Abha Meena

Reliable encoding of stimulus intensities within random sequences of intracellular Ca2+ spikes.

Mathematical analysis of Ca2+ signals in single cells reveals how cells can encode stimulus intensity in the frequency of Ca2+ spikes. Apparently Random Signals Encode Information Reliably Repetitive Ca2+ spikes occur in many cells in response to stimuli that activate an intracellular signaling cascade that involves Ca2+ released from internal stores. These repetitive spikes are believed to represent the intensity of the stimulus, such that increasing the stimulus increases the frequency of the spikes. But the time between spikes (interspike interval) is random within a cell, and cells in a population exhibit variable spiking frequencies. Thurley et al. performed single-cell Ca2+ imaging of primary liver cells and human embryonic kidney (HEK) 293 cells to examine the properties of Ca2+ spikes in response to extracellular ligands under various conditions. Mathematical analysis revealed that, although the interspike interval had a random element, there was a consistent fold change in this interval across populations of cells responding to different amounts of the ligands. Thus, a common change to a random element enables the cells to properly interpret signal intensity from the frequency of repetitive Ca2+ spikes. Ca2+ is a ubiquitous intracellular messenger that regulates diverse cellular activities. Extracellular stimuli often evoke sequences of intracellular Ca2+ spikes, and spike frequency may encode stimulus intensity. However, the timing of spikes within a cell is random because each interspike interval has a large stochastic component. In human embryonic kidney (HEK) 293 cells and rat primary hepatocytes, we found that the average interspike interval also varied between individual cells. To evaluate how individual cells reliably encoded stimuli when Ca2+ spikes exhibited such unpredictability, we combined Ca2+ imaging of single cells with mathematical analyses of the Ca2+ spikes evoked by receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP3). This analysis revealed that signal-to-noise ratios were improved by slow recovery from feedback inhibition of Ca2+ spiking operating at the whole-cell level and that they were robust against perturbations of the signaling pathway. Despite variability in the frequency of Ca2+ spikes between cells, steps in stimulus intensity caused the stochastic period of the interspike interval to change by the same factor in all cells. These fold changes reliably encoded changes in stimulus intensity, and they resulted in an exponential dependence of average interspike interval on stimulation strength. We conclude that Ca2+ spikes enable reliable signaling in a cell population despite randomness and cell-to-cell variability, because global feedback reduces noise, and changes in stimulus intensity are represented by fold changes in the stochastic period of the interspike interval.

Development of QSAR model for immunomodulatory activity of natural coumarinolignoids

Immunomodulation is the process of alteration in immune response due to foreign intrusion of molecules inside the body. Along with the available drugs, a large number of herbal drugs are promoted in traditional Indian treatments, for their immunomodulating activity. Natural coumarinolignoids isolated from the seeds of Cleome viscose have been recognized as having hepatoprotective action and have recently been tested preclinically for their immunomodulatory activity affecting both cell-mediated and humoral immune response. To explore the immunomodulatory compound from derivatives of coumarinolignoids, a quantitative structure activity relationship (QSAR) and molecular docking studies were performed. Theoretical results are in accord with the in vivo experimental data studied on Swiss albino mice. Immunostimulatory activity was predicted through QSAR model, developed by forward feed multiple linear regression method with leave-one-out approach. Relationship correlating measure of QSAR model was 99% (R2 = 0.99) and predictive accuracy was 96% (RCV2 = 0.96). QSAR studies indicate that dipole moment, steric energy, amide group count, lambda max (UV-visible), and molar refractivity correlates well with biological activity, while decrease in dipole moment, steric energy, and molar refractivity has negative correlation. Docking studies also showed strong binding affinity to immunomodulatory receptors.

In silico exploration of anti-inflammatory activity of natural coumarinolignoids.

Natural coumarinolignoids isolated from the seeds of Cleome viscosa consist of a racemic mixture of cleomiscosins A, B and C. To screen out potential lead, anti‐inflammatory activity of the isolated compounds was evaluated through molecular docking and QSAR studies by using reported in vivo activity of Swiss albino mice. Based on docking binding affinity, a possible mechanism of action has been hypothesized which constitute toll‐like receptors (TLR‐4), cluster of differentiation molecules (CDs), iNOS, COX‐2 and STAT‐6 proteins. It was very interesting to find that the 3D topology of the active site of COX‐2 from the docking was in good agreement with QSAR model and in silico ADME/T parameters. A forward feed multiple linear regression model was developed with r2 = 0.92 and rCV2 = 0.87. This study showed that chemical descriptors, for example dipole vector‐X, dipole vector‐Y, steric energy, LUMO energy, size of smallest ring, size of largest ring and carboxyl group count, correlate reasonably well with experimental in vivo activity (logLD50). QSAR study indicates that dipole vector‐Y and carboxyl group count have negative correlation with activity. Cleomiscosins also showed compliance with 95% of in silico ADME/T properties of available drugs, e.g. serum protein binding, blood–brain barrier, CNS activity, HERG K+ channel activity, apparent Caco‐2 permeability, apparent MDCK permeability, skin permeability and human oral absorption in GI. Besides, toxicity screening study suggests that cleomiscosin molecules possess no toxicity risk parameters. This study offer useful references for understanding and molecular design of inhibitors with improved anti‐inflammatory activity.

