncertainty. Often the likely performance, cost or environmental impacts of a product proposal could be estimated only with certain confidence, which may vary from one proposal to another. In such situations, the classical approaches to evaluation can give misleading results. There is a need for a method that can aid in decision making by supporting quantitative comparison of alternatives to identify the most promising alternative, under uncertain information about the alternatives. A method called confidence weighted objectives method is developed to compare the whole life cycle of product proposals using multiple evaluation criteria under various levels of uncertainty with non crisp values. It estimates the overall worth of proposal and confidence on the estimate, enabling deferment of decision making when decisions cannot be made using current information available.

Issues in Achievement of Haptic Realism

This paper deals with haptic realism related to Kinematic capabilities of the devices used in manipulation of virtual objects in virtual assembly environments and its effect on achieving haptic realism. Haptic realism implies realistic touch sensation. In virtual world all the operations are to be performed in the same way and with same level of accuracy as in the real world .In order to achieve realism there should be a complete mapping of real and virtual world dimensions. Experiments are conducted to know the kinematic capabilities of the device by comparing the dimensions of the object in the real and virtual world. Registered dimensions in the virtual world are found to be approximately 1.5 times that of the real world. Dimensional variations observed were discrepancy due to exoskeleton and discrepancy due to real and virtual hands. Experiments are conducted to know the discrepancy due to exoskeleton and this discrepancy can be taken care of by either at the hardware or software level. A Mathematical model is proposed to know the discrepancy between real and virtual hands. This could not give a fixed value and can not be taken care of by calibration. Experiments are conducted to figure out how much compensation can be given to achieve haptic realism.

Assessing Degree of Novelty of Products to Ascertain Innovative Products

Substantial increase in competition compels design firms to develop new products at an increasingly rapid pace. This situation pressurizes engineering teams to develop better products and at the same time develop products faster [1]. Continuous innovation is a key factor to enable a company to generate profit on a continued basis, through the introduction of new products in the market – a prime intention for Product Lifecycle Management. Creativity, affecting a wide spectrum of business portfolios, is regarded as the crucial factor for designing products. A central goal of product development is to create products that are sufficiently novel and useful. This research focuses on the determination of novelty of engineering products. Determination of novelty is important for ascertaining the newness of a product, to decide on the patentability of the design, to compare designers' capability of solving problems and to ascertain the potential market of a product. Few attempts at measuring novelty is available in literature [2, 3, 4], but more in-depth research is required for assessing degree of novelty of products. This research aims to determine the novelty of a product by enabling a person to determine the degree of novelty in a product. A measure of novelty has been developed by which the degree of ''novelty'' of products can be ascertained. An empirical study has been conducted to determine the validity of this method for determining the 'novelty' of the products.

Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Ribosome-RNA interaction: a potential target for developing antiviral against hepatitis C virus

Hepatitis C virus (HCV), a member of Flaviviridae, encoding a positive-sense single-stranded RNA translates by cap-independent mechanism using the internal ribosome entry site (IRES) present in the 5' UTR of the virus. The IRES has complex stem loop structures and is capable of recruiting the 40S ribosomal subunit in a factor-independent fashion. As the IRES sequence is highly conserved throughout the HCV genotypes and the translation is the first obligatory step of the HCV life cycle, the IRE'S-mediated translation, or more specifically, the ribosome HCV RNA interaction is an attractive target to design effective antivirals. This article will focus on the mechanism of the HCV IRES translation and the various ways in which the interaction of ribosome and IRES has been targeted.

Rural electrification: Optimising the choice between decentralised renewable energy sources and grid extension

The study presents an analysis aimed at choosing between off-grid solar photovoltaic, biomass gasifier based power generation and conventional grid extension for remote village electrification. The model provides a relation between renewable energy systems and the economical distance limit (EDL) from the existing grid point, based on life cycle cost (LCC) analysis, where the LCC of energy for renewable energy systems and grid extension will match. The LCC of energy feed to the village is arrived at by considering grid availability and operating hours of the renewable energy systems. The EDL for the biomass gasifier system of 25 kW capacities is 10.5 km with 6 h of daily operation and grid availability. However, the EDL for a similar 25 kW capacity photovoltaic system is 35 km for the same number of hours of operation and grid availability. The analysis shows that for villages having low load demand situated far away from the existing grid line, biomass gasification based systems are more cost competitive than photovoltaic systems or even compared to grid extension. (C) 2012 Elsevier Inc. All rights reserved.

Is land really a constraint for the utilization of solar energy in India?

This article compares the land use in solar energy technologies with conventional energy sources. This has been done by introducing two parameters called land transformation and land occupation. It has been shown that the land area transformed by solar energy power generation is small compared to hydroelectric power generation, and is comparable with coal and nuclear energy power generation when life-cycle transformations are considered. We estimate that 0.97% of total land area or 3.1% of the total uncultivable land area of India would be required to generate 3400 TWh/yr from solar energy power systems in conjunction with other renewable energy sources.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule

Translation initiation of hepatitis C virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the internal ribosome entry site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

Evidence for the role of Mycobacteriumtuberculosis RecG helicase in DNA repair and recombination

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacteriumtuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichiacoli recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.