RETRACTED ARTICLE: Virus-induced gene silencing for functional analysis of selected genes

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism and has been shown to be of great potential in plant reverse genetics. Circumvention of plant transformation, methodological simplicity, robustness, and speedy results makes VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is well established in Nicotiana benthamiana, and efforts are being made to improve VIGS in other species, including monocots. Here, we discuss the issues specific to the application of VIGS technology to determine gene function, which has revealed the roles of a variety of genes in disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis. M. R. Godge and A. Purkayastha made equal contributions and hence should be treated as joint first authors for this paper.     

Grid cellware: the first grid-enabled tool for modelling and simulating cellular processes

Modelling and simulation of complex cellular transactions involve development of platforms that understand diverse mathematical representations and are capable of handling large backend computations. Grid Cellware, an integrated modelling and simulation tool, has been developed to precisely address these niche requirements of the modelling community. Grid Cellware implements various pathway simulation algorithms along with adaptive Swarm algorithm for parameter estimation. For enchanced computational productivity Grid Cellware uses grid technology with Globus as the middleware.     

Biodegradation of Allethrin, a Pyrethroid Insecticide, by an Acidomonas Sp.

Allethrin is a major mosquito repellent agent. To degrade allethrin present in used mats and the environment, a bacterium capable of utilizing allethrin was isolated. This isolate, an Acidomonas sp., grew in minimal medium with 16 mM: allethrin as sole source of carbon and degraded >70% of it in 72 h, with negligible residual metabolites in the medium. Culture filtrates collected after 48 h and 72 h showed presence of (i) cyclopropanecarboxylic acid, 2,2-dimethyl-3-(2-methyl-1-propenyl), (ii) 2-ethyl-1,3-dimethyl-cyclopent-2-ene-carboxylic acid (iii) chrysanthemic acid and (iv) allethrolone [2-cyclopenten-l-one, 4-hydroxy-3-methyl-2(-2-propenyl)] as the major metabolites with 2 minor metabolites. Allethrin is thus metabolized by a hydrolytic pathway followed by oxidation and dehydrogenation.     

A dynamical-systems model for Parkinson's disease

The juxtaposition of hypokinetic and hyperkinetic symptoms in Parkinson's disease (PD) presents a challenge in modeling the basal ganglia. We propose a model of the striatum that can account for the mixture of symptoms seen in PD. In the model, the problem of motor planning is cast in terms of a particle in a potential, where potentials are generated internally in striatal modules, subject to afferent control. Planned movement is governed by Hamilton's equations, where potential energy is supplied by potentials expressed in the striatum. To test the model in realistic situations, a dynamic simulation of a two-link robot arm was used. Normal movement is modeled and shown to exhibit observed experimental properties. Symptoms of PD are reproduced by modeling hypothetical consequences of PD pathology. Received: 17 February 1999 / Accepted in revised form: 21 December 1999     

5‐Fluorouracil efficacy requires anti‐tumor immunity triggered by cancer‐cell‐intrinsic STING

5‐Fluorouracil (5‐FU) is a widely used chemotherapeutic drug, but the mechanisms underlying 5‐FU efficacy in immunocompetent hosts in vivo remain largely elusive. Through modeling 5‐FU response of murine colon and melanoma tumors, we report that effective reduction of tumor burden by 5‐FU is dependent on anti‐tumor immunity triggered by the activation of cancer‐cell‐intrinsic STING. While the loss of STING does not induce 5‐FU resistance in vitro, effective 5‐FU responsiveness in vivo requires cancer‐cell‐intrinsic cGAS, STING, and subsequent type I interferon (IFN) production, as well as IFN‐sensing by bone‐marrow‐derived cells. In the absence of cancer‐cell‐intrinsic STING, a much higher dose of 5‐FU is needed to reduce tumor burden. 5‐FU treatment leads to increased intratumoral T cells, and T‐cell depletion significantly reduces the efficacy of 5‐FU in vivo. In human colorectal specimens, higher STING expression is associated with better survival and responsiveness to chemotherapy. Our results support a model in which 5‐FU triggers cancer‐cell‐initiated anti‐tumor immunity to reduce tumor burden, and our findings could be harnessed to improve therapeutic effectiveness and toxicity for colon and other cancers.     

Impaired growth and neurological abnormalities in branched-chain alpha-keto acid dehydrogenase kinase-deficient mice

The BCKDH (branched-chain alpha-keto acid dehydrogenase complex) catalyses the rate-limiting step in the oxidation of BCAAs (branched-chain amino acids). Activity of the complex is regulated by a specific kinase, BDK (BCKDH kinase), which causes inactivation, and a phosphatase, BDP (BCKDH phosphatase), which causes activation. In the present study, the effect of the disruption of the BDK gene on growth and development of mice was investigated. BCKDH activity was much greater in most tissues of BDK-/- mice. This occurred in part because the E1 component of the complex cannot be phosphorylated due to the absence of BDK and also because greater than normal amounts of the E1 component were present in tissues of BDK-/- mice. Lack of control of BCKDH activity resulted in markedly lower blood and tissue levels of the BCAAs in BDK-/- mice. At 12 weeks of age, BDK-/- mice were 15% smaller than wild-type mice and their fur lacked normal lustre. Brain, muscle and adipose tissue weights were reduced, whereas weights of the liver and kidney were greater. Neurological abnormalities were apparent by hind limb flexion throughout life and epileptic seizures after 6-7 months of age. Inhibition of protein synthesis in the brain due to hyperphosphorylation of eIF2alpha (eukaryotic translation initiation factor 2alpha) might contribute to the neurological abnormalities seen in BDK-/- mice. BDK-/- mice show significant improvement in growth and appearance when fed a high protein diet, suggesting that higher amounts of dietary BCAA can partially compensate for increased oxidation in BDK-/- mice. Disruption of the BDK gene establishes that regulation of BCKDH by phosphorylation is critically important for the regulation of oxidative disposal of BCAAs. The phenotype of the BDK-/- mice demonstrates the importance of tight regulation of oxidative disposal of BCAAs for normal growth and neurological function.     

Synthesis and therapeutic evaluation of pyridyl based novel mTOR inhibitors

A series of novel cyanopyridyl based molecules (1–14) were designed, synthesized and probed for inhibition of mammalian target of rapamycin (mTOR) activity. Compound 14 was found to be a potent inhibitor of mTOR activity as assessed by enzyme-linked immunoassays and Western blot analysis. Most importantly, systemic application (intraperitoneal; ip) of compound 14 significantly suppressed macroscopic and histological abnormalities associated with chemically-induced murine colitis.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.