Aiyagari Ramesh

NIR- and FRET-based sensing of Cu2+ and S2- in physiological conditions and in live cells.

We have synthesized a new indole functionalized rhodamine derivative L(1) which specifically binds to Cu(2+) in the presence of large excess of other competing ions with visually observable changes in their electronic and fluorescence spectral behavior. These spectral changes are significant enough in the NIR and visible region of the spectrum and thus enable naked eye detection. The receptor, L(1), could be employed as a resonance energy transfer (RET) based sensor for detection of Cu(2+) based on the process involving the donor indole and the acceptor Cu(2+) bound xanthene fragment. Studies reveal that L(1)-Cu complex is selectively and fully reversible in presence of sulfide anions. Further, fluorescence microscopic studies confirmed that the reagent L(1) could also be used as an imaging probe for detection of uptake of these ions in HeLa cells.

Adenoviral vectors: a promising tool for gene therapy.

Gene therapy is a promising tool for treatment of the human diseases that cannot be cured by rational therapies, and its primary success depends on suitable vectors to deliver therapeutic genes. Adenoviruses (Ads) are among the most commonly used vectors for gene therapy, second only to retroviruses. During the last decade, remarkable progress has been made in the development of Ad vectors and in the understanding of the toxicity related to the Ad vector system. Ad vector has certain advantages such as high transduction efficiency for different quiescent and dividing cell types and high levels of short-term expression to provide therapeutic benefits. However, researchers are facing the challenges associated with tissue-specific targeting of vectors and the vector-mediated immunogenicity. This review mainly focuses on the studies that have employed methods to improve Ad vectors and reduce viral toxicity for different applications. These methods include minimization or elimination of viral genes, retargeting of vector to the tissue of interest, and generation of immunocompromised recombinant vectors that lead to safer use of Ad vector systems that improve persistence of transgene expression. Moreover, the therapeutic applications of Ad vectors for liver-targeted gene therapy, suicide gene therapy, delivery of small interfering RNA, and production of recombinant vaccine under regulated conditions used in clinical trials are discussed.

Zn2+ and Pyrophosphate Sensing: Selective Detection in Physiological Conditions and Application in DNA-Based Estimation of Bacterial Cell Numbers

A diformyl-quinoline based receptor (L1) exhibits selective colorimetric and fluorometric sensing of Zn(2+) in aqueous medium at pH 7.4 based on the intraligand charge transfer (ICT) process. The in situ formed phenoxo-bridged complex, L1·2Zn can selectively and specifically sense PPi among all the other biologically important anions including ATP through reversible binding. The detection limit for Zn(2+) and PPi were found to be approximately 56 and 2 ppb, respectively. The unique selectivity of the PPi by the L1-Zn ensemble could be used as an analytical tool to probe PPi generation in a prototype polymerase chain reaction (PCR) setup and track DNA amplification with higher sensitivity as compared to conventional agarose gel electrophoresis. Interestingly, the principle of PPi estimation in PCR rendered rapid estimation of bacterial cell numbers with a limit of detection of 10 CFU of Escherichia coli MTCC 433 in as early as 10 PCR cycles. The proposed method of PPi sensing offers interesting application potential in PCR-based rapid diagnostics for pathogenic agents and microbiological quality control.

Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: Insights from a PCR-based approach

The goal of the investigation was to study the succession of major groups of lactic acid bacteria (LAB) and their antagonism in salt-fermented cucumber using PCR. In a direct detection method as well as a short enrichment process, PCR enabled detection of Leuconostoc and Lactobacillus during early hours of fermentation. Subsequently, Lactobacillus and Pediococcus emerged as the dominant genera. Nucleic acid sequence of culture-independent clones confirmed the detection of Pediococcus as a dominant genera emerging during late stages of fermentation. PCR also revealed time-dependent emergence of mesentericin, pediocin and plantaricin A producers and accounted for the LAB succession in the fermenting samples. A total of 328 LAB isolates were obtained collectively from 30 cucumber samples, of which PCR could identify an overwhelming 186 Lactobacillus isolates followed by 113 Pediococcus and 29 Leuconostoc isolates, respectively. Based on antimicrobial assay against target strain Leuconostoc mesenteroides NRRL B640, 28% of the LAB were bacteriocin producers, of which pediocin producers were substantial, followed by plantaricin A and mesentericin producers. The bacteriocins elaborated by the isolates were active against a large number of Gram-positive target LAB strains and pathogenic bacteria including Bacillus cereus, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

Selective sensing and efficient separation of Hg2+ from aqueous medium with a pyrene based amphiphilic ligand

We have synthesized a new fluorogenic compound L, which can selectively bind and sense Hg2+ in aqueous medium over a broad pH range. It exhibits excellent selectivity for Hg2+ over a large number of competitive cations (Fe3+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Pb2+, Ca2+, Mg2+, Na+, K+). The sensing ability of the ligand is studied by fluorescence and UV-vis spectroscopy. Hg2+ present even in a nanomolar range can be detected. The effectiveness of L for detecting Hg2+ inside live human cancer cells (HeLa) is also examined. The hydrophobic part of L is efficiently employed for the quantitative extraction of Hg2+ from an aqueous medium into the organic layer. The extraction ability of L is also estimated by NMR, fluorescence and atomic absorption spectroscopy showing that approximately 99% of the Hg2+ ions are extracted. These results imply that the compound has potential application for sensing and removal of Hg2+ ions from waste water in a large cross-section of mercury threatened zones around the world.

