Nandan Bhattacharyya

ESIPT based Hg2+ and fluoride chemosensor for sensitive and selective ‘turn on’ red signal and cell imaging

An excited state intramolecular proton transfer (ESIPT) enabled fluorescent sensor phenanthroline diimino phenol (PDP) for Hg2+ has been designed and synthesized. PDP acts as a dual sensor and selectively detects only Hg2+ in mixed aqueous medium and fluoride in acetonitrile medium over other competing metal ions and anions. The binding of PDP with Hg2+ is supported by DFT. The ESIPT phenomenon in PDP is favored in the presence of Hg2+, which is rarely reported, along with an intense red fluorescence suppressing other competing metal ions. Among different anionic analytes, only fluoride shows a visually detectable exciting color change from pale yellow to pink with almost similar emission characteristics. PDP also demonstrates its importance in the fluorescent imaging of Hg2+ ions in human cancer cells.

A Novel Nuclear Protein, MGC5306 Interacts with DNA Polymerase β and Has a Potential Role in Cellular Phenotype

A novel protein MGC5306 has been identified in yeast–two-hybrid analysis by screening a HeLa cDNA library with a truncated DNA polymeraseβ (polβΔ) as bait. The polβΔ is expressed in various types of cancers. Co-immunoprecipitation–Western blot analysis confirms not only its interaction with polβΔ but also with wild-type polβ. Binding to polβ indicates potential function of MGC5306 in repair pathway. Transfection of cells with MGC5306-GFP and Western blot analysis with anti-MGC5306 antibody reveal its nuclear localization. MGC5306 is expressed in human carcinomas and tumor cell lines but not in normal tissues, suggesting MGC5306 is most likely involved in carcinogenesis. An antigrowth activity and modulations of cell cycle events are identified in cells expressing siRNAMGC5306.

Association of Two Polymorphisms of DNA Polymerase Beta in Exon-9 and Exon-11 with Ovarian Carcinoma in India

BACKGROUND DNA polymerase beta (polβ ) is a key enzyme in the base excision repair pathway. It is 39 kDa protein, with two subunits, one large subunit of 31 kDa having catalytic activity between exon V to exon XIV, and an 8 kDa smaller subunit having single strand DNA binding activity. Exons V to VII have double strand DNA binding activity, whereas exons VIII to XI account for the nucleotidyl transferase activity and exons XII to XIV the dNTP selection activity. AIM To examine the association between polβ polymorphisms and the risk of ovarian cancer, the present case control study was performed using 152 cancer samples and non-metastatic normal samples from the same patients. In this study, mutational analysis of polβ genomic DNA was undertaken using primers from exons IX to XIV - the portion having catalytic activity. RESULTS We detected alteration in DNA polymerase beta by SSCP. Two specific heterozygous point mutations of polβ were identified in Exon 9:486, A->C (polymorphism 1; 11.18%) and in Exon 11:676, A->C (polymorphism 2; 9.86%). The correlation study involving polymorphism 1 and 4 types of tissue showed a significant correlation between mucinous type with a Pearson correlation value of 4.03 (p=0.04). The association among polymorphism 2 with serous type and stage IV together have shown Pearson χ2 value of 3.28 with likelihood ratio of 4.4 (p=0.07) with OR =2.08 (0.3- 14.55). This indicates that there is a tendency of correlation among polymorphism 2, serous type and stage IV, indicating a risk factor for ovarian cancer. CONCLUSION Hence, the results indicate that there is a tendency for polβ polymorphisms being a risk factor for ovarian carcinogenesis in India.

Biological application of green silver nanoparticle synthesized from leaf extract of Rauvolfia serpentina Benth

Abstract Objective To synthesize silver nanoparticles (AgNPs) from the leaf extract of Rauvolfia serpentina Benth and examination of their various biological activities. Methods An ecofriendly, easy, one step, non-toxic and inexpensive approach is used, where aqueous plant extract acts as a reducing as well as stabilizing agent of AgNPs. The nanoparticles were characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy analysis. Results Surface plasmon resonance of the nanoparticles was observed at 427 nm in UV-vis spectroscopy. Fourier transform infrared spectroscopy result confirms that the plant extract acts as the reducing as well as the capping agent of the AgNPs. Transmission electron microscopy indicated that the synthesized nanoparticles are spherical in shape and approximately 7–10 nm in size, whereas the crystalline nature with face-centered cubic structure of the AgNPs was detected by X-ray diffraction analysis. Presence of silver in the AgNPs is 31.43% by weight, as confirmed by energy-dispersive X-ray spectroscopy. The synthesized AgNPs have antimicrobial activities against human pathogenic microorganisms. It also shows larvicidal activity and cytotoxicity against HeLa, MCF-7 cell lines. Conclusions Synthesized spherical shaped AgNPs from the leaf extract of Rauvolfia serpentina Benth have antimicrobial and larvicidal activities as well as cytotoxicity against HeLa and MCF-7 cell lines.

