Optimization of BRAF V600 Assay as A 2-Step Real-time PCR Protocol

The QClamp® BRAF Codon Specific Mutation Detection Kit is a real-time PCR assay for the detection of somatic mutations in codon 600 Valine at exon 15 in the BRAF gene which encodes the serine/threonine protein kinase, using purified DNA. The V600E mutation is the most common BRAF gene mutation found in human cancers. This mutation leads to production of a BRAF protein that is abnormally active, which disrupts regulation of cell growth and division. Mutations in this gene have been found in cancers, including non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, papillary thyroid carcinoma, non-small-cell lung carcinoma, gastric cancer, and even prostate cancer. Currently, the established qPCR protocol for the QClamp® BRAF Mutation Detection Assay is comprised of a 4-step procedure: Denaturation, XNA Annealing, Primer Annealing and Extension. The purpose of this experiment was to test the feasibility of optimizing this assay to a more efficient and faster 2-step Real-time PCR which has just the Denaturation and the Primer Annealing/Extension steps. Optimization was attempted on both the ABI-QS5 and LC480 thermocycling instruments using parallel testing. The newly established 2-step thermocycling parameters were successfully tested and validated on the ABI-QS5 instrument. For the LC480, however, the experiment was not successful. This result might be due to the different platforms and technologies of the two instruments. Further research is needed to develop the mutational status scoring and acceptance criteria for clinical samples on the ABI-QS5, and to complete the development of the 2-step qPCR protocol individually on the LC480; and also to study the effects of factors such as temperature, ramp rates, PCR enzymes/master mix, primer/probes and XNA concentrations for both ABI-QS5, and the LC480

Estrogen Receptor Activation Function 1 Works by Binding p160 Coactivator Proteins

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize

Estrogen Receptor Activation Function 1 Works by Binding p160 Coactivator Proteins

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize

Differential anti-viral response to respiratory syncytial virus A in preterm and term infantsResearch in context

Summary: Background: Preterm infants are more likely to experience severe respiratory syncytial virus (RSV) disease compared to term infants. The reasons for this are multi-factorial, however their immature immune system is believed to be a major contributing factor. Methods: We collected cord blood from 25 preterm (gestational age 30.4–34.1 weeks) and 25 term infants (gestation age 37–40 weeks) and compared the response of cord blood mononuclear cells (CBMCs) to RSVA and RSVB stimulation using neutralising assays, high-dimensional flow cytometry, multiplex cytokine assays and RNA-sequencing. Findings: We found that preterm and term infants had similar maternally derived neutralising antibody titres to RSVA and RSVB. Preterm infants had significantly higher myeloid dendritic cells (mDC) RSV infection compared to term infants. Differential gene expression analysis of RSVA stimulated CBMCs revealed enrichment of genes involved in cytokine production and immune regulatory pathways involving IL-10, IL-36γ, CXCL1, CXCL2, SOCS1 and SOCS3 in term infants, while differentially expressed genes (DEGs) in preterm infants were related to cell cycle (CDK1, TTK, ESCO2, KNL1, CDC25A, MAD2L1) without associated expression of immune response genes. Furthermore, enriched genes in term infants were highly correlated suggesting an increased co-ordination of their immune response to RSVA. When comparing DEGs in preterm and term infants following RSVB stimulation, no differences in immune response genes were identified. Interpretation: Overall, our data suggests that preterm infants have a more restricted immunological response to RSVA compared with term infants. While further studies are required, these findings may help to explain why preterm infants are more susceptible to severe RSV disease and identify potential therapeutic targets to protect these vulnerable infants. Funding: Murdoch Children's Research Institute Infection and Immunity theme grant