Membrane-Directed High Bactericidal Activity of (Gold Nanoparticle)–Polythiophene Composite for Niche Applications Against Pathogenic Bacteria

The use of nanoscale materials as bactericidal agents represents a novel paradigm in the development of therapeutics against drug-resistant pathogenic bacteria. In this paper the antimicrobial activity of a water soluble (gold nanoparticle)-polythiophene (AuNP-PTh) composite against common bacterial pathogens is reported. The nanocomposite is broad-spectrum in its bactericidal activity and exhibits a membrane-directed mode of action on target pathogens. The therapeutic potency of AuNP-PTh is demonstrated by experiments which reveal that the nanocomposite can breach the outer membrane defense barrier of Gram-negative pathogens for subsequent killing by a hydrophobic antibiotic, inhibit the growth of model gastrointestinal pathogens in simulated gastric fluid, and significantly eradicate bacterial biofilms. The high bacterial selectivity and lack of cytotoxicity on human cells augers well for future therapeutic application of the nanocomposite against clinically relevant pathogenic bacteria.

A simple and efficient fluorophoric probe for dual sensing of Fe3+ and F−: application to bioimaging in native cellular iron pools and live cells

A new quinoline functionalized fluorophoric Schiff base L1 was synthesized and its colorimetric and fluorescence responses toward various metal ions in mixed aqueous media were explored. The ligand exhibited high selectivity towards Fe3+ in the presence of a large excess of other competing ions with certain observable optical and fluorescence changes. These spectral changes are significant enough in the visible region of the spectrum and thus enable naked eye detection. The efficiency of L1 in detecting Fe3+ ions was also checked in the presence of relevant complex biomacromolecules viz. methaemoglobin, fetal bovine serum and human serum albumin. L1 was also found to be sensitive enough for visual detection of Fe3+ ions in native iron pools of banana pith. Studies revealed that L1–Fe complex formation is fully reversible in the presence of the fluoride anion with very high selectivity. Furthermore, fluorescence microscopic studies demonstrated that compound L1 could also be used as an imaging probe for detection of uptake of these ions in model human cells. This selective sensing behaviour of L1 towards Fe3+ was explained via the CHEF process where theoretical calculations also supported the premise.

Synthesis, crystal structure and bio-macromolecular interaction studies of pyridine-based thiosemicarbazone and its Ni(II) and Cu(II) complexes

A new pyridine-based heterocyclic thiosemicarbazone ligand and its Ni(II) and Cu(II) complexes have been synthesized and characterized by structural, analytical and spectral methods. The mono-deprotonated anionic form of the ligand coordinates via NNS donor atoms to yield an octahedral Ni(II) complex and distorted square planar Cu(II) complex. UV-visible and fluorescence-based spectroscopic techniques revealed that both metal complexes interact with double stranded DNA via intercalation. A comparative assessment indicated that the Ni(II) complex displayed superior DNA binding. The interaction of these compounds with bovine serum albumin (BSA) suggested that the ligand and its Cu(II) complex quenched the intrinsic fluorescence of BSA in a static quenching process, whereas for the Ni(II) complex, fluorescence quenching of BSA was a combination of both static and collision/dynamic quenching processes. The quenching of the fluorescence of BSA is owing to energy transfer from the tryptophan residues of BSA to the compounds bound to BSA. Cytotoxicity tests based on the standard MTT assay revealed that the Cu(II) complex displayed prominent anti-proliferative activity against HeLa cells.

Rapid Estimation of Bacteria by a Fluorescent Gold Nanoparticle−Polythiophene Composite

Herein we present a facile method for rapid quantitation of bacterial cells over several logarithmic dilutions. The quantitation is based on loss of the fluorescence intensity of a positively charged Au nanoparticle-polythiophene composite in the presence of bacterial cells. The present method allowed estimation of both Gram-positive and Gram-negative bacteria with cells as low as 1000. Transmission electron microscopic investigations revealed attachment of the composite with bacteria with no discernible change in the morphology of the cells. Further, dynamic light scattering experiments indicated preferential attachment of smaller composite particles over larger ones, which were also attached at higher bacterial concentrations. The ease of operation with minimal sample manipulation steps, high sensitivity, quantitative detection, and its generality offer specific advantages over conventional methods.

Biocompatible nanocarrier fortified with a dipyridinium-based amphiphile for eradication of biofilm.

Annihilation of bacterial biofilms is challenging owing to their formidable resistance to therapeutic antibiotics and thus there is a constant demand for development of potent antibiofilm agents that can abolish established biofilms. In the present study, the activity of a dipyridinium-based cationic amphiphile (compound 1) against established bacterial biofilms and the subsequent development of a compound 1-loaded nanocarrier for potential antibiofilm therapy are highlighted. Solution-based assays and microscopic analysis revealed the antagonistic effect of compound 1 on biofilms formed by Staphylococcus aureus MTCC 96 and Pseudomonas aeruginosa MTCC 2488. In combination studies, compound 1 could efficiently potentiate the action of tobramycin and gentamicin on P. aeruginosa and S. aureus biofilm, respectively. A human serum albumin (HSA)-based nanocarrier loaded with compound 1 was generated, which exhibited sustained release of compound 1 at physiological pH. The compound 1-loaded HSA nanocarrier (C1-HNC) displayed the signature membrane-directed activity of the amphiphile on target bacteria, efficiently eliminated established bacterial biofilms, and was observed to be nontoxic to a model human cell line. Interestingly, compound 1 as well as the amphiphile-loaded HSA nanocarrier could eradicate established S. aureus biofilm from the surface of a Foleys urinary catheter. On the basis of its biocompatibility and high antibiofilm activity, it is conceived that the amphiphile-loaded nanocarrier may hold potential in antibiofilm therapy.