Precise determination of plutonium in nuclear fuel process samples.

A REDOX-based analytical method was developed for determining the plutonium concentration. In this method, plutonium was oxidized to the +6-oxidation state using of ceric ammonium nitrate solution. The interference from ceric(IV) nitrate was suppressed by reducing its oxidation state from +4 to +3 with sodium nitrite. Hexavalent plutonium in this sample was then reduced to be tetravalent by adding a known volume of excess standard ferrous ammonium sulphate. The dichromate equivalence required for unreacted ferrous ammonium sulphate was determined to obtain the concentration of plutonium. Interference studies from chemicals envisaged to be present in the PUREX process stream, such as dissolved tri-n-butyl phosphate, uranium, and various reagents employed during analysis, were performed for the determination of plutonium concentration. The relative standard deviation was found and it is within ± 1.0% for an aliquot containing plutonium in a range of 0.7-2.5 mg.

Single nucleotide polymorphism in hMLH1 promoter and risk of tobacco-related oral carcinoma in high-risk Asian Indians.

hMLH1 is a member of mismatch repair genes (MMR) that plays a crucial role in correcting replication errors, cell cycle arrest, apoptosis and oxidative stress. We explored the risk associated with hMLH1 -93 A>G (rs 1800734) single nucleotide polymorphism (SNP) with the oral squamous cell carcinoma (OSCC) in Asian Indians. We genotyped 242 patients with tobacco-related OSCC and 205 healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The frequency of AA genotype was found to be significantly (Pc<0.0006) lower in patients as compared to the controls (21.49% vs. 47.8%) while GG genotype showed significantly higher (Pc<0.0006) prevalence in patients as compared to the healthy controls (41.32% vs. 13.66%). In logistic regression analysis AG (adjusted OR=1.95, 95% CI=0.72-5.26) and GG genotype (adjusted OR=4.5, 95% CI=1.54-13.16, P=0.006) appeared susceptible when compared with the wild-type AA genotype. The allelic distribution showed that variant G allele is significantly higher (Pc<0.0004) in patients and associated with increased risk (adjusted OR=2.36, 95% CI=1.33-4.19, P=0.003) as compared to the wild-type A allele. Altogether, our results suggest that the hMLH1 -93 A>G polymorphism is associated with the higher risk of tobacco-related OSCC in Asian Indians and could be useful in screening population at a higher risk.

Genetic polymorphisms in FAS (CD95) and FAS ligand (CD178) promoters and risk of tobacco-related oral carcinoma: gene-gene interactions in high-risk Indians.

We assessed the association of functional single nucleotide polymorphisms (SNP) in FAS -1377, -670 and FAS ligand (FASL) -844 promoters in 139 oral cancer patients and 126 normal subjects by PCR-RFLP. In logistic regression analysis FAS -1377 GA genotype appeared to marginally increase the risk while FASL -844 TC genotype appeared as low risk factor. The combined genotypes FAS -1377 GA or AA and FASL -844 TT (p <0.03), and FAS -670 AG or GG and FASL -844 TT (p <0.007) appeared to double the risk. FAS and FASL gene-gene and gene-environment interactions seems to modulate susceptibility/resistance to tobacco-related oral cancer in Indians.

Functional genetic variants of CTLA-4 and risk of tobacco-related oral carcinoma in high-risk North Indian population.

Single nucleotide polymorphisms (SNPs) of the CTLA-4 gene have been implicated in susceptibility to different cancer in different ethnic populations. We assessed the association of five SNPs [-1722C/T, -1661A/G and -318C/T in the promoter region49A/G in exon 1 and CT60A/G in the 3'untranslated region (UTR)] with tobacco-related oral squamous cell carcinoma (OSCC) in North Indian subjects. We genotyped 130 OSCC patients and 180 normal subjects by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) using BbvI, MseI, NcoI and BstEII restriction endonucleases. Among these SNPs, -1722CC, -1661AG and CT60AA genotypes were more prevalent in OSCC patients as compared to controls and in the logistic regression analysis with odd ratio (OR) 2.85, 95% CI (0.69-11.68); OR 2.48, 95% CI (1.29-4.78) and OR 3.0, 95% CI (1.43-6.28) respectively, these genotypes showed strong association with OSCC risk. With higher prevalence in controls 49GG genotype and G allele (OR 0.57, 95% CI 0.40-0.81) appeared to be protective. Moreover, TACAG, TACGA and TATAG appeared as susceptible while TACGG and CACGG appeared as protective haplotypes. These results suggest significant risk modifying effects of CTLA-4 -1722C/T, -1661A/G, -318T/C, CT60 A/G and 49A/G SNPs in tobacco-related OSCC in North Indian population.

Skewed immunological balance between Th17 (CD4(+)IL17A (+)) and Treg (CD4 (+)CD25 (+)FOXP3 (+)) cells in human oral squamous cell carcinoma.

