Mukund V. Deshpande

Perspectives for nano-biotechnology enabled protection and nutrition of plants.

Indiscriminate use of pesticides and fertilizers causes environmental pollution, emergence of agricultural pests and pathogens, and loss of biodiversity. Nanotechnology, by virtue of nanomaterial related properties, has potential agro-biotechnological applications for alleviation of these problems. The literature pertaining to the role of nanotechnology in plant and soil systems demonstrates that nanomaterials may assist in a) the controlled release of agrochemicals for nutrition and protection against pests and pathogens, b) delivery of genetic material, c) sensitive detection of plant disease and pollutants and d) protection and formation of soil structure. For instance, porous silica (15nm) and biodegradable, polymeric chitosan (78nm) nanoparticles displayed slow release of encapsulated pesticide and fertilizer, respectively. Further, nanosized gold (5-25nm) delivered DNA to plant cells while iron oxide (30nm) based nanosensors detected pesticides at minute levels. These functions assist the development of precision farming by minimizing pollution and maximizing the value of farming practice.

Synthesis and antimicrobial activity of β-lactam-bile acid conjugates linked via triazole

Synthesis of novel 1,2,3-triazole-linked beta-lactam-bile acid conjugates 17-24 using 1,3-dipolar cycloaddition reaction of azido beta-lactam and terminal alkyne of bile acids in the presence of Cu(I) catalyst (click chemistry) have been realized. These molecules were evaluated in vitro for their antifungal and antibacterial activities. Most of the compounds exhibited significant antifungal and moderate antibacterial activity against all the tested strains.

Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC(80) values ranging from 15.62 μM to 250 μM. Synergy experiments showed that MIC(80) of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.

Synthesis and biological evaluation of bile acid dimers linked with 1,2,3-triazole and bis-β-lactam

We report herein the synthesis and biological evaluation of bile acid dimers linked through 1,2,3-triazole and bis-beta-lactam. The dimers were synthesized using 1,3-dipolar cycloaddition reaction of diazido bis-beta-lactams , and terminal alkynes derived from cholic acid/deoxycholic acid in the presence of Cu(i) catalyst (click chemistry). These novel molecules were evaluated in vitro for their antifungal and antibacterial activity. Most of the compounds exhibited significant antifungal as well as antibacterial activity against all the tested fungal and bacterial strains. Moreover, their in vitro cytotoxicities towards HEK-293 and MCF-7 cells were also established.

Natural yeast flora of different varieties of grapes used for wine making in India

The natural Saccharomyces and non-Saccharomyces yeast flora present on the grape berries significantly affect wine production. Six grape varieties, Bangalore blue, Zinfandel, Cabernet, Chenin Blanc, Sauvignon Blanc and Shiraz are being used in India for wine making. The yeast diversity was studied on the basis of morphological, colony, physiological characteristics and 5.8S-ITS sequencing of rDNA of the isolates. Eleven different species belonging to seven genera were identified as: Candida azyma, Candida quercitrusa, Debaryomyces hansenii, Hanseniaspora guilliermondii, Hanseniaspora viniae, Hanseniaspora uvarum, Issatchenkia orientalis, Issatchenkia terricola, Pichia membranifaciens, Saccharomyces cerevisiae and Zygoascus steatolyticus. H. guilliermondii was the predominant species while S. cerevisiae was observed occasionally in the six vine varieties. For the first time, C. azyma was isolated from Bangalore blue and Cabernet varieties grown in different localities. This association may be attributed to the change in cropping pattern from sugarcane to viticulture in the vine growing regions and the known association of C. azyma with sugarcane phylloplane. Further analysis of the indigenous strains and the qualitative and quantitative changes in the flora during fermentation will be useful to understand wine quality and to design preservation strategies to control wine spoilage.

Chitin Synthase Inhibitors as Antifungal Agents

Increased risk of fungal diseases in immunocompromised patients, emerging fungal pathogens, limited repertoire of antifungal drugs and resistance development against the drugs demands for development of new and effective antifungal agents. With greater knowledge of fungal metabolism efforts are being made to inhibit specific enzymes involved in different biochemical pathways for the development of antifungal drugs. Chitin synthase is one such promising target as it is absent in plants and mammals. Nikkomycin Z, a chitin synthase inhibitor is under clinical development. Chitin synthesis in fungi, chitin synthase as a target for antifungal agent development, different chitin synthase inhibitors isolated from natural sources, randomly synthesized and modified from nikkomycin and polyoxin are discussed in this review.

Mechanism of action of novel synthetic dodecapeptides against Candida albicans

BACKGROUND Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,β-didehydrophenylalanine (∆Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated. METHODS The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy. RESULTS AND CONCLUSIONS The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250μM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans. GENERAL SIGNIFICANCE The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.

Stereoselective synthesis and antimicrobial activity of steroidal C-20 tertiary alcohols with thiazole/pyridine side chain

Stereoselective synthesis of novel steroidal C-20 tertiary alcohols with thiazole and pyridine side chain using Grignard reaction of steroidal ketones and thiazole/pyridine magnesium bromide have been realized. These molecules were evaluated in vitro for their antifungal and antibacterial activities. Most of the compounds exhibited significant antifungal and antibacterial activity against all the tested strains.

Synthesis of chimeric tetrapeptide-linked cholic acid derivatives: impending synergistic agents

Tetrapeptides derived from glycine and beta-alanine were hooked at the C-3beta position of the modified cholic acid to realize novel linear tetrapeptide-linked cholic acid derivatives. All the synthesized compounds were tested against a wide variety of microorganisms (gram-negative bacteria, gram-positive bacteria and fungi) and their cytotoxicity was evaluated against human embryonic kidney (HEK293) and human mammary adenocarcinoma (MCF-7) cell lines. While relatively inactive by themselves, these compounds interact synergistically with antibiotics such as fluconazole and erythromycin to inhibit growth of fungi and bacteria, respectively, at 1-24 microg/mL. The synergistic effect shown by our novel compounds is due to their inherent amphiphilicity. The fractional inhibitory concentrations reported are comparable to those reported for Polymyxin B derivatives.

Possible mechanism of antifungal phenazine-1-carboxamide from Pseudomonas sp. against dimorphic fungi Benjaminiella poitrasii and human pathogen Candida albicans

Investigation of antifungal mechanism of phenazine 1‐carboxamide (PC) produced by a Pseudomonas strain MCC2142.