Parimal Karmakar

In Vitro Structural and Functional Evaluation of Gold Nanoparticles Conjugated Antibiotics

Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains,Escherichia coli DH5α,Micrococcus luteus andStaphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications.

A novel study of antibacterial activity of copper iodide nanoparticle mediated by DNA and membrane damage.

In this article potential activity of nanoparticles (NPs) of copper iodide (CuI) as an antibacterial agent has been presented. The nano particles are synthesized by co-precipitation method with an average size of 8 nm as determined by Transmission Electron Microscope (TEM). The average charge of the NPs is -21.5 mV at pH 7 as obtained by zeta potential measurement and purity is determined by XRD. These NPs are able to kill both gram positive and gram negative bacteria. Among the bacteria tested, DH5α is more sensitive but Bacillus subtilis is more resistant to NPs of CuI. Consequently, the MIC and MBC values of DH5α is least (0.066 mg/ml and 0.083 mg/ml respectively) and B. subtilis is highest (0.15 mg/ml and 0.18 mg/ml respectively) among the tested bacterial strains. From our studies it is inferred that CuI NPs produce reactive oxygen species (ROS) in both gram negative and gram positive bacteria and it also causes ROS mediated DNA damage for the suppression of transcription as revealed by reporter gene assay. Probably ROS is formed on the surface of NPs of CuI in presence of amine functional groups of various biological molecules. Furthermore they induce membrane damage as determined by atomic force microscopy (AFM). Thus production of ROS and membrane damage are major mechanisms of the bactericidal activity of these NPs of CuI.

The involvement of human RECQL4 in DNA double-strand break repair

Rothmund–Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double‐strand break (DSB) repair. The results show that RECQL4‐deficient fibroblasts are moderately sensitive to γ‐irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4‐deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser‐induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N‐terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser‐induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser‐induced DSB and that it might play important roles in efficient repair of DSB’s.

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7.

BACKGROUND Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses. METHODS In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~30nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD50 doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM. RESULTS This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis. CONCLUSIONS A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells. GENERAL SIGNIFICANCE CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.

Synthesis of highly fluorescent nitrogen and phosphorus doped carbon dots for the detection of Fe3+ ions in cancer cells

Highly fluorescent nitrogen and phosphorus-doped carbon dots with a quantum yield 59% have been successfully synthesized from citric acid and di-ammonium hydrogen phosphate by single step hydrothermal method. The synthesized carbon dots have high solubility as well as stability in aqueous medium. The as-obtained carbon dots are well monodispersed with particle sizes 1.5-4 nm. Owing to a good tunable fluorescence property and biocompatibility, the carbon dots were applied for intercellular sensing of Fe(3+) ions as well as cancer cell imaging.

Characterization of VP1, VP2 and VP3 gene segments of a human rotavirus closely related to porcine strains.

Long RNA electropherotype rotavirus strains with subgroup I specificity predominated the infantile gastroenteritis outbreak in Manipur, India, in 1987–88. One such strain (RMC321) was found to possess porcine characteristics in 7 out of 8 genes sequenced. Partial characterization of its remaining VP1, VP2 and VP3 genes along with a porcine rotavirus strain (HP140) uncovered their close genetic relation to porcine strains. VP7 was the only gene segment of this strain with significant genetic identity to human strains. This indicates that a rotavirus reassortant strain with most of its genetic material derived from a porcine strain may cause symptomatic infection in a human host.

Al3+ selective coumarin based reversible chemosensor: application in living cell imaging and as integrated molecular logic gate

An efficient coumarin based fluorescent ‘turn-on’ receptor (H2L) for the detection of Al3+ has been synthesized following simple Schiff base condensation of 4-hydroxy-3-acetylcoumarin with 2-amino-4-methylphenol. The receptor H2L shows about 21 fold increase in fluorescence intensity upon addition of Al3+ than in the case of other metals. The limit of detection is 0.39 μM. H2L is efficient in detecting Al3+ in the intracellular region of human cervical cancer cells and also exhibits an INHIBIT logic gate with Al3+ and EDTA as chemical inputs by monitoring both the absorption as well as emission mode. Theoretical calculations (DFT and TDDFT) are applied to interpret the sensing mechanism of the synthesized receptor.

