Deciphering growth abilities of fusarium oxysporum f. sp. pisi under variable temperature, pH and nitrogen.

Fusarium wilt caused by Fusarium oxysporum f. sp. pisi (Fop) is an important disease and major obstacle to pea production, causing huge losses to growers. The focus of this study was on isolation followed by morphological, molecular characterization and analyzing the growth of the casual agent under variable temperature, pH and Nitrogen levels. The morphological features of radial growth, sporulation, pigmentation and mycelial characterization were examined and the variability of all isolates was presented. Molecular characterization of the fungus by ITS rDNA sequencing revealed that all 13 isolates belong to Fusarium oxysporum species. Six isolates were tested for temperature, pH and nitrogen dosage optimization studies. Seven different temperatures, viz., 21, 23, 25, 27, 29, 31, 33°C and pH values, having 3, 4, 5, 6, 7, 8, and 9 pH, as well as nitrogen dosage levels of 0 g, 3 g, 5 g, 7 g, 9 g, 11 g, and 13 g were tested against all six isolates, respectively. The results showed that all isolates exhibited the highest growth at a temperature of 25°C and the optimal temperature range for growth of Fusarium oxysporum was 23-27°C. All isolates showed the highest growth at pH5. Change in the nitrogen doses of the base ended in formation of thick, dense, fluffy mycelium of the casual agent. Six isolates were used for combination studies with seven different levels of temperatures, pH levels and nitrogen dosages. The density plots revealed the variations in the growth of the isolates with changes in temperature, pH and nitrogen levels, which can lead to mutations or genetic changes in the pathogens that could potentially introduce new threats to pea cultivation.

Probing the origins of programmed death ligand-1 inhibition by implementing machine learning-assisted sequential virtual screening techniques.

PD-L1 is a key immunotarget involved in binding to its receptor PD-1. PD-L1/PD-1 interface blocking using antibodies (or small molecules) is the central area of interest for tumor suppression in various cancers. Blocking the PD-L1/PD-1 pathway in the tumor cells results in its immune activation and destruction, and thereby restoring the T-cell proliferation and cytokine production. The active binding site interface residues of PD-L1/PD-1 were experimentally known and proven by structural biology and site-directed mutagenesis studies. Structure-based molecular design technique was employed to identify the inhibitors for blocking the PD-L1/PD-1 interface. Nine hits to leads were identified from the SPECS small molecule database by machine learning, molecular docking, and molecular dynamics simulation techniques. Following this, a machine learning-assisted QSAR modeling approach was implemented using ChEMBL database to gain insights into the inhibitory potential of PD-L1 inhibitors and predict the activity of our previously screened nine hit molecules. The best leads identified in the present study bind strongly with the active sites of PD-L1/PD-1 interface residues, which include A121, M115, I116, S117, I54, Y56, D122, and Y123. These computational leads are considered promising molecules for further in vitro and in vivo analysis to be developed as potential PD-L1 checkpoint inhibitors to cure different types of cancers.

Synthesis, Molecular Docking and Preliminary Antileukemic Activity of 4-Methoxybenzyl Derivatives Bearing Imidazo[2,1-b][1,3,4]thiadiazole.

In this study, we synthesized 22 compounds in a series with various substitution on imidazo[2,1-b][1,3,4]thiadiazole. The potential cytotoxic activity of these compounds investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identified two compounds, 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate and 6-(4-chlorophenyl)-2-(4-methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde, exhibited the most cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa) with IC50 values ranging between 0.79 and 1.6 µM. The results indicate that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-ß) type I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.

Generation of humanized single-chain fragment variable immunotherapeutic against EGFR variant III using baculovirus expression system and in vitro validation.

Epidermal growth factor receptor variant III (EGFRvIII) is known to be specifically expressed in cancer cells and associated with tumor virulence. The receptor provides an opportunity for both specifically targeting the tumor cells as well as for potentially controlling and inhibiting tumor progression. In this study, humanized anti-EGFRvIII single-chain fragment variable (hscFv) was expressed in insect cell culture system to accommodate post-translational glycosylations crucial for the fragment stability and efficacy. Target specific binding of the developed fragment to EGFRvIII expressing cell lines and EGFRvIII positive glioblastoma patient samples was evaluated by immunocytochemistry and immunohistochemistry respectively. Downstream intracellular signaling mechanisms related to the action of the developed antibody fragment on growth/metabolism of the cell was evaluated in U87-EGFRvIII human glioblastoma cell lines. It was observed that the hscFv bound specifically to EGFRvIII in mutant expressing cells. Functionally, hscFv was found to confer anti-proliferative properties in EGFRvIII expressing cell lines by downregulating phosphorylation of EGFR/EGFRvIII, Lyn, PI3K and GLUT3 involved in proliferation and metabolism. This study demonstrated the significance of hscFv as a potential immunotherapeutic agent as well as a targeting agent for specific delivery of drugs to EGFRvIII expressing cancer cells.

Predictive models for designing potent tyrosine kinase inhibitors in chronic myeloid leukemia for understanding its molecular mechanism of resistance by molecular docking and dynamics simulations.

BCR-ABL fusion protein drives chronic myeloid leukemia (CML) which constitutively activates tyrosine kinase involved in the initiation and maintenance of CML phenotype. Ponatinib, an oral drug, was discovered as an efficient BCR-ABL inhibitor by addressing imatinib drug resistance arising due to the point mutations at its active sites. In this study, 44 BCR-ABL kinase inhibitors, which are derivatives of ponatinib, were used to develop a robust two-dimensional quantitative structure-activity relationship (2D-QSAR) and 3D-Pharmacophore models by dividing dataset into 32 training sets and 12 test set molecules. 2D-QSAR model was developed using Genetic Function Approximation (GFA) algorithm consisting of four types of information-rich molecular descriptors, electrotopological (ES_Count_aasN and ES_Sum_aaaC), electronic (Dipole_X), spatial (PMI_Y) and thermodynamic (LogD), primarily contributing to BCR-ABL kinase inhibitory activity. For the best 2D-QSAR model, the statistics were R2 = 0.8707, R2pred = 0.8142 and N = 32 for the training set molecules. Phase module of Schrödinger suit was employed for 3D-Pharmacophore model development showing five different pharmacophoric features - ADHHPRR with good R2 of 0.9629, F of 175.3, Q2 of 0.645 and root-mean-square error (RMSE) of 0.214 that are essential for an effective BCR-ABL kinase inhibition. These two models were further validated by cross-validation, test set predictions, enrichment factor calculations and predictions based on the external dataset. The molecular mechanism of resistance arising due to gate keeper mutation T315I of ABL kinase in complex with its inhibitors was also studied using molecular docking and molecular dynamics simulations. Our developed models predicted key chemical features for designing potent inhibitors against BCR-ABL kinase activity and its resistance mechanism to CML disease therapy. Communicated by Ramaswamy H. Sarma.

Structure-function studies of prothrombin Amrita, a dysfunctional prothrombin characterized by point mutation at Arg553 → Gln.

A dysfunctional prothrombin gene characterized by novel point mutation at Arg553 to Gln residue in Deep vein thrombosis (DVT) patient which we designated as "Prothrombin Amrita" was previously reported from our lab. The mutation occurred at nucleotide 20030 in exon 14 and was confirmed by restriction enzyme digestion. Arg553 has been reported as one of the key residues for the binding of cofactor Na+ ion in the thrombin protein. Structural analysis revealed the molecular mechanism behind the coagulant form of thrombin due to point Arg553Gln mutation near the cofactor Na+ ion region. Molecular electrostatic potential maps and molecular dynamics (MD) simulation of the wild type and mutated thrombin showed the key role played by the Na+ ion for its coagulant mechanism by analysing the charge distribution and nature of the hydrogen bonding at the mutated region of interest. We observed maintenance of the fast or procoagulant form of dysfunctional prothrombin due to changes in the charge distribution by this mutation and thereby also keeping strong hydrogen bonding network revealed by MD simulation between prothrombin and Na+ ion. This molecular mechanism might be the main cause for DVT in patients with this dysfunctional prothrombin gene.

Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus.

Lysostaphin (LST) is a bacteriocin that cleaves within the pentaglycine cross bridge of Staphylococcus aureus peptidoglycan. Previous studies have reported the high efficiency of LST even against multi drug resistant S. aureus including methicillin resistant S. aureus (MRSA). In this study, we have developed a new chitosan based hydrogel formulation of LST to exploit its anti-staphylococcal activity. The atomic interactions of LST with chitosan were studied by molecular docking studies. The rheology and the antibacterial properties of the developed LSTC gel were evaluated. The developed LST containing chitosan hydrogel (LSTC gel) was flexible, flows smoothly and remains stable at physiological temperature. The in vitro studies by agar well diffusion and ex vivo studies in porcine skin model exhibited a reduction in S. aureus survival by ∼3 Log10CFU/mL in the presence of LSTC gel. The cytocompatibility of the gel was tested in vitro using macrophage RAW 264.7 cell line and in vivo in Drosophila melanogaster. A gradual disruption of S. aureus biofilms with the increase of LST concentrations in the LSTC gel was observed which was confirmed by SEM analysis. We conclude that LSTC gel could be highly effectual and advantageous over antibiotics in treating staphylococcal-topical and biofilm infections.

Carboxymethylated É©-carrageenan conjugated amphotericin B loaded gelatin nanoparticles for treating intracellular Candida glabrata infections.

Intercellular Candida glabrata infections are difficult to treat due to poor penetration of drugs into the fungal niche. Delivering amphotericin B (Amp B) into the macrophages where the pathogen inhabits is an effective solution. We are studying the macrophage targeting proficiency of É©-carrageenan for the delivery of Amp B using gelatin A nanoparticles (GNPs). The choice of gelatin A was the outcome of in silico inspections where the amino functionalized polymer having the best docking score with Amp B was selected. We prepared a sustained release formulation of amp B loaded carboxymethyl É©-carrageenan conjugated gelatin nanoparticles (CMC-Amp B-GNPs) with size 343±12nm and -25±5.3mV zeta potential. The formulations were found to be stable, biocompatible and non-haemolytic. Flow cytometry analysis showed 3 fold higher uptake of CMC-GNPs compared to the GNPs by RAW 264.7 cells. CMC-Amp B-GNPs showed enhanced antifungal activity than bare Amp B and Amp B-GNPs.

In silico identification of T-type calcium channel blockers: A ligand-based pharmacophore mapping approach.

Limited progress has been made in the quest to identify both selective and non-toxic T-type calcium channel blocking compounds. The present research work was directed toward slaking the same by identifying the selective three dimensional (3D) pharmacophore map for T-type calcium channel blockers (CCBs). Using HipHop module in the CATALYST 4.10 software, both selective and non-selective HipHop pharmacophore maps for T-type CCBs were developed to identify its important common pharmacophoric features. HipHop pharmacophore map of the selective T-type CCBs contained six different chemical features, namely ring aromatic (R), positive ionizable (P), two hydrophobic aromatic (Y), hydrophobic aliphatic (Z), hydrogen bond acceptor (H) and hydrogen bond donor (D). However, non-selective T-type CCBs contain all the above mentioned features except ring aromatic (R). The present ligand-based pharmacophore mapping approach could thus be utilized in classifying selective vs. non-selective T-type CCBs. Further, the model can be used for virtual screening of several small molecule databases.

Integration of in silico modeling, prediction by binding energy and experimental approach to study the amorphous chitin nanocarriers for cancer drug delivery.

In silico modeling of the polymer-drug nanocarriers have now days became a powerful virtual screening tool for the optimization of new drug delivery systems. The interactions between amorphous chitin nanoparticles (AC-NPs) with three different types of anti-cancer drugs such as curcumin, docetaxel and 5-flurouracil were studied by integration of computational and experimental techniques. The drug entrapment and drug loading efficiency of these three drugs with AC-NPs were (98±1%), (77±2%), and (47±12%), respectively. Further, cytotoxicity and cellular uptake studies of drug loaded AC-NPs on Gastric adenocarcinoma (AGS) cells showed enhanced drug uptake and cancer cell death. In silico binding energy (BE) between AC-NPs with these anti-cancer drugs were studied by molecular docking technique. Computational drug's BEs are in excellent agreement with experimental AC-NPs drug loading (R(2)=0.9323) and drug entrapment (R(2)=0.9741) efficiencies. Thus, present integrated study revealed significant insight on chemical nature, strength, and putative interacting sites of anti-cancer drugs with AC-NPs.

Antibiotic resistance in Pseudomonas aeruginosa and alternative therapeutic options.

Pseudomonas aeruginosa is a leading cause of nosocomial infections and is responsible for ∼10% of all hospital-acquired infections worldwide. It continues to pose a therapeutic challenge because of the high rate of morbidity and mortality associated with it and the possibility of development of drug resistance during therapy. Standard antibiotic regimes against P. aeruginosa are increasingly becoming ineffective due to the rise in drug resistance. With the scope for developing new antibiotics being limited, alternative treatment options are gaining more and more attention. A number of recent studies reported complementary and alternative treatment options to combat P. aeruginosa infections. Quorum sensing inhibitors, phages, probiotics, anti-microbial peptides, vaccine antigens and antimicrobial nanoparticles have the potential to act against drug resistant strains. Unfortunately, most studies considering alternative treatment options are still confined in the pre-clinical stages, although some of these findings have tremendous potential to be turned into valuable therapeutics. This review is intended to raise awareness of several novel approaches that can be considered further for combating drug resistant P. aeruginosa infections.

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: the case of chelerythrine.

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aß) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aß aggregation as well as the ability to disaggregate already preformed Aß aggregates in an experimental set-up using HFIP as promotor of Aß aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aß aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aß aggregation as well as to disaggregate preformed Aß aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Antibacterial potential of sponge endosymbiont marine Enterobacter sp at Kavaratti Island, Lakshadweep archipelago.

OBJECTIVE: To isolate antibacterial potential of sponge endosymbiotic bacteria from marine sponges at Lakshadweep archipelago. Also to identify the potent bacteria by 16s rDNA sequencing and determine the antibacterial activity against clinical pathogens by MIC. METHODS: Sponge samples was collected from sub-tidal habitats at Kavaratti Island and identified. The endosymbiotic bacteria were isolated and selected potential bacteria which show antibacterial activity in preliminary screening against clinical pathogens Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Salmonella typhi (S. typhi), Klebsiella pneumoniea (K. pneumoniea) and Streptococcus sp. by disc diffusion assay. The crude extracts of potential bacteria LB3 was tested against clinical pathogens by MIC. The LB3 strain was identified by 16s rDNA sequencing, 1 111 bp was submitted in NCBI (HQ589912) and constructed phylogenetic tree. RESULTS: Sponge sample was identified as Dysidea granulosa (D. granulosa) and potential bacteria LB3 identified as Enterobacter sp TTAG. Preliminary screening of sponge isolates against clinical pathogens, LB3 strain was selected as potential producer of secondary metabolites and crude extract was implies on MIC of LB3 have confirmed with lowest concentration of 5.0 mg/mL in broth medium influence of crude extract on growth inhibitory activity after 5 h of incubation period and completed the inhibitory activity at 15 h. CONCLUSIONS: The present study concluded that phylogenetic analysis of endosymbiotic bacteria Enterobacter sp from sponge D. granulosa of Lakshadweep islands showed significant antibacterial activity against clinical bacterial pathogens.

