Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host's immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.     

Fetal exposure to high-avidity TCR ligand enhances expansion of peripheral T regulatory cells

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.     

Caffeic acid affects early growth, and morphogenetic response of hypocotyl cuttings of mung bean (Phaseolus aureus)

Caffeic acid (CA) is one of the most common cinnamic acids ubiquitously present in plants and implicated in a variety of interactions including allelopathy among plants and microbes. This study investigated the possible interference of CA with root growth and the process of rhizogenesis in hypocotyl cuttings of mung bean (Phaseolus aureus=Vigna radiata). Results indicated that CA (0-1000 microM) significantly suppressed root growth of mung bean, and impaired adventitious root formation and root length in the mung bean hypocotyl cuttings. Further investigations into the role of CA in hampering root formation indicated its interference with the biochemical processes involved in rooting process at the three stages - root initiation (third day; RI), root expression (fifth day; RE), and post-expression (seventh day; PE) - of rhizogenesis. CA caused significant changes in the activities of proteases, peroxidases (PODs), and polyphenol oxidases (PPOs) during root development and decreased the content of total endogenous phenolics (TP) in the hypocotyl cuttings. The enhanced activity of PODs and PPOs, though, relates to lignification and/or phenolic metabolism during rhizogenesis; yet their protective role to CA-induced stress, especially during the PE phase, is not ruled out. At 1000 microM CA, where rooting was significantly affected, TP content was very high during the RI phase, thus indicating its non-utilization. The study concludes that CA interferes with the rooting potential of mung bean hypocotyl cuttings by altering the activities of PODs and PPOs and the endogenous TP content that play a key role in rhizogenesis.     

Time resolved secretion of chloride from a monolayer of mucin-secreting epithelial cells

Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride ion-selective electrode (ISE) at a short distance (∼50 μm) from the monolayer. The recorded concentrations are transformed to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875–2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl⁻ flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl⁻ may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution of seconds provides unique information about the dynamics of solute fluxes across the apical membrane. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and Pathogenic Relationships of Rhizoctonia solani Isolates in a Potato–Rice System and their Sensitivity to Fungicides

Potato is planted after rice in several parts of Punjab in India and both crops are attacked by Rhizoctonia solani Kühn. Potato tubers showing black scurf and rice plants affected by sheath blight were collected from different regions of the state and the isolates of R. solani so obtained were studied to determine their variability and to ascertain their cross-infectivity and response to fungicides. Potato isolates of R. solani did not infect rice plants but some rice isolates were weakly pathogenic on potato, the sclerotia being less firmly attached on tuber surface, indicating a possible unsuccessful attempt of rice isolates to infect potato. Rice isolates (66.6%) grew faster (>20 mm colony growth per 24 h) than those of the potato isolates (15–20 mm growth rate per 24 h). Hyphal width of isolates from both hosts varied from 7.2 to 12.1 μm. Colony growth of most potato isolates (61.2%) was appressed, whereas that of most rice isolates (53.3%) was fluffy. Rice isolates (73.3%) formed larger sclerotia (1.5–2.0 mm in diameter) than those of the potato isolates (0.5–1.0 mm in diameter). Anastomosis studies indicated that potato isolates belonged to AG-3 and AG-5 groups while rice isolates belonged to the AG-1-1-A group. Representative R. solani isolates from the two hosts showed significant variation in response to fungicides (i.e. carbendazim, carboxin, pencycuron, propiconazole and validamycin) based on their ED₅₀ and ED₉₀ values.     

