Forensic evaluation of mitochondrial DNA heteroplasmy in Gujarat population, India.

BACKGROUND: Owing to its high copy number and its small size, mtDNA analysis is the most reliable choice when biological materials from crime scenes are degraded or have mixed STR profiles. AIM: To examine the occurrence of heteroplasmy along with its frequency and pattern in both HV1 and HV2 regions of the mtDNA among unrelated individuals from India. SUBJECTS AND METHODS: Mitochondrial DNA control region [hypervariable region one (HV1) and hypervariable region two (HV2)] were analysed in blood and buccal tissues of 104 unrelated individuals from the Indian state of Gujarat. RESULTS: A high frequency of point heteroplasmy (PH) and length heteroplasmy (LH) was revealed. PH was detected in 7.69% of the population, with a higher frequency observed in blood than in buccal samples. However, there were no statistically significant differences in PH between the two tissues (Chi-square = 0.552, p ≥ 0.05). A total of six PH positions were detected: three at HV1, and another three at HV2. The studied population showed 46.15% LH in the HV1 and HV2 regions of both tissues. The LH positions observed in the Gujarat population were the same as those previously reported at HV1 np16184-16193 and HV2 np303-315. CONCLUSIONS: Our findings suggest that differences in the pattern of heteroplasmy found in different tissues can complicate the forensic analysis, on the other hand, the probability of a match between the questioned and reference samples increases when the heteroplasmy is identical in both tissues. Variability of PH among persons and even within tissues recommends analysing multiple tissue samples before drawing a conclusion in forensic mtDNA analyses.

Comparative evaluation of different human dental tissues and alveolar bone for DNA quantity and quality for forensic investigation.

In this study, the efficacy of dental tissues (cementum, dentine and pulp) and alveolar bone as a potential source of DNA was tested in terms of the quality and quantity using nuclear and mitochondrial markers for forensic investigation.This study found dentine as the best source of DNA with only 5.36% imbalanced (PHR<0.7) heterozygous loci. Pulp showed the highest quantity of DNA but exhibited 22.3% imbalanced (PHR<0.7) heterozygous loci. Cementum with highest (46.67%) heterozygote imbalance proved to be the last choice as a source of DNA. Alveolar bone exhibited the second-highest total yield of DNA/mg of tissue. All Global Filer™ STR loci were amplified in 70% samples of fresh alveolar bone whereas for 30% samples, only partial profile was generated along with successful sex determination. All the dental tissues and alveolar bone samples amplified non STR markers (D-loop, Cytochrome Oxidase I, SRY, AMEL). Of the alveolar bones from archival samples, one sample exhibited full STR profile whereas other alveolar bone samples gave partial profiles. This study substantiates alveolar bone as an alternate source of nuclear and mitochondrial DNA.

A systematic evaluation of 'Bidi - a hand-rolled cigarette' as a forensic DNA evidence.

Bidis are small handmade cigarettes consisting of ~0.2 g of tobacco flakes rolled in a dried leaf of 'Tendu' (Diospyros melanoxylon) or Piliostigma racemosum and tied with a thin thread. They have gained worldwide popularity among smokers and are often collected as forensic DNA evidence from crime scenes and processed similarly to cigarette butts. However, bidi's composition and manufacturing process differs distinctly from the cigarette, and hence the optimal processing for DNA analysis should not be assumed to be similar to cigarette butts. In the present study, the methodical evaluation of the bidi for DNA analysis is reported and an additional process of cell elution from bidi stubs prior to DNA extraction is systematically compared with the direct lysis of bidi stubs which is identical to the standard practice in forensic labs for cigarette butts. In terms of cell recovery from bidi stubs, the SDS based Cell Elution Buffer (CEB) proved to be better than Tween20 based CEB. The three components (cell-elute, supernatant, and processed stub) obtained after the cell elution process of smoked bidi stubs showed varying amounts of DNA. The cell-elute and processed stub exhibited high quality DNA, resulting in 90-100% of the samples giving a full STR profile. On the contrary, the directly lysed stubs yielded high quantity but low quality of DNA, resulting in only 40% of the samples with full STR profiles. The cell elution process enables three components namely cell-elute, supernatant and processed stub from the same bidi to be used for forensic DNA analysis, although the cell-elute proved to be the best source of DNA for STR profiling. The current study demonstrates that the additional cell elution process proved to be an essential step prior to DNA extraction procedure for bidi stubs.

Kinetics and mechanisms of mercury biosorption by an exopolysaccharide producing marine isolate Bacillus licheniformis.