Determination of metal levels in thirteen fish species from Lakshadweep Sea.

A bio-monitoring study was performed to investigate the concentration of certain metals (cadmium, cobalt, copper, iron, magnesium, manganese, nickel, lead and zinc) in gill, skin and muscle of thirteen demersal fish species of Agatti Coast of Lakshadweep Sea. All the metal concentrations in gill are higher than skin and muscle. Concentrations of toxic metals such as Cd (0.61 ± 0.67 µg g(-1) in Gerres longirostris), Mn (0.83 ± 1.21 µg g(-1) in Lutjanus fulvus) and Ni (0.56 ± 0.83 µg g(-1) in L. bengalensis) were well above the permissible limits suggested by World Health Organization and Food and Agricultural Organizations.

Harnessing Human N-type Ca(2+) Channel Receptor by Identifying the Atomic Hotspot Regions for Its Structure-Based Blocker Design.

The voltage dependent N-type Ca(2+) channel (NCC) receptor was identified to have therapeutic potential for the treatment of neuropathic pain and stroke disease. The Ca(2+) ion transport through the transmembrane influx is mainly dependent on the closing, opening, or intermediate state gating mechanism of NCC. Harnessing this dynamic gating mechanism at the structural level is an important and challenging physiological phenomenon. The three dimensional (3D) structure of this membrane receptor is not yet experimentally determined to understand its mechanism of action. Based on these observations, we have developed for the first time the structure of the closed state of the NCC receptor at the pore forming domains which mainly involve three transmembrane helices (TMhs) S5, P and S6. Hot-spot binding site residues of this receptor model were identified by molecular docking technique using amlodipine, cilnidipine and nifedipine compounds known to be potent Ca(2+) channel antagonists. Further, the Ca(2+) ion permeability and the hydrophobic gating mechanism provided better structural and functional insights on the NCC receptor. These results are in consonance with other Ca(2+) channel receptors and would provide guidance for further biochemical investigations.

Exploring QSTR and toxicophore of hERG K+ channel blockers using GFA and HypoGen techniques.

Predictive quantitative structure-toxicity and toxicophore models were developed for a diverse series of hERG K+ channel blockers, acting as anti-arrhythmic agents using QSAR+ module in Cerius2 and HypoGen module in Catalyst software, respectively. The 2D-QSTR analysis has been performed on a dataset of 68 molecules carefully selected from literature for which IC50 values measured on hERG K+ channels expressed in mammalian cells lines using the voltage patch clamp assay technique were reported. Their biological data, expressed as IC50, spanned from 7.0nM to 1.4mM, with 7 orders difference. Several types of descriptors including electrotopological, thermodynamic, ADMET, graph theoretical (topological and information content) were used to derive a quantitative relationship between the channel blockers and its physico-chemical properties. Statistically significant QSTR model was obtained using genetic function approximation methodology, having seven descriptors, with a correlation coefficient (r2) of 0.837, cross-validated correlation coefficient (q2) of 0.776 and predictive correlation coefficient (r2 pred) of 0.701, indicating the robustness of the model. Toxicophore model generated using HypoGen module in Catalyst, on these datasets, showed three important features for hERG K+ channel blockers, (i) hydrophobic group (HP), (ii) ring aromatic group (RA) and (iii) hydrogen bond acceptor lipid group (HBAl). The most predictive hypothesis (Hypo 1), consisting of these three features had a best correlation coefficient of 0.820, a low rms deviation of 1.740, and a high cost difference of 113.50, which represents a true correlation and a good predictivity. The hypothesis, Hypo 1 was validated by a test set consisting of 12 molecules and by a cross-validation of 95% confidence level. Accordingly, our 2D-QSTR and toxicophore model has strong predictivity to identify structurally diverse hERG K+ channel blockers with desired biological activity. These models provide a useful framework for understanding binding, and gave structural insight into the specific protein-ligand interactions responsible for affinity, and how one might modify any given structure to mitigate binding.