Exon 8-9 Mutations of DNA Polymerase β in Ovarian Carcinoma Patients from Haldia, India

BACKGROUND Ovarian cancer is the number one killer among all the gynecological cancers. We undertook association study to identify potential alterations in the genomic DNA of a DNA repair gene, DNA polymerase beta (polβ), involved in base excision repair (BER), in ovarian carcinomas of patients from Haldia, India. Mutations, splice variants have been reported earlier in different tumors other than ovarian tumors. AIM In this study we explored the possibility of association of any mutation of pol beta (Exon 8) with prognosis in 152 ovarian cancer samples. RESULTS Alteration in the exon 8 region (Exon 8:468, AgC; 15.1%) was noted among fifty seven polymorphism positive samples. Alteration in the intervening sequence 8 (IVS8, -25, AgC; 3.9%) was also noted. All alterations are heterozygous in nature. CONCLUSIONS We found no significant association among the samples from serous type, stage IV, and the polβ mutations (P ≤ 0.01). Only a slight tendency of association was evident between IVS8, -25, A to C; and stage III. Further analysis with a larger number of samples is needed.

Association of a newly identified variant of DNA polymerase beta (polβΔ63-123, 208-304) with the risk factor of ovarian carcinoma in India.

BACKGROUND DNA polymerase is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. The encoded enzyme is a key to the base excision repair (BER) pathway. It is evident that pol beta has mutations in various cancer samples, but little is known about ovarian cancer. AIM Identification of any variant form of polβ cDNA in ovarian carcinoma and determination of association between the polymorphism and ovarian cancer risk in Indian patients. We used 152 samples to isolate and perform RT-PCR and sequencing. RESULTS A variant of polymerase beta (deletion of exon 4-6 and 11-13, comprising of amino acid 63-123, and 208-304) is detected in heterozygous condition. The product size of this variant is 532 bp while wild type pol beta is 1 kb. Our study of association between the variant and the endometrioid type shows that it is a statistically significant factor for ovarian cancer [OR=31.9 (4.12-246.25) with p<0.001]. The association between variant and stage IV patients further indicated risk (χ2 value of 29.7, and OR value 6.77 with 95% CI values 3.3-13.86). The correlation study also confirms the association data (Pearson correlation values for variant/stage IV and variant/endometrioid of 0.44 and 0.39). CONCLUSION Individuals from this part of India with this type of variant may be at risk of stage IV, endometrioid type ovarian carcinoma.

HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polβ Δ208-304) specific for ovarian cancer. Pol β Δ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. Polβ Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol β Δ208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

Identification of an endoplasmic reticulum membrane protein interacting with DNA polymerase beta by a yeast two-hybrid screen.

Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polβ). It has been shown that more than 11% of breast, bladder, esophageal, colon, and gastric cancer samples studied so far exhibit polβ mutation. A truncated form of polβ, polβΔ (exon 11 deletion), identified in a colon tumour sample, exhibited dominant negative activity. Using this poβΔ as bait, we screened a HeLa cDNA library for any interacting protein(s) in the yeast two-hybrid (Y2H) system. PolβΔ was cloned into a pGBKT7 vector (pGBKT7-polβΔ). pGBKT7-polβΔ was transformed into the yeast strain AH109. Then the cDNA library was co-transformed into AH109=pGBKT7-polβΔ and screened by the selection procedure. The yeast-purified plasmids were transformed into Escherichia coli. Plasmid DNA was isolated from the colonies, purified, digested with Sma I and Sal I, and the fragments were sequenced. Four positive clones were obtained. Out of these, three proteins were already known to interact with polβ (XRCC1, MGC5306, and AP endonuclease 1). The only member previously not known to interact with polβ was phosphatidylinositol glycosylase type S (PIGS). PIGS is a 64-kDa membrane protein, encoded in chromosome 17. The PIGS protein interacts also with wild-type polβwhich was confirmed by co-immunoprecipitation and Western blot analysis. The role of the newly identified protein in the dominant negative function of the variant form of polβ remains to be seen.

Association between newly identified variant form of DNA polymerase betaΔ208–304 and ovarian cancer

BACKGROUND Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polβ), which removes the deoxyribose phosphate group (dRP) and fills in the gap with a nucleotide after the DNA lesion is excised. It has been shown that more than thirty percent of breast, bladder, esophageal, colon, and gastric cancer samples studied so far have exhibited DNA polymerase beta mutation. AIM To examine the association between polβ polymorphism and ovarian cancer, case control study was performed using one hundred fifty two cancer samples and non-metastatic normal samples from the same patients in Indian population. DESIGN The polβ polymorphism was studied in ovarian carcinoma tissues samples initially by RT-PCR followed by sequencing and then by western blot analysis. RESULT A new type of variant was detected along with the WT allele (polβΔ _{208-304}). Stage IV samples have shown a significant factor for cancer progression in ovarian cancer patients of India [OR=3.58; 95% CI (1.6-7.9); and p=0.001]. The association study involving serous type and the variant showed a tendency towards ovarian carcinogenesis [OR=1.57; 95% CI (0.8-3.1); p=0.19]. The western blot analysis result indicates that the specific deletion appears to be associated with disease progression. CONCLUSION The result reveals that this variant form of polβ is a predisposing factor for stage IV ovarian cancer samples in Indian population.

Bio-efficacy of nanoparticles in tea garden prepared from Heliotropium indicum

Silver nanoparticles prepared from aqueous extract of Heliotropium indicum leaves showed the potentiality to control the pests in tea garden. The nanoparticles controlled all of the insects including looper and semi-looper, red slug caterpillars and aphids. At 1 DAS (day after spraying), the winged and wingless aphids and termites died at a concentration of 20 mg/l of water. At 2 DAS, 80% semi-looper died and at 3 DAS 50% red slug beetles died. The nanoparticles were less toxic to human cells. This approach was eco-friendly and cost effective as compared to the chemical pesticides use in the tea gardens.