Regulatory options for genetically modified crops in India

The introduction of semi‐dwarfing, high‐yielding and nutrients‐responsive crop varieties in the 1960s and 1970s alleviated the suffering of low crop yield, food shortages and epidemics of famine in India and other parts of the Asian continent. Two semi‐dwarfing genes, Rht in wheat and Sd‐1 in rice heralded the green revolution for which Dr. Norman Borlaug was awarded the Nobel Peace Prize in 1970. In contrast, the revolutionary new genetics of crop improvement shamble over formidable obstacles of regulatory delays, political interferences and public misconceptions. India benefited immensely from the green revolution and is now grappling to deal with the nuances of GM crops. The development of GM mustard discontinued prematurely in 2001 and insect‐resistant Bt cotton varieties were successfully approved for commercial cultivation in 2002 in an evolving nature of regulatory system. However, the moratorium on Bt brinjal by MOEF in 2010 meant a considerable detour from an objective, science‐based, rigorous institutional process of regulatory approval to a more subjective, nonscience‐driven, political decision‐making process. This study examines what ails the regulatory system of GM crops in India and the steps that led to the regulatory logjam. Responding to the growing challenges and impediments of existing biosafety regulation, it suggests options that are critical for GM crops to take roots for a multiplier harvest.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

First Systematic Plant Proteomics Workshop in Botany Department,University of Delhi: Transferring Proteomics Knowledge to Next‐generation Researchers and Students

International Plant Proteomics Organization (INPPO) outlined ten initiatives to promote plant proteomics in each and every country. With greater emphasis in developing countries, one of those was to “organize workshops at national and international levels to train manpower and exchange information”. This third INPPO highlights covers the workshop organized for the very first time in a developing country, India, at the Department of Botany in University of Delhi on December 26–30, 2013 titled – “1^st Plant Proteomics Workshop / Training Program” under the umbrella of INPPO India‐Nepal chapter. Selected 20 participants received on‐hand training mainly on gel‐based proteomics approach along with manual booklet and parallel lectures on this and associated topics. In house, as well as invited experts drawn from other Universities and Institutes (national and international), delivered talks on different aspects of gel‐based and gel‐free proteomics. Importance of gel‐free proteomics approach, translational proteomics, and INPPO roles were presented and interactively discussed by a group of three invited speakers Drs. Ganesh Kumar Agrawal (Nepal), Randeep Rakwal (Japan), and Antonio Masi (Italy). Given the output of this systematic workshop, it was proposed and thereafter decided to be organized every alternate year; the next workshop will be held in 2015. Furthermore, possibilities on providing advanced training to those students / researchers / teachers with basic knowledge in proteomics theory and experiments at national and international levels were discussed. INPPO is committed to generating next‐generation trained manpower in proteomics, and it would only happen by the firm determination of scientists to come forward and do it.     

The OMSSAPercolator: An automated tool to validate OMSSA results

Protein identification by MS/MS is an important technique in proteome studies. The Open Mass Spectrometry Search Algorithm (OMSSA) is an open‐source search engine that can be used to identify MS/MS spectra acquired in these experiments. Here, we present a software tool, termed OMSSAPercolator, which interfaces OMSSA with Percolator, a post‐search machine learning method for rescoring database search results. We demonstrate that it outperforms the standard OMSSA scoring scheme, and provides reliable significant measurements. OMSSAPercolator is programmed using JAVA and can be readily used as a standalone tool or integrated into existing data analysis pipelines. OMSSAPercolator is freely available and can be downloaded at http://sourceforge.net/projects/omssapercolator/ .     

Live Imaging Provides New Insights on Dynamic F-Actin Filopodia and Differential Endocytosis during Myoblast Fusion in Drosophila

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.     

Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

Chemotherapy and anti-hormonal therapies are the most common treatments for non-organ-confined prostate cancer (PCa). However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches. The present study focused on analyzing the role of pterostilbene (PTER)-isothiocyanate (ITC) conjugate – a novel class of hybrid compound synthesized by appending an ITC moiety on PTER backbone – in regulating the functions of androgen receptor (AR), thereby causing apoptosis of PCa cells. The conjugate molecule caused 50% growth inhibition (IC₅₀) at 40±1.12 and 45±1.50 μM in AR positive (LNCaP) and negative (PC-3) cells, respectively. The reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis. Both PI3K/Akt and MAPK/ERK pathways played an important and differential role in conjugate-induced apoptosis of these PCa cells. While the inhibitor of Akt (A6730) or Akt-specific small interference RNA (siRNA) greatly sensitized PC-3 cells to conjugate-induced apoptosis, on the contrary, apoptosis was accelerated by inhibition of ERK (by PD98059 or ERK siRNA) in case of LNCaP cells, both ultimately culminating in the expression of cleaved caspase-3 protein. Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR. All these data suggests that conjugate-induced inhibition of cell proliferation and induction of apoptosis are partly mediated by the down regulation of AR, Akt, and ERK signaling. These observations provide a rationale for devising novel therapeutic approaches for treating PCa by using conjugate alone or in combination with other therapeutics.     

Protein Distributions from a Stochastic Model of the lac Operon of E. coli with DNA Looping: Analytical solution and comparison with experiments

Although noisy gene expression is widely accepted, its mechanisms are subjects of debate, stimulated largely by single-molecule experiments. This work is concerned with one such study, in which Choi et al., 2008, obtained real-time data and distributions of Lac permease in E. coli. They observed small and large protein bursts in strains with and without auxiliary operators. They also estimated the size and frequency of these bursts, but these were based on a stochastic model of a constitutive promoter. Here, we formulate and solve a stochastic model accounting for the existence of auxiliary operators and DNA loops. We find that DNA loop formation is so fast that small bursts are averaged out, making it impossible to extract their size and frequency from the data. In contrast, we can extract not only the size and frequency of the large bursts, but also the fraction of proteins derived from them. Finally, the proteins follow not the negative binomial distribution, but a mixture of two distributions, which reflect the existence of proteins derived from small and large bursts.     

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.     

Role of isothiocyanate conjugate of pterostilbene on the inhibition of MCF-7 cell proliferation and tumor growth in Ehrlich ascitic cell induced tumor bearing mice

Naturally occurring pterostilbene (PTER) and isothiocyanate (ITC) attract great attention due to their wide range of biological properties, including anti-cancer, anti-leukemic, anti-bacterial and anti-inflammatory activities. A novel class of hybrid compound synthesized by introducing an ITC moiety on PTER backbone was evaluated for its anti-cancer efficacy in hormone-dependent breast cancer cell line (MCF-7) in vitro and Ehrlich ascitic tumor bearing mice model in vivo. The novel hybrid molecule showed significant in vitro anti-cancer activity (IC₅₀=25±0.38) when compared to reference compound PTER (IC₅₀=65±0.42). The conjugate molecule induced both S and G2/M phase cell cycle arrest as indicated by flow cytometry analysis. In addition, the conjugate induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-9, release of cytochrome-c into cytosol and increased Bax: Bcl-2 ratio. The conjugate also suppressed the phosphorylation of Akt and ERK. The conjugate induced cell death was significantly increased in presence of A6730 (a potent Akt1/2 kinase inhibitor) and PD98059 (a specific ERK inhibitor). Moreover, the conjugated PTER inhibited tumor growth in Ehrlich ascitic cell induced tumor bearing mice as observed by reduction in tumor volume compared to untreated animals. Collectively, the pro-apoptotic effect of conjugate is mediated through the activation of caspases, and is correlated with the blockade of the Akt and ERK signaling pathways in MCF-7 cells.     

Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14: RELEVANCE TO ITS STORAGE AND SECRETION*?

The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys³–Cys¹⁴) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.