Eight exopolysaccharide (EPS) producing metal-removing marine bacteria were screened for mercury (Hg) sorption. Bacillus licheniformis with the highest MIC values and Hg sorption ability was selected for further study. Biosorption of Hg from aqueous solution by Bacillus licheniformis was studied with respect to the metal concentration, adsorbent concentration, pH, different contact times, and in the presence of other metal ions. Under optimum conditions, more than 70% mercury was removed by 25 mg dried biomass of Bacillus licheniformis at pH 7.0 after 1 h of contact time. Freundlich adsorption isotherm was acceptable at studied Hg concentrations as compared to Langmuir isotherm model. Pseudo-second-order kinetic model was found to be more suitable for data presentation in contrast to pseudo-first-order kinetic model. Involvement of external mass transfer was prominent as compared to intraparticle diffusion model. Desorption of Hg was more effective with acids from all the studied eluents, showing 49.36 and 33.8% eluting capacity for 0.1 N HCL and 0.1 N HNO3, respectively. Scanning electron microscopy exhibited altered cell surface morphology of the cells under the influence of mercury. The spectral images of energy dispersive spectroscopy showed the presence of metal ions on the surface of cells.

Application of novel consortium TSR for treatment of industrial dye manufacturing effluent with concurrent removal of ADMI, COD, heavy metals and toxicity.

The present study was aimed towards the effective bio-treatment of actual industrial effluent containing as high as 42,000 mg/L COD (chemical oxygen demand), >28,000 ADMI (American Dye Manufacturers Institute) color value and four heavy metals using indigenous developed bacterial consortium TSR. Mineral salt medium supplemented with as low as 0.02% (w/v) yeast extract and glucose was found to remove 70% ADMI, 69% COD and >99% sorption of heavy metals in 24 h from the effluent by consortium TSR. The biodegradation of effluent was monitored by UV-vis light, HPLC (high performance liquid chromatography), HPTLC (high performance thin layer chromotography) and FTIR (Fourier transform infrared spectroscopy) and showed significant differences in spectra of untreated and treated effluent, confirming degradation of the effluent. Induction of intracellular azoreductase (107%) and NADH-DCIP reductase (128%) in addition to extracellular laccase (489%) indicates the vital role of the consortium TSR in the degradation process. Toxicity study of the effluent using Allium cepa by single cell gel electrophoresis showed detoxification of the effluent. Ninety per cent germination of plant seeds, Triticum aestivum and Phaseolus mungo, was achieved after treatment by consortium TSR in contrast to only 20% and 30% germination of the respective plants in case of untreated effluent.

A novel biphasic leaching approach for the recovery of Cu and Zn from polymetallic bulk concentrate.

In scale-up biphasic leaching process of polymetallic concentrate, the ferric bioregeneration cycles were performed in 15.0L down flow packed bed reactor; whereas the chemical leaching cycles were done using the biogenerated ferric in an indigenously designed 10.0L stirred tank reactor. The consortium took 25cycles for proper biofilm formation. It showed highest iron oxidation rate (IOR) of 3908.21mg/L/h at 25thcycle under no polymetallic stress. Even under stressed conditions, it was 2650-558mg/L/h. Cu extractions were 86.63-46.51 and Zn extractions were 67.89-14.74% in 1st-4thcycle, respectively. The developed consortium exhibited 17-51times higher IOR compared to original wild type consortium. Extraction isotherm for zinc with 30% Cyanex® 301 indicated that a total of two stages are required for its complete extraction using the phase ratio of 2:1 at equilibrium pH 1.5, leaving behind Fe(II) in the raffinate.

Development of Leptospirillum ferriphilum dominated consortium for ferric iron regeneration and metal bioleaching under extreme stresses.

Activated iron oxidizing consortium SR-BH-L enriched from Rajpardi lignite mine soil sample gave iron oxidation rate 1954 mg/L/h. Developed novel polystress resistant consortium oxidized ferrous iron under 11cP viscosity, 7.47 M ionic strength, 2.3 pH and g/L of 0.50 cadmium, 3.75 copper, 0.20 lead, 92.00 zinc, 6.4 sodium, 5.5 chloride, 154 sulphate and 393.8 TDS. The developed consortium showed 78.0% and 70.0% copper and zinc extraction from polymetallic bulk concentrate in monophasic bioleaching process. The bioregenerated ferric by the consortium in leachate showed 80.81% and 54.0% copper and zinc leaching in only 30 and 90 min. The DGGE analysis indicated the presence of 11 OTUs in the consortium. 16S rRNA gene sequence (JN797729) of the dominant band on DGGE shared >99% similarity with Leptospirillum ferriphilum. RE digestion analysis of the total 16S rRNA gene also illustrated the dominance of L. ferriphilum in the consortium.