Benefits of Implementing Reflex Genomic Analysis for Nonsmall Cell Lung Cancer.

Background: Molecular biomarker analysis is standard of care in advanced nonsmall cell lung cancer (NSCLC). Pathologist-driven reflex testing protocols are one approach to initiating this analysis. Two years after insourcing genomic analysis at our institution, a reflex testing protocol for advanced NSCLC was initiated.

Comprehensive Validation of Cytology Specimens for Next-Generation Sequencing and Clinical Practice Experience.

Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutation profiling via next-generation sequencing (NGS). Specimens derived via fine-needle aspiration, a common biopsy technique, are subjected to a variety of cytopreparatory methods compared with surgical biopsies that are almost uniformly processed as formalin-fixed, paraffin-embedded tissue. Therefore, the fine-needle aspiration-derived specimens most commonly accepted for molecular analysis are cell blocks (CBs), because they are processed most similarly to surgical biopsy tissue.

Variant call concordance between two laboratory-developed, solid tumor targeted genomic profiling assays using distinct workflows and sequencing instruments.

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability.

Characterization of the GbdR regulon in Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the host-derived quaternary amines choline and glycine betaine (GB).

Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds.

Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa.

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P.

Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements.

The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5' UTR of the glmS mRNA, resulting in downregulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured.

A rate-limiting conformational step in the catalytic pathway of the glmS ribozyme.

The glmS ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located upstream of the glucosamine-6-phosphate (GlcN6P) synthetase reading frame. Binding of GlcN6P activates site-specific self-cleavage of the glmS mRNA, resulting in the downregulation of glmS gene expression. Unlike other riboswitches, the glmS ribozyme does not undergo structural rearrangement upon metabolite binding, indicating that the metabolite binding pocket is preformed in the absence of ligand.

An ontological knowledge framework for adaptive medical workflow.

As emerging technologies, semantic Web and SOA (Service-Oriented Architecture) allow BPMS (Business Process Management System) to automate business processes that can be described as services, which in turn can be used to wrap existing enterprise applications. BPMS provides tools and methodologies to compose Web services that can be executed as business processes and monitored by BPM (Business Process Management) consoles. Ontologies are a formal declarative knowledge representation model.

Evidence for preorganization of the glmS ribozyme ligand binding pocket.

We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of methods with the aim of evaluating the magnitude of RNA conformational change induced by binding of the cofactor, glucosamine 6-phosphate (GlcN6P). Hydroxyl radical footprinting of a trans-acting ribozyme complex identifies several sites of solvent protection upon incubation of the RNA in Mg(2+)-containing solutions, providing initial evidence of the tertiary fold of the ribozyme. Under these folding conditions and at GlcN6P concentrations that saturate the ligand-induced cleavage reaction, we do not observe changes to this pattern.

In vitro selection of second site revertants analysis of the hairpin ribozyme active site.

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction.

Solvent protection of the hammerhead ribozyme in the ground state: evidence for a cation-assisted conformational change leading to catalysis.

Tertiary folding of the hammerhead ribozyme has been analyzed by hydroxyl radical footprinting. Three hammerhead constructs with distinct noncore sequences, connectivities, and catalytic properties show identical protection patterns, in which conserved core residues (G5, A6, U7, G8, and A9) and the cleavage site (C17, G1.1, and U1.