From cheek swabs to consensus sequences : an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

Background: Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results: Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions: All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources

Neolithic Mitochondrial Haplogroup H Genomes and the Genetic Origins of Europeans

Haplogroup H dominates present-day Western European mitochondrial DNA variability (\u3e40%), yet was less common (~19%) among Early Neolithic farmers (~5450 BC) and virtually absent in Mesolithic hunter-gatherers. Here we investigate this major component of the maternal population history of modern Europeans and sequence 39 complete haplogroup H mitochondrial genomes from ancient human remains. We then compare this ‘real-time’ genetic data with cultural changes taking place between the Early Neolithic (~5450 BC) and Bronze Age (~2200 BC) in Central Europe. Our results reveal that the current diversity and distribution of haplogroup H were largely established by the Mid Neolithic (~4000 BC), but with substantial genetic contributions from subsequent pan-European cultures such as the Bell Beakers expanding out of Iberia in the Late Neolithic (~2800 BC). Dated haplogroup H genomes allow us to reconstruct the recent evolutionary history of haplogroup H and reveal a mutation rate 45% higher than current estimates for human mitochondria

Pharmacological evaluations of ethanol extract of Ficus benghalensis L. seeds for its antiulcer and antimicrobial efficacy

To evaluate the gastroprotective effect and antimicrobial efficacy of the seeds of Ficus benghalensis L. Acute oral toxicity study was performed according to the OECD test guidelines and the antiulcer assay was performed using ethanol-induced gastric ulcer model in albino rats. The antimicrobial activity and HPTLC analysis were also performed. The seed extract did not show any sign of toxicity at a dose of 4000 mg/kg body weight. Reductions in the ulcer index and gastric acid volume with increase in the pH of the gastric fluid in extract treated rats proved antiulcer activity. Increased levels of endogenous antioxidant enzymes superoxide dismutase and catalase with a decrease in lipid peroxidation in the extract treated animals demonstrated its antioxidant effect. The zone of inhibition was extremely great for all the tested microorganisms. HPTLC chromatogram showed a total of 9 peaks at different Rf values and peak area at 366 nm whereas seven peaks were observed at 254 nm. The number of peaks indicates the presence of constituents in the extract. Pre-treatment with F. benghalensis seed extract showed significant antiulcer activity that might be attributed to its antioxidant potential. F. benghalensis showed significant activity against bacteria and fungi. The extract containing phytoconstituents must have contributed to its antimicrobial activity

Pharmacological evaluation of ethanol extract of <em>Ficus benghalensis</em> seeds for antiulcer and antimicrobial efficacy

329-334The alcoholic extract of the seeds of the plant Ficus benghalensis L. has been screened for acute toxicity, gastroprotective effect, antimicrobial efficacy, antioxidant potential and HPTLC analysis.Toxicity study was performed according to the OECD test guidelines and the antiulcer assay was performed using ethanol-induced gastric ulcer model in albino rats. The antimicrobial activity and HPTLC analysis were also performed. The seed extract did not show any sign of toxicity upto dose of 2000 mg/kg body weight. Reductions in the ulcer index and gastric acid volume with increase in the pH of the gastric fluid in extract treated rats proved antiulcer activity. Increased levels of endogenous antioxidant enzymes superoxide dismutase and catalase with a decrease in lipid peroxidation in the extract treated animals demonstrated its antioxidant effect. The zone of inhibition was significant in all the tested microorganisms. HPTLC chromatogram showed a total of 9 peaks at different Rf values and peak area at 366 nm whereas seven peaks were observed at 254 nm. The number of peaks indicates the presence of constituents in the extract. Pre-treatment with F. benghalensis seed extract showed appreciable antiulcer activity that might be attributed to its antioxidant potential. The extract also showed approciable activity against the bacteria and fungi tested. The extract containing phytoconstituents must have contributed to this property