Design and implementation of a novel pharmacogenetic assay for the identification of the CYP2D6*10 genetic variant.

OBJECTIVES: Tamoxifen is considered to be the most widely used adjuvant therapy for hormone receptor positive breast cancer in premenopausal women. However, it is reported that nearly 30% of patients receiving tamoxifen therapy have shown reduced or no benefits. This may be due to the high inter-individual variations in the CYP2D6 gene that is involved in tamoxifen metabolism. The CYP2D6*10 gene variant (rs1065852C>T) is reported to be commonly found in Asian and South Asian populations. The present study was undertaken to design a novel pharmacogenetic assay (Single step-Tetra Arms Polymerase Chain Reaction) for the identification of the CYP2D6*10 variant and implement the designed assay by genotyping a cohort of breast cancer patients. RESULTS: The novel assay was successfully designed, optimized and validated using Sanger sequencing. Blood samples from 70 patients were genotyped. The following bands were observed in the gel image: Control band at 454 bp; band for C allele at 195 bp; band for T allele at 300 bp. The genotype frequencies for the CYP2D6*10 (rs1065852C>T) variant were: CC-24.28% (17/70), CT-75.71% (53/70), TT-0% (0/70). The allele frequencies were: T-allele-37.86% and C-allele-62.14%.

Ethical Responses to the COVID-19 Pandemic-Lessons from Sri Lanka.

The COVID-19 pandemic has undoubtedly become an era-defining challenge for the entire world. It has implications not only in the public health sector but also in the global economy and political landscape. The prevention strategy that has been followed in Sri Lanka is unique. Early action taken by the government and the ministry of health, being one of pre-emptive quarantining and isolation of suspected contacts even before they developed symptoms, was vital to contain the spread of the disease. During the early phase, a nationwide lockdown in the form of a curfew was imposed which helped mitigate the spread of the virus. However, due to several lapses, there was a threat of community transmission; this was swiftly brought under control through ongoing government interventions. Thus, strict social/physical distancing measures enforced by the government, together with an increase in testing capacity, prevented widespread community transmission. Strictly containing the outbreaks as and when they were identified made it easier to bring the spread under control through contact tracing. In this article, we give an account of the strategy taken by Sri Lanka to mitigate the pandemic and comment on the lessons learned concerning the ethical responses to the COVID-19 crisis.

Glucose-induced nuclear shuttling of ChREBP is mediated by sorcin and Ca(2+) ions in pancreatic ß-cells.

Carbohydrate-responsive element-binding protein (ChREBP) is a regulator of pancreatic ß-cell gene expression and an important mediator of glucotoxicity. Glucose increases the activity and nuclear localization of ChREBP by still ill-defined mechanisms. Here we reveal, using both MIN6 and primary mouse ß-cells, a unique mechanism behind ChREBP nuclear translocation. At low glucose concentrations, ChREBP interacts with sorcin, a penta EF hand Ca(2+) binding protein, and is sequestered in the cytosol. Sorcin overexpression inhibits ChREBP nuclear accumulation at high glucose and reduced the activity of L-type pyruvate kinase (L-PK) and TxNIP promoters, two well-characterized ChREBP target genes. Sorcin inactivation by RNA interference increases ChREBP nuclear localization and in vivo binding to the L-PK promoter at low glucose concentrations. Ca(2+) influx was essential for this process since Ca(2+) chelation with EGTA, or pharmacological inhibition with diazoxide and nifedipine, blocked the effects of glucose. Conversely, mobilization of intracellular Ca(2+) with ATP caused the nuclear accumulation of ChREBP. Finally, sorcin silencing inhibited ATP-induced increases in intracellular Ca(2+) and glucose-stimulated insulin secretion. We therefore conclude that sorcin retains ChREBP in the cytosol at low glucose concentrations and may act as a Ca(2+) sensor for glucose-induced nuclear translocation and the activation of ChREBP-dependent genes.

Roles of Ca2+ ions in the control of ChREBP nuclear translocation.

Carbohydrate-responsive element binding protein (ChREBP (MLXIPL)) is emerging as an important mediator of glucotoxity both in the liver and in the pancreatic ß-cells. Although the regulation of its nuclear translocation and transcriptional activation by glucose has been the subject of intensive research, it is still not fully understood. We have recently uncovered a novel mechanism in the excitable pancreatic ß-cell where ChREBP interacts with sorcin, a penta-EF-hand Ca(2)(+)-binding protein, and is sequestered in the cytosol at low glucose concentrations. Upon stimulation with glucose and activation of Ca(2)(+) influx, or application of ATP as an intracellular Ca(2)(+)-mobilising agent, ChREBP rapidly translocates to the nucleus. In sorcin-silenced cells, ChREBP is constitutively present in the nucleus, and both glucose and Ca(2)(+) are ineffective in stimulating further ChREBP nuclear shuttling. Whether an active Ca(2)(+)-sorcin element of ChREBP activation also exists in non-excitable cells is discussed.

Carbohydrate-responsive element-binding protein (ChREBP) is a negative regulator of ARNT/HIF-1beta gene expression in pancreatic islet beta-cells.

OBJECTIVE: Carbohydrate-responsive element-binding protein (ChREBP) is a transcription factor that has been shown to regulate carbohydrate metabolism in the liver and pancreatic beta-cells in response to elevated glucose concentrations. Because few genes have been identified so far as bona fide ChREBP-target genes, we have performed a genome-wide analysis of the ChREBP transcriptome in pancreatic beta-cells. RESEARCH DESIGN AND METHODS: Chromatin immunoprecipitation and high-density oligonucleotide tiling arrays (ChIP-chip; Agilent Technologies) using MIN6 pancreatic beta-cell extracts were performed together with transcriptional and other analysis using standard techniques. RESULTS: One of the genes identified by ChIP-chip and linked to glucose sensing and insulin secretion was aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor-1beta (HIF-1beta), a transcription factor implicated in altered gene expression and pancreatic-islet dysfunction in type 2 diabetes. We first confirmed that elevated glucose concentrations decreased ARNT/HIF-1beta levels in INS-1 (832/13) cells and primary mouse islets. Demonstrating a role for ChREBP in ARNT gene regulation, ChREBP silencing increased ARNT mRNA levels in INS-1 (832/13) cells, and ChREBP overexpression decreased ARNT mRNA in INS-1 (832/13) cells and primary mouse islets. We demonstrated that ChREBP and Max-like protein X (MLX) bind on the ARNT/HIF-1beta promoter on the proximal region that also confers the negative glucose responsiveness. CONCLUSIONS: These results demonstrate that ChREBP acts as a novel repressor of the ARNT/HIF-1beta gene and might contribute to beta-cell dysfunction induced by glucotoxicity.

A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1.

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.