Recruitment and retention dynamics of RECQL5 at DNA double strand break sites

RECQL5 is one of the five human RecQ helicases, involved in the maintenance of genomic integrity. While much insight has been gained into the function of the Werner (WRN) and Bloom syndrome proteins (BLM), little is known about RECQL5. We have analyzed the recruitment and retention dynamics of RECQL5 at laser-induced DNA double strand breaks (DSBs) relative to other human RecQ helicases. RECQL5-depleted cells accumulate persistent 53BP1 foci followed by γ-irradiation, indicating a potential role of RECQL5 in the processing of DSBs. Real time imaging of live cells using confocal laser microscopy shows that RECQL5 is recruited early to laser-induced DSBs and remains for a shorter duration than BLM and WRN, but persist longer than RECQL4. These studies illustrate the differential involvement of RecQ helicases in the DSB repair process. Mapping of domains within RECQL5 that are necessary for recruitment to DSBs revealed that both the helicase and KIX domains are required for DNA damage recognition and stable association of RECQL5 to the DSB sites. Previous studies have shown that MRE11 is essential for the recruitment of RECQL5 to the DSB sites. Here we show that the recruitment of RECQL5 does not depend on the exonuclease activity of MRE11 or on active transcription by RNA polymerase II, one of the prominent interacting partners of RECQL5. Also, the recruitment of RECQL5 to laser-induced damage sites is independent of the presence of other DNA damage signaling and repair proteins BLM, WRN and ATM.

Morphology-Directing Synthesis of Rhodamine-Based Fluorophore Microstructures and Application toward Extra- and Intracellular Detection of Hg2+

A new, easily synthesizable rhodamine-based chemosensor with potential N2O2 donor atoms, L(3), has been characterized by single-crystal X-ray diffraction together with (1)H NMR and high-resolution mass spectrometry (HRMS) studies. L(3) was found to bind selectively and reversibly to the highly toxic Hg(2+) ion. The binding stoichiometry and formation constant of the sensor toward Hg(2+) were determined by various techniques, including UV-vis, fluorescence, and Jobs studies, and substantiated by HRMS methods. None of the biologically relevant and toxic heavy metal ions interfered with the detection of Hg(2+) ion. The limit of detection of Hg(2+)calculated by the 3σ method was 1.62 nM. The biocompatibility of L(3) with respect to its good solubility in mixed organic/aqueous media (MeCN/H2O) and cell permeability with no or negligible cytotoxicity provides good opportunities for in vitro/in vivo cell imaging studies. As the probe is poorly soluble in pure water, an attempt was made to frame nano/microstructures in the absence and in the presence of sodium dodecyl sulfate (SDS) as a soft template, which was found to be very useful in synthesizing morphologically interesting L(3) microcrystals. In pure water, micro-organization of L(3) indeed occurred with block-shaped morphology very similar to that in the presence of SDS as a template. However, when we added Hg(2+) to the solution of L(3) under the above two conditions, the morphologies of the microstructures were slightly different; in the first case, a flowerlike structure was observed, and in second case, a simple well-defined spherical microstructure was obtained. Optical microscopy revealed a dotlike microstructure for L(3)-SDS assemblies, which changed to a panicle microstructure in the presence of Hg(2+). UV-vis absorption and steady-state and time-resolved fluorescence studies were also carried out in the absence and presence of Hg(2+), and also the SDS concentration was varied at fixed concentrations of the receptor and guest. The results revealed that the fluorescence intensity increased steadily with [SDS] until it became saturated at ∼7 mM SDS, indicating that the extent of perturbation to the emissive species increases with the increase in [SDS] until it becomes thermodynamically stable. There was also an increase in anisotropy with increasing SDS concentration, which clearly manifests the restriction of movement of the probe in the presence of SDS.

Induction of apoptosis by Phenothiazine derivatives in V79 cells.

Phenothiazine derivatives chlorpromazine (cpz) and trifluoperazine (tfp) were found to induce apoptosis, abnormal cell cycle and expression of p53 in Chinese hamster lung fibroblast V79 cells. Both the drugs can induce apoptosis when cells are treated with drug at a concentration of 10 microg/ml within 4 h, as detected by propidium iodide staining and DNA fragmentation analysis. Flow cytometric analysis revealed that the apoptotic response is mediated by a loss of G(1) population of cells. In Western blot analysis, p21 is induced and p53 is accompanied by additional bands. Also indirect immunolabeling of single cells revealed that p21 is accumulated from cytoplasm into nucleus after the drug treatment and the intensities of p53 increased. Our findings demonstrate for the first time that phenothiazine derivatives, in addition to their cytotoxic effects, could induce apoptosis, an observation that has important clinical implications.