Enhancing Cohort PASA Efficiency from Lessons Assimilated by Mutant Genotyping in C. elegans.

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.

Systematic and comprehensive investigation of the toxicity of curcuminoid­essential oil complex: A bioavailable turmeric formulation.

Curcumin, the active component present in Curcuma longa of the family Zingiberaceae, has a number of pharmacological effects, including potential anti­inflammatory activity. One of the major limitations of curcumin/turmeric extract is its poor absorption through the gastrointestinal tract. Several approaches have been adopted to increase the bioavailability of curcumin, including loading curcumin into liposomes or nanoparticles, complexation with phospholipids, addition of essential oils and synthesizing structural analogues of curcumin. In the present study, the toxicity and safety of one such bioavailable turmeric formulation, curcuminoid­essential oil complex (CEC), the toxicity profile of which has not been reported, were examined using in vivo and in vitro models, as per the guidelines of the Organisation for Economic Co-operation and Development. Investigations of acute toxicity study were performed in rats and mice, and the results revealed no signs and symptoms or toxicity or mortality in any of the animals at the maximum recommended dose level of 5,000 mg/kg body weight. The repeated administration of CEC for 90 days in Wistar rats at a dose of 1,000 mg/kg body weight did not induce any observable toxic effects, compared with corresponding control animals. Mutagenicity/genotoxicity investigations were also performed using a bacterial reverse mutation assay (Ames test), a mammalian bone marrow chromosome aberration test and a mammalian erythrocyte micronucleus test in mice. CEC was found to be non­mutagenic in all three mutagenic investigations. Consequently, the present study indicated that CEC elicited no toxic effects in animals or in vitro. Therefore, following investigations of acute toxicity, repeated dose toxicity and mutagenicity, CEC was deemed a safe, non­toxic pharmacological formulation.