BACKGROUND: Several studies have documented modulation of Th17 and T regulatory (Treg) cells in various human malignancies which may vary with the type and extent of the disease. However, such data in patients with oral cancer is scarce and hence the current study was designed to elaborate the immunological balance between these two T cell subsets in oral cancer. METHODS AND RESULTS: We analyzed various T cell subsets in the peripheral blood of 45 oral squamous cell carcinoma (OSCC) patients and 40 healthy volunteers. We found that, compared with the healthy controls, patients had a significantly (p < 0.0001) higher proportion of both Th17 (CD4(+)IL17A(+)) and Treg (CD4(+)CD25(+)FOXP3(+)) cells, which further showed a reciprocal balance in relation to clinico-pathological parameters in patients. We also detected a circulating CD8(+) subset of these cells in both patients and healthy controls, although the difference between the two groups was statistically insignificant. Higher frequencies of Th17 cells were found in patients with early stages and without lymph node involvement, while an increased prevalence of Tregs was associated with higher clinical stages and lymph node involvement. Moreover, Th17 cells were quantitatively and positively correlated to CD4(+)T and CD8(+)T cells and inversely correlated with Tregs. Contrarily, Tregs showed a negative association with CD4(+)T and CD8(+)T cells. CONCLUSIONS: Our results suggest an increase in Th17/Tregs ratio in early stages and a decrease in this ratio in higher stages of oral cancer. Such counter regulation of Th17 and Tregs may be a significant prognostic factor in oral cancer patients.

Deregulation of Beclin 1 in patients with tobacco-related oral squamous cell carcinoma.

Autophagy is a physiologically regulated and evolutionary conserved process that plays a critical role in degradation of cytoplasmic proteins and other macromolecules within the lysosomes. Beclin-1, the mammalian orthologue of yeast Atg6, is an important mediator of autophagy that has been studied in many human cancers. However, the expression of Beclin-1 has not yet been investigated in oral cancer. We for the first time investigated the expression of Beclin-1 in serum and tissues and correlated it with the clinic-pathological features of oral cancer patients. m-RNA expression of Beclin-1 was evaluated in tumor and normal areas of surgical specimens from 10 oral cancer patients by real-time PCR. Approximately, 8-fold lower expression (p<0.001) of Beclin-1 mRNA was observed in tumor tissue as compared to the normal tissue. Serum levels of Beclin-1 were evaluated by SPR and ELISA. No significant difference was observed in serum Beclin-1 levels in patients as compared to healthy subjects, similarly no correlation was found between serum levels and clinic-pathological parameters such as stage, lymph node involvement and tumor size. Our results demonstrate that down-regulation of Beclin-1 may play an important role in the development and progression of oral cancer possibly by dysregulation of autophagy in tumor cells.

Functional genetic variants of TGF-ß1 and risk of tobacco-related oral carcinoma in high-risk Asian Indians.

Transforming growth factor (TGF)-ß1, the most abundant isoform of TGF-ß have been implicated in various stages of carcinogenesis such as epithelial to mesenchymal transition, enhanced expression of metalloproteases, down-regulation of cellular adhesion molecule, increased tumor motility and angiogenesis as well as local and systemic immunosuppression leading to a more aggressive and metastatic behavior. We assessed the association of TGF-ß1 functional genetic polymorphisms at codon 10 (869 T>C) and 25 (915 G>C) of exon 1 in 140 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 120 normal subjects by PCR-RFLP. The frequency of 869 CC genotype and C allele were significantly higher in patients as compared to controls (P(c), 0.024 and 0.0004, respectively) while no significant difference was observed in the frequency of 915 CC genotype and C allele. In logistic regression analysis CC genotype (OR, 3.87; 95% CI, 1.78-8.41) and C allele (OR, 2.20; 95% CI 1.51-3.20) appeared as susceptible while TT genotype and T allele as protective. In addition C(869)-C(915) haplotype with OR of 2.48 at 95% CI, 1.51-4.06 significantly (P=0.0003) increased the risk of tobacco-related OSCC in Asian Indians.

Functional variants of COX-2 and risk of tobacco-related oral squamous cell carcinoma in high-risk Asian Indians.

We assessed the association of COX-2 polymorphisms at promoter sites -1195, -765 and at 3'UTR (8473) in 193 patients with oral squamous cell carcinoma (OSCC) and 137 normal subjects by PCR-RFLP. Although no significant difference was observed in the frequency of COX-2 -1195G>A, -765G>C and 8473C>T single nucleotide polymorphisms (SNPs) between patients and controls, COX-2 -1195G/A genotype showed higher while -765G/C and 8473C/T showed lower prevalence in patients as compared to normal subjects. Logistic regression analysis of these three polymorphisms revealed a quantitative risk associated with -1195G>A SNP (OR 3.07 at 95%CI) while -765G>C and 8473C>T appeared to be protective. Results of the present study indicate that these three functional variants in the COX-2 regulatory region may contribute to risk modification of tobacco-related oral squamous cell carcinoma in Asian Indians.

Enumeration of lymphocyte subsets using flow cytometry: Effect of storage before and after staining in a developing country setting.

Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38-45°C). In the second experiment, blood samples were stained within 1-2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38-45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.