Understanding pluripotency under folic acid deficiency using embryonic stem cells as an in vitro model.

Innumerable studies have been carried out by people to trace out those elements, find out the mechanisms that are vital to the maintenance of stemness. Amongst them, the one which is gaining importance in the recent past is the metabolism of stem cells, especially One carbon metabolism. Being associated directly or indirectly, with DNA and Histone methylation, One carbon metabolism has an important role to play as far as epigenetic regulation of stemness is concerned. Folic acid being a methyl donor, forms an important component of One Carbon metabolism, and thus its availability has a significant effect on the methylation reactions. The present hypothesis says, using embryonic stem cells grown in vitro as a model system, one can study how deficiency of Folate during pre implantation embryonic development influence the self renewal as well as pluripotency of stem cells, from the perspective of One carbon metabolism and epigenetic mechanisms. Findings from such a study can possibly provide an explanation for the observed intra uterine growth retardation seen under maternal micronutrient deficiency.

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus.

AIM: The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. MATERIALS AND METHODS: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription-PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV. RESULTS: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269E×103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. CONCLUSION: Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.