Genomic Identification of Potential Targets Unique to Candida albicans for the Discovery of Antifungal Agents

Despite of modern antifungal therapy, the mortality rates of invasive infection with human fungal pathogen Candida albicans are up to 40%. Studies suggest that drug resistance in the three most common species of human fungal pathogens viz., C. albicans, Aspergillus fumigatus (causing mortality rate up to 90%) and Cryptococcus neoformans (causing mortality rate up to 70%) is due to mutations in the target enzymes or high expression of drug transporter genes. Drug resistance in human fungal pathogens has led to an imperative need for the identification of new targets unique to fungal pathogens. In the present study, we have used a comparative genomics approach to find out potential target proteins unique to C. albicans, an opportunistic fungus responsible for severe infection in immune-compromised human. Interestingly, many target proteins of existing antifungal agents showed orthologs in human cells. To identify unique proteins, we have compared proteome of C. albicans [SC5314] i.e., 14,633 total proteins retrieved from the RefSeq database of NCBI, USA with proteome of human and non-pathogenic yeast Saccharomyces cerevisiae. Results showed that 4,568 proteins were identified unique to C. albicans as compared to those of human and later when these unique proteins were compared with S. cerevisiae proteome, finally 2,161 proteins were identified as unique proteins and after removing repeats total 1,618 unique proteins (42 functionally known, 1,566 hypothetical and 10 unknown) were selected as potential antifungal drug targets unique to C. albicans.

Impact of biochar amendment on enzymatic resilience properties of mine spoils.

Soil enzymes are crucial for soil nutrient cycling function. Understanding of the factors that control their response to major disturbances such as dumping of environmentally toxic acidic waste remains limited. We evaluated the effect of dumping of overburden (OB) and their amendments using biochar, on the resistance and resilience of soil enzyme activities involved in phosphorus, nitrogen, sulphur and carbon cycling (acid & alkaline phosphatase, urease, arylsulphatase, dehydrogenase, phenol oxidases, cellulase and β-glucosidase). For investigation the soils treated with OB and with the mixture of OB and biochar were used for the cultivation of bacopa were used. We assessed 0 day, 45 day and 90 days activities of the target soil enzymes, available phosphorus, nitrogen, sulphur, soil organic carbon and microbial identification. The resilience and resistance index of all the treatments were calculated. We found that phyto-remediated OB-contaminated soil has its own resilience power. However, biochar addition enhanced the enzyme resistance and resilience of OB contaminated soil. In silico study indicates that biochar-Fe complex play a significant role in enzymatic activities. Overall, the results indicate a significant influence of phytoremediation and biochar addition on soil enzymatic activity that is extremely resistant to OB. This study provides insight on how biochar addition modulates soil biochemical and microbiological response to OB affected soils.

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana.

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition.

D-MATRIX: a web tool for constructing weight matrix of conserved DNA motifs.

Despite considerable efforts to date, DNA motif prediction in whole genome remains a challenge for researchers. Currently the genome wide motif prediction tools required either direct pattern sequence (for single motif) or weight matrix (for multiple motifs). Although there are known motif pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a D-MATRIX tool which predicts the different types of weight matrix based on user defined aligned motif sequence set and motif width. For retrieval of known motif sequences user can access the commonly used databases such as TFD, RegulonDB, DBTBS, Transfac. DMATRIX program uses a simple statistical approach for weight matrix construction, which can be converted into different file formats according to user requirement. It provides the possibility to identify the conserved motifs in the coregulated genes or whole genome. As example, we successfully constructed the weight matrix of LexA transcription factor binding site with the help of known sosbox cisregulatory elements in Deinococcus radiodurans genome. The algorithm is implemented in C-Sharp and wrapped in ASP.Net to maintain a user friendly web interface. DMATRIX tool is accessible through the CIMAP domain network. Availability http://203.190.147.116/dmatrix/

Neoflavonoids and tetrahydroquinolones as possible cancer chemopreventive agents

Several lactone‐ and lactam‐based neoflavonoids and tetrahydroquinolones were synthesized and evaluated for cancer chemopreventive studies using cell and molecular target‐based in vitro bioassays, namely NFκB, aromatase, and quinone reductase 1. These analogs blocked TNF‐α‐induced NFκB activation in a dose‐dependent manner with IC50 values in the range of 0.11–3.2 μm. In addition, compound 8 inhibited aromatase activity with an IC50 value of 12.12 μm, and compound 10 affected quinone reductase 1 induction (IR, 3.6; CD, 19.57 μm). Neoflavonoids 8 and 10 exhibiting good results can further be optimized for improved therapeutic profiles. However, investigations into the actions of neoflavonoids and tetrahydroquinolones, especially those related to the NFκB signaling pathway, aromatase inhibition, induction of quinone reductase 1 expression, and in vivo studies could provide new insights into the cancer chemopreventive ability of these molecules.

Suppression of tumor necrosis factor-α-induced nuclear factor κB activation and aromatase activity by capsaicin and its analog capsazepine.

Target-specific drugs, including natural products, offer promise for the amelioration of cancer and other human ailments. Capsaicin, the pungent ingredient present in chilies (Capsicum annuum L.), and capsazepine, a synthetic analog of capsaicin (collectively referred to as vanilloids), are known to possess a variety of pharmacological and physiological properties. In our continuous effort to discover and characterize cancer chemopreventive agents from natural products, we investigated the effect of vanilloids on nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activation using stably transfected 293/NFκB-Luc human embryonic kidney cells induced by treatment with tumor necrosis factor-α (TNFα) and on aromatase activity. Capsaicin and capsazepine blocked TNFα-induced NFκB activation in a dose-dependent manner with 50% inhibitory concentration (IC(50)) values of 0.68 and 4.2 μM, respectively. No significant cytotoxicity was observed at the highest concentrations tested (53.1 μM for capsazepine and 65.5 μM for capsaicin). In addition, these vanilloids inhibited aromatase activity with IC(50) values of 13.6 and 8.8 μM, respectively. Computer-aided molecular docking studies showed docking scores indicative of good binding affinity of vanilloids with aromatase and NFκB. The highly conserved residues for capsaicin and capsazepine binding with NFκB p50 were Ser299 and Ile278 (H-bond 2.81Å) and with NFκB p100 were Ser6, Arg82, Val86, Arg90 (H-bond 2.89Å), Gly4, and Ser2 (H-bond 2.81Å). The amino acids Trp224, Arg435, and Val373 (H-bond 2.80Å) were found to be important for the binding of capsaicin and capsazepine with aromatase. Based on these findings, aromatase and NFκB are suggested as valid targets for these compounds; additional investigation of chemopreventive or chemotherapeutic potential is required.

Sustained signalling by PTH modulates IP3 accumulation and IP3 receptors through cyclic AMP junctions

ABSTRACT Parathyroid hormone (PTH) stimulates adenylyl cyclase through type 1 PTH receptors (PTH1R) and potentiates the Ca2+ signals evoked by carbachol, which stimulates formation of inositol 1,4,5-trisphosphate (IP3). We confirmed that in HEK cells expressing PTH1R, acute stimulation with PTH(1-34) potentiated carbachol-evoked Ca2+ release. This was mediated by locally delivered cyclic AMP (cAMP), but unaffected by inhibition of protein kinase A (PKA), exchange proteins activated by cAMP, cAMP phosphodiesterases (PDEs) or substantial inhibition of adenylyl cyclase. Sustained stimulation with PTH(1-34) causes internalization of PTH1R–adenylyl cyclase signalling complexes, but the consequences for delivery of cAMP to IP3R within cAMP signalling junctions are unknown. Here, we show that sustained stimulation with PTH(1-34) or with PTH analogues that do not evoke receptor internalization reduced the potentiated Ca2+ signals and attenuated carbachol-evoked increases in cytosolic IP3. Similar results were obtained after sustained stimulation with NKH477 to directly activate adenylyl cyclase, or with the membrane-permeant analogue of cAMP, 8-Br-cAMP. These responses were independent of PKA and unaffected by substantial inhibition of adenylyl cyclase. During prolonged stimulation with PTH(1-34), hyperactive cAMP signalling junctions, within which cAMP is delivered directly and at saturating concentrations to its targets, mediate sensitization of IP3R and a more slowly developing inhibition of IP3 accumulation.