Gas Chromatography- Mass Spectrometry Based Metabolomic Approach for Optimization and Toxicity Evaluation of Earthworm Sub-Lethal Responses to Carbofuran

Despite recent advances in understanding mechanism of toxicity, the development of biomarkers (biochemicals that vary significantly with exposure to chemicals) for pesticides and environmental contaminants exposure is still a challenging task. Carbofuran is one of the most commonly used pesticides in agriculture and said to be most toxic carbamate pesticide. It is necessary to identify the biochemicals that can vary significantly after carbofuran exposure on earthworms which will help to assess the soil ecotoxicity. Initially, we have optimized the extraction conditions which are suitable for high-throughput gas chromatography mass spectrometry (GC-MS) based metabolomics for the tissue of earthworm, Metaphire posthuma. Upon evaluation of five different extraction solvent systems, 80% methanol was found to have good extraction efficiency based on the yields of metabolites, multivariate analysis, total number of peaks and reproducibility of metabolites. Later the toxicity evaluation was performed to characterize the tissue specific metabolomic perturbation of earthworm, Metaphire posthuma after exposure to carbofuran at three different concentration levels (0.15, 0.3 and 0.6 mg/kg of soil). Seventeen metabolites, contributing to the best classification performance of highest dose dependent carbofuran exposed earthworms from healthy controls were identified. This study suggests that GC-MS based metabolomic approach was precise and sensitive to measure the earthworm responses to carbofuran exposure in soil, and can be used as a promising tool for environmental eco-toxicological studies.     

Tumor versus Stromal Cells in Culture—Survival of the Fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.     

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete‐expressing local interneurons in development of the adult olfactory circuitry. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance.     

High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex

Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.     

Ligand-Induced Protein Mobility in Complexes of Carbonic Anhydrase II and Benzenesulfonamides with Oligoglycine Chains

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1–5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with ¹⁵N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even “static” proteins in studies of protein-ligand binding, including rational ligand design approaches.     

TDP-43, an ALS Linked Protein,Regulates Fat Deposition and Glucose Homeostasis

The identification of proteins which determine fat and lean body mass composition is critical to better understanding and treating human obesity. TDP-43 is a well-conserved RNA-binding protein known to regulate alternative splicing and recently implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). While TDP-43 knockout mice show early embryonic lethality, post-natal conditional knockout mice show weight loss, fat depletion, and rapid death, suggesting an important role for TDP-43 in regulating energy metabolism. Here we report, that over-expression of TDP-43 in transgenic mice can result in a phenotype characterized by increased fat deposition and adipocyte hypertrophy. In addition, TDP-43 over-expression in skeletal muscle results in increased steady state levels of Tbc1d1, a RAB-GTPase activating protein involved in Glucose 4 transporter (Glut4) translocation. Skeletal muscle fibers isolated from TDP-43 transgenic mice show altered Glut4 translocation in response to insulin and impaired insulin mediated glucose uptake. These results indicate that levels of TDP-43 regulate body fat composition and glucose homeostasis in vivo.     

Enhanced N-metabolites,ABA and IAA-conjugate in anthers instigate heat sensitivity in spring wheat

Unraveling the metabolic and phytohormonal changes in anthers exposed to heat stress would help identify mechanisms regulating heat stress tolerance during the sensitive reproductive stage. Two spring wheat genotypes contrasting for heat tolerance were exposed to heat stress during heading in controlled environment chambers. Anthers were collected from main and primary spikes for metabolic and phytohormonal profiling. A significant reduction in seed set (38%), grain number (54%) and grain weight (52%) per plant was recorded in the sensitive (KSG1177) but not in the tolerant (KSG1214) genotype under heat stress compared to control. Anther metabolite accumulation did not vary quantitatively between main and primary spikes. Hierarchical clustering of the genotypes and treatments using metabolites and phytohormones revealed a distinct cluster for KSG1177 under heat stress from that of control and KSG1214. A significant increase in N-based amino acids, ABA, IAA-conjugate and a decrease in polyamines and organic acids were observed in wheat anthers exposed to heat stress. Unlike KSG1214, a significantly higher accumulation of amino acids, ABA and IAA-conjugate in anthers of the sensitive KSG1177 was recorded under heat stress. These findings provide the rationale and direction towards developing molecular markers for enhancing heat stress tolerance in wheat.