Dedifferentiation-mediated changes in transposition behavior make the Activator transposon an ideal tool for functional genomics in rice

There is an inverse relationship between the level of cytosine methylation in genomic DNA and the activity of plant transposable elements. Increased transpositional activity is seen during early plant development when genomic methylation patterns are first erased and then reset. Prolonging the period of hypomethylation might therefore result in an increased transposition frequency, which would be useful for rapid genome saturation in transposon-tagged plant lines. We tested this hypothesis using transgenic rice plants containing Activator (Ac) from maize. R₁ seeds from an Ac-tagged transgenic rice line were either directly germinated and grown to maturity, or induced to dedifferentiate in vitro, resulting in cell lines that were subsequently regenerated into multiple mature plants. Both populations were then analyzed for the presence, active reinsertion and amplification of Ac. Plants from each population showed excision-reinsertion events to both linked and unlinked sites. However, the frequency of transposition in plants regenerated from cell lines was more than nine-fold greater than that observed in plants germinated directly from seeds. Other aspects of transposon behavior were also markedly affected. For example, we observed a significantly larger proportion of transposition events to unlinked sites in cell line-derived plants. The tendency for Ac to insert into transcribed DNA was not affected by dedifferentiation. The differences in Ac activity coincided with a pronounced reduction in the level of genomic cytosine methylation in dedifferentiated cell cultures. We used the differential transposon behavior induced by dedifferentiation in the cell-line derived population for direct applications in functional genomics and validated the approach by recovering Ac insertions in a number of genes. Our results demonstrate that obtaining multiple Ac insertions is useful for functional annotation of the rice genome.These authors contributed equally to the work     

Cr(VI) Imposed Toxicity in Maize Seedlings Assessed in Terms of Disruption in Carbohydrate Metabolism

The present study examined the toxic effects of Cr(VI; 100, 250 and 500 μM) in maize seedlings by investigating the changes in carbohydrate metabolism after 48, 96, and 144 h of exposure. Cr-stress results in severe alterations in the contents of carbohydrates and reducing sugars and the activities of carbohydrate metabolizing enzymes, amylases, phosphatases and phosphorylases, and invertases in maize seedlings. Under Cr stress, the contents of carbohydrates and reducing sugars declined in roots, whereas an increase was noticed in leaves. The catalytic activity of carbohydrate metabolizing enzymes, except invertases, in roots declined in the presence of Cr(VI) in a concentration- and exposure time-dependent manner. In contrast, the activities of these enzymes were enhanced in leaves under Cr(VI) stress. The activity of invertases increased with increasing amount of Cr(VI) but declined with an increase in the time interval. In conclusion, our results show that carbohydrate metabolism is severely affected under Cr(VI) toxicity. The study suggests that Cr-induced perturbations in the carbohydrate metabolism are one of the factors resulting in growth inhibition under Cr(VI) stress.     

Differences in biophysical properties of nucleus accumbens medium spiny neurons emerging from inactivation of inward rectifying potassium currents

Inward rectifying potassium (K_IR) currents in medium spiny (MS) neurons of nucleus accumbens inactivate significantly in ~40% of the neurons but not in the rest, which may lead to differences in input processing by these two groups. Using a 189-compartment computational model of the MS neuron, we investigate the influence of this property using injected current as well as spatiotemporally distributed synaptic inputs. Our study demonstrates that K_IR current inactivation facilitates depolarization, firing frequency and firing onset in these neurons. These effects may be attributed to the higher input resistance of the cell as well as a more depolarized resting/down-state potential induced by the inactivation of this current. In view of the reports that dendritic intracellular calcium levels depend closely on burst strength and spike onset time, our findings suggest that inactivation of K_IR currents may offer a means of modulating both excitability and synaptic plasticity in MS neurons.     

A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast.

The point mutation M26 in the ade6 gene of Schizosaccharomyces pombe increases recombination frequency by an order of magnitude in comparison with other mutations in the same gene. The hypothesis is tested that this hot spot of recombination requires a specific nucleotide sequence at the M26 site. The DNA sequence is altered systematically by in vitro mutagenesis, and the resulting sequences are introduced into the ade6 gene in vivo by gene replacement. It results that any change of the heptanucleotide ATGACGT leads to loss of high frequency of recombination. Thus this oligonucleotide sequence is necessary for high frequency of recombination, but it seems not to be sufficient.     

The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP₆). We extend the applicability of our results to show that IP₆ also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP₆-induced conformational change in AvrA suggests that IP₆ acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP₆; once injected into mammalian cells by the pathogen these toxins bind host cell IP₆, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity.