Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci.

Chlamydia psittaci is a zoonotic pathogen mainly transmitted by psittacine birds and poultry. The low shedding rate of the pathogen in the apparently healthy birds and human clinical cases may result in false-negative results. In the present study, a droplet digital PCR (ddPCR) assay was developed and compared with optimized quantitative PCR (qPCR) for the detection of C. psittaci from the clinical samples. The ddPCR assay was found to be comparatively more sensitive than the qPCR, wherein the limit of detection (LOD) of ddPCR was upto 2.4 copies of the DNA template, whereas, the qPCR could detect upto 38 copies of the DNA template in the reaction mixture. Overall, the developed ddPCR assay was found to be robust, specific, and could reliably quantify up to 17.8 copies of the DNA template. Finally, the applicability of the developed ddPCR assay was tested by screening the field samples (n = 124), comprising lung tissues from dead poultry and feral birds; pooled faecal samples from the free-living birds, commercial and backyard poultry farms; pharyngeal and cloacal swabs collected from the duck farms. Of these, a total of seven samples were found to be positive by the ddPCR, whereas, three samples could be detected as positive using the qPCR. The developed ddPCR could serve as a reliable screening tool, particularly in those clinical samples wherein the shedding of C. psittaci is substantially very low.

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435 bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448 bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448 bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210 bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.

Effect of Benzothiazole based conjugates in causing apoptosis by Regulating p53, PTEN and MAP Kinase proteins affecting miR-195a and miR-101-1.

BACKGROUND: Hepatocellular carcinoma (HCC) accounts for majority of liver cancers and is the leading cause of cancer related death in Asia. Like any other cancer, HCC develops when there is a mutation to the cellular machinery that causes the cell to replicate at a higher rate and results in the loss of apoptosis. Therefore, a delicate balance between the expression of various genes involved in proliferation and apoptosis decide the ultimate fate of the cell to undergo rapid proliferation (cancer) or cell death. RESULTS: The benzothiazole based compounds exhibited effective cytotoxicity at 4 µM concentration and have shown G1 cell cycle arrest with decrease in levels of G1 cell cycle proteins such as cyclin D1 and Skp2. Involvement of tumour suppressor proteins such as PTEN and p53 was studied. Interestingly these compounds displayed decrease in the phosphorylated forms of AKT, p38 MAPK and ERK1/2 which play a vital role in cell proliferation. Compounds have exhibited strong and significant effect on the expression of micro RNAs such as miR-195a & miR-101-1 which regulate hepatic cell proliferation. CONCLUSIONS: The cell cycle arrest and apoptotic inducing nature of these compounds was revealed by FACS, BrdU cell proliferation and tunel assays. Compounds affected both tumour suppressor proteins as well as proteins that are involved in active cell proliferation. Micro RNAs whose target is Cyclin D1 such as miR-195a and miR-101-1 that is required for growth of hepatoma cells was drastically affected. These compounds caused apoptosis by activating caspase-3 and PARP.

First stereoselective total synthesis and anticancer activity of new amide alkaloids of roots of pepper.

The first stereoselective total synthesis of new natural amide alkaloids 1-3 have been achieved from commercially available starting materials. Wittig olefination, Sharpless asymmetric dihydroxylation, epoxidation, a trans regioselective opening of 2,3-epoxy alcohol, Horner-Wadsworth-Emmons (HWE) olefination and amide coupling are the key steps. The amide alkaloids 1-3 are evaluated for their anticancer activity against colon (HT-29), breast (MCF-7) and lung (A-549) human cancer cell lines for the first time.