Rita Mukhopadhyaya

BRAF V600E mutation in papillary thyroid carcinoma: significant association with node metastases and extra thyroidal invasion.

B-Raf (BRAF) is the strongest activator in the downstream of MAP kinase signaling. The somatic point mutation of BRAF gene (V600E) is the most common and specific event in papillary thyroid carcinoma (PTC). However, its prevalence is variable among different studies and its association with clinico-pathological features is controversial. This study tests the prevalence of BRAFV600E mutation in thyroid cancer patients in Indian subcontinental population. We analyzed 140 thyroid tumor specimens for BRAF gene mutation at codon 600 using mutant-allele-specific amplification, single-strand conformation polymorphism, Mutector assay, and DNA sequencing of the PCR-amplified exon 15. BRAF mutation at codon 600 was detected in 46 of 86 PTC patients (53.4%) from Indian subcontinental cohort. Frequency of mutation varied across the subtypes of PTCs. BRAFV600E mutation was more common in the conventional PTC (38 out of 62; 61%) than in the follicular variant of PTC (2 out of 17; 11.7%). None of the 8 follicular thyroid adenomas, 14 follicular thyroid carcinomas, 16 medullary thyroid carcinomas, and 16 benign hyperplasia patients showed any exon 15 mutation. We found significant correlation between BRAF mutation status and extra-thyroidal invasion, lymph node metastasis, and tumor stage. However no correlation was observed with gender, age, and tumor size of the patients. Thus our findings suggest that BRAFV600E is a prevalent genetic alteration in adult sporadic PTCs in Indian cohort and it may be responsible for the progression of classic variant of PTC to metastatic and poorly differentiated subtype and likely to have significant impact on its diagnostic and prognostic management.

Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation

A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insects capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD(20) dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chironomus in radioactively contaminated regions.

Radiation Desiccation Response Motif-Like Sequences Are Involved in Transcriptional Activation of the Deinococcal ssb Gene by Ionizing Radiation but Not by Desiccation

Single-stranded-DNA binding protein (SSB) levels during poststress recovery of Deinococcus radiodurans were significantly enhanced by (60)Co gamma rays or mitomycin C treatment but not by exposure to UV rays, hydrogen peroxide (H₂O₂), or desiccation. Addition of rifampin prior to postirradiation recovery blocked such induction. In silico analysis of the ssb promoter region revealed a 17-bp palindromic radiation/desiccation response motif (RDRM1) at bp -114 to -98 and a somewhat similar sequence (RDRM2) at bp -213 to -197, upstream of the ssb open reading frame. Involvement of these cis elements in radiation-responsive ssb gene expression was assessed by constructing transcriptional fusions of edited versions of the ssb promoter region with a nonspecific acid phosphatase encoding reporter gene, phoN. Recombinant D. radiodurans strains carrying such constructs clearly revealed (i) transcriptional induction of the ssb promoter upon irradiation and mitomycin C treatment but not upon UV or H₂O₂ treatment and (ii) involvement of both RDRM-like sequences in such activation of SSB expression, in an additive manner.

Hsp70 expression in Chironomus ramosus exposed to gamma radiation

Purpose: A tropical species of midge, Chironomus ramosus has been recently reported to be one of the radio-tolerant groups of organisms. The present study was undertaken to examine the protein profile and expression of Heat shock protein-70 (Hsp70) in gamma radiation stress, which has also been reported as a common biomarker for different type of stressors. Materials and methods: Metabolic labelling of salivary gland (SG) proteins with [35S]-methionine showed over-expression of a 70 kDa protein band up to 4 hours (h) of observation in the post exposure recovery period. For confirmation of the expression of Hsp70 in SG cells after gamma radiation exposure, semi-quantitative real-time-polymerase chain reaction (RT-PCR), Western blotting and immuno-fluorescence detection of Hsp70 were carried out. Results: Results showed elevated levels of Hsp70 mRNA and protein in SG cells of larvae immediately after gamma radiation exposure. The levels dropped to basal values by 48 h in the recovery period. Conclusions: The present study confirmed that radio-tolerant midge, C. ramosus expressed Hsp70 upon gamma radiation exposure and Hsp70 might be one of the gamma radiation-induced stress proteins required during the early stages of radiation stress management in aquatic midge larvae. This is the first report of its kind from the juvenile stage of any aquatic insect group.

Gamma radiation tolerance of a tropical species of midge, Chironomus ramosus Chaudhuri (Diptera: Chironomidae).

