Villain of Molecular Biology: Why are we not reproducible in research?

Worldwide, there is an issue of  irreproducibility in life science research. In the USA alone $28 billion per year spent on preclinical research is not reproducible. Within this opinion article, I provide a brief historical account of the discovery of the Watson-Crick DNA model and introduce another neglected model of DNA. This negligence may be one of the fundamental reasons behind irreproducibility in molecular biology research.

Parallel DNA polymerase chain reaction: Synthesis of two different PCR products from a DNA template.

Conventionally, in a polymerase chain reaction (PCR), oligonucleotide primers bind to the template DNA in an antiparallel complementary way and the template DNA is amplified as it is. Here we describe an approach in which the first primer binds in a parallel complementary orientation to the single-stranded DNA, leading to synthesis in a parallel direction. Further reactions happened in a conventional way leading to the synthesis of PCR product having polarity opposite to the template used. This is the first study showing that synthesis of DNA can happen also in a parallel direction. We report that from a single-stranded DNA template, two different but related PCR products can be synthesized.

Tumour necrosis factor-alpha attenuates insulin action on phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis by altering the cellular localization of Foxa2 in HepG2 cells.

Circulating tumour necrosis factor-alpha (TNFalpha) levels, which are elevated in obesity-associated insulin resistance and diabetes, inhibit insulin signalling at several points in the signalling cascade. The liver is critical in maintaining circulating glucose levels and, in a preliminary investigation using the human hepatoma (HepG2) cell line in this study, we demonstrated the role of TNFalpha in the regulation of this phenomenon and determined the underlying molecular mechanisms. As the transcription factor Foxa2 has been implicated, in part, in the regulation of gluconeogenic genes, we studied the effects of TNFalpha and/or insulin on its cellular status in hepatocytes, followed by an assessment of its occupancy on the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Preincubation of cells with TNFalpha, followed by insulin, significantly prevented insulin-mediated nuclear exclusion of Foxa2 and substantially increased its nuclear concentration. Foxa2 was subsequently found to occupy its binding element on the PEPCK promoter. TNFalpha alone, however, did not alter the status of cellular Foxa2 or its occupancy on the PEPCK promoter. TNFalpha preincubation also significantly attenuated insulin-induced inhibition of the expression of gluconeogenic enzymes and hepatic glucose production. Insulin inhibition of PEPCK expression and the preventive effect of TNFalpha could be partially but significantly restored in the presence of Foxa2 siRNA. Several other well-known mediators of insulin action in the liver in general and of gluconeogenic genes in particular include Foxo1, PGC-1 and SREBP-1c. Our results indicate that another transcription factor, Foxa2, is at least partly responsible for the attenuating effect of TNFalpha on insulin action on PEPCK expression and glucose production in HepG2 cells.