PharmVar Tutorial on CYP2D6 Structural Variation Testing and Recommendations on Reporting.

The Pharmacogene Variation Consortium (PharmVar) provides nomenclature for the highly polymorphic human CYP2D6 gene locus and a comprehensive summary of structural variation. CYP2D6 contributes to the metabolism of numerous drugs and, thus, genetic variation in its gene impacts drug efficacy and safety. To accurately predict a patient's CYP2D6 phenotype, testing must include structural variants including gene deletions, duplications, hybrid genes, and combinations thereof. This tutorial offers a comprehensive overview of CYP2D6 structural variation, terms, and definitions, a review of methods suitable for their detection and characterization, and practical examples to address the lack of standards to describe CYP2D6 structural variants or any other pharmacogene. This PharmVar tutorial offers practical guidance on how to detect the many, often complex, structural variants, as well as recommends terms and definitions for clinical and research reporting. Uniform reporting is not only essential for electronic health record-keeping but also for accurate translation of a patient's genotype into phenotype which is typically utilized to guide drug therapy.

PharmVar GeneFocus: CYP3A5.

The Pharmacogene Variation Consortium (PharmVar) catalogs star (*) allele nomenclature for the polymorphic human CYP3A5 gene. Genetic variation within the CYP3A5 gene locus impacts the metabolism of several clinically important drugs, including the immunosuppressants tacrolimus, sirolimus, cyclosporine, and the benzodiazepine midazolam. Variable CYP3A5 activity is of clinical importance regarding tacrolimus metabolism. This GeneFocus provides a CYP3A5 gene summary with a focus on aspects regarding standardized nomenclature. In addition, this review also summarizes recent changes and updates, including the retirement of several allelic variants and provides an overview of how PharmVar CYP3A5 star allele nomenclature is utilized by the Pharmacogenomics Knowledgebase (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC).

The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

Growing and analyzing biofilms in fermenters.

One of the most daunting challenges of biofilm research is comparing experimental results produced by multiple laboratories, each of which uses different techniques to generate, analyze, and interpret biofilm data. The heterogeneity inherent to biofilm communities contributes to the difficulty in obtaining reproducible results between experiments within a single laboratory, but the problem is compounded further by a lack of standardization in techniques. A number of biofilm culture methods are presented in this unit to provide a set of standards for biofilm study. Each model system differs in growth conditions, applied variables, and experimental output, all of which must be carefully considered when designing an experiment and, most critically, during data interpretation. In this unit, two methods of biofilm culture that are known to reliably provide reproducible, statistically clean results in determining the viability and antimicrobial susceptibility of biofilm communities are described. The spinning disc model provides multiple biofilm samples from the same biofilm reactor, significantly reducing data variability. The tube biofilm method, in addition to providing this benefit, can be used for expression analysis, and thus can yield informative data on both macro- and micro-scales. These methods also utilize continuous culture, or chemostat, conditions to maintain a quasi-steady state.

Biofilm formation in plant-microbe associations.

Bacteria adhere to environmental surfaces in multicellular assemblies described as biofilms. Plant-associated bacteria interact with host tissue surfaces during pathogenesis and symbiosis, and in commensal relationships. Observations of bacteria associated with plants increasingly reveal biofilm-type structures that vary from small clusters of cells to extensive biofilms. The surface properties of the plant tissue, nutrient and water availability, and the proclivities of the colonizing bacteria strongly influence the resulting biofilm structure. Recent studies highlight the importance of these structures in initiating and maintaining contact with the host by examining the extent to which biofilm formation is an intrinsic component of plant-microbe interactions.

The FNR-type transcriptional regulator SinR controls maturation of Agrobacterium tumefaciens biofilms.

Agrobacterium tumefaciens is a plant pathogen that persists as surface-associated populations on plants or soil particles. A genetic screen for A. tumefaciens mutants deficient for surface interactions identified a mutant that forms thin, sparsely populated biofilms, but is proficient for initial attachment. The mutant is disrupted in a gene designated sinR, encoding a member of the DNR subfamily of FNR-type transcription regulators. SinR is required for normal maturation of A. tumefaciens biofilms on both inert surfaces and plant tissues, and elevated sinR expression results in accelerated biofilm formation. Expression of sinR is increased close to 30-fold in cultures grown in oxygen-limited environments and is also induced within biofilms grown under oxic conditions. A consensus FNR box, the presumptive binding site for FNR-type proteins, is located upstream of the sinR promoter. FnrN, a second A. tumefaciens FNR-like regulator, is required for induction of sinR in oxygen-limited cultures, whereas SinR negatively influences its own expression. FnrN influences biofilm formation, but its effects are less dramatic than those of SinR. We propose a model in which a signal cascade, responsive to oxygen limitation and initiated by FnrN, activates sinR expression in response to decreased oxygen levels, and influences the formation of A. tumefaciens biofilms.