Purpose: The Chironomid midges are known to thrive well under adverse environmental conditions and are even found inhabiting in areas contaminated by radioactive wastes. Studies were therefore undertaken to find out the radiosensitivity of different developmental stages of the Indian tropical midge, Chironomus ramosus. Materials and methods: In order to determine the threshold levels of lethality, eggs, larvae, pupae and adults of C. ramosus were exposed to varying dosages of gamma radiation (60Co radiation source) ranging from 0–3500 Gray (Gy) at dose-rate of 5.5 Gy/minute. The post-irradiation studies were conducted at three different time points: (a) Immediately after the end of irradiation, (b) 24 hours (h), and (c) 48 h after the end of radiation treatments. Determination of the lethal dose required to kill 50% (LD50), 90% (LD90) and 100% population was carried out using the log-probit analysis. Results: Different developmental stages showed variable threshold levels of radiosensitivity. The radiation doses required to cause 100% mortality immediately after radiation exposure of egg, larva, pupa and adult stages were 1000 Gy, 3000 Gy, 3200 Gy and 3500 Gy, respectively, indicating eggs as the most sensitive stage. Detailed analysis of the LD50 values of different post-irradiation time points indicated that pupal stages were also sensitive at 48 h post-irradiation amongst all the post-embryonic stages as described in many other insects. Interestingly detailed analysis of data indicated that amongst the adult population, females were the most radioresistant, compared to the males as reported in many other insect groups in the literature. Conclusions: The Indian tropical midge C. ramosus was found to tolerate higher dose of gamma radiation as compared to other known dipteran insects. It is evident from the present findings that C. ramosus falls in the category of radiation-tolerant group of insects.

Unfolding and inactivation of proteins by counterions in protein-nanoparticles interaction.

In this work, the structure and activity of proteins; such as, hen egg lysozyme (HEWL) and calf intestine alkaline phosphatase (CIAP); have been investigated after incubation with surface coated iron oxide nanoparticles (IONPs) in water. IONPs were coated with counterions bound charge-ligands and were named as the charge-ligand counterions iron oxide nanoparticles (CLC-IONPs). The coating was done with tri-lithium citrate (TLC) and tri-potassium citrate (TKC) to have negative surface charge of CLC-IONPs and Li(+) and K(+), respectively, as counterions. To have positive surface charge, IONPs were coated with cetylpyridinium chloride (CPC) and cetylpyridinium iodide (CPI) having Cl(-) and I(-), respectively, as counterions. The secondary structure of proteins was measured using far ultraviolet circular dichroism (CD) spectroscopy which showed that both proteins were irreversibly unfolded after incubation with CLC-IONPs. The unfolded proteins were seen to be functionally inactive, as confirmed through their activity assays, i.e., HEWL with Escherichia coli (E. coli) and CIAP with para-nitrophenyl phosphate (pNPP). Additionally, we have observed that monomeric hemoglobin (Hb) from radio-resistant insect Chironomus ramosus (ChHb) was also partially unfolded upon interaction with CLC-IONPs. This work clearly shows the role of counterions in protein inactivation via protein-nanoparticles interaction and, therefore, CLC-IONPs could be used for therapeutic purpose.

Harnessing a radiation inducible promoter of Deinococcus radiodurans for enhanced precipitation of uranium

Bioremediation is an attractive option for the treatment of radioactive waste. We provide a proof of principle for augmentation of uranium bioprecipitation using the radiation inducible promoter, Pssb from Deinococcus radiodurans. Recombinant cells of D. radiodurans carrying acid phosphatase gene, phoN under the regulation of Pssb when exposed to 7 kGy gamma radiation at two different dose rates of 56.8 Gy/min and 4 Gy/min, showed 8-9 fold increase in acid phosphatase activity. Highest whole cell PhoN activity was obtained after 2h in post irradiation recovery following 8 kGy of high dose rate radiation. Such cells showed faster removal of high concentrations of uranium than recombinant cells expressing PhoN under a radiation non-inducible deinococcal promoter, PgroESL and could precipitate uranium even after continuous exposure to 0.6 Gy/min gamma radiation for 10 days. Radiation induced recombinant D. radiodurans cells when lyophilized retained high levels of PhoN activity and precipitated uranium efficiently. These results highlight the importance of using a suitable promoter for removal of radionuclides from solution.

Chironomus ramosus larvae exhibit DNA damage control in response to gamma radiation.

Purpose: Chironomus ramosus is one of the recently reported radiotolerant insects. Salivary gland cells of fourth instar larvae respond to ionizing radiations with increases in the levels of antioxidant enzymes and chaperone proteins. Here we made an attempt to study the state of nuclear DNA after exposure of larvae to a lethal dose for 20% of the population (LD20) of gamma radiation (2200 Gy, at a dose rate 5.5 Gy/min). Materials and methods: Genomic DNA preparations were subjected to competitive ELISA (Enzyme linked immunosorbent assay) for detection of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and dynamic light scattering (DLS) to monitor any radiation-induced damage. Single salivary gland cells were subjected to alkaline single cell gel electrophoresis (ASCGE), comet assay and pulsed field gel electrophoresis (PFGE) to check for DNA double-strand breaks. Results: Results from all four experimental procedures confirmed damage of nucleobases and fragmentation of nuclear DNA immediately after radiation. Some 48 h after radiation exposure, modified 8-oxodG residues returned to basal level, homodispersity of genomic DNA reappeared, the length of comet tail regressed significantly (ASCGE) and PFGE pattern matched with that of high molecular weight unirradiated DNA. Conclusion: Chironomus ramosus larvae showed control of DNA damage as observed over 48 h in post irradiation recovery which could be attributed to their ability to tolerate gamma radiation stress.

Differential sensitivity of Chironomus and human hemoglobin to gamma radiation.

Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study.