Use of cytology fluid samples for predictive biomarker testing in lung cancer patients.

OBJECTIVE: To provide a method of directly using cytology fluid samples for predictive biomarker testing in lung cancer patients and to determine the efficacy of a variety of fluid sample types. METHOD: A review of our in-house data from a range of cytology samples including endobronchial ultrasound (EBUS) fine-needle aspirate (FNA) needle washings (NW) and serous effusions tested on the Biocartis Idylla platform. All fluid samples were originally tested using Sanger sequencing. RESULTS: Using our method for fluid samples all of our cytology samples tested for epithelial growth factor receptor (EGFR) yielded valid results on this platform and all variant cases identified. The data showed serous fluids provided the best quality DNA, and variant genotype reports were obtained within 150 minutes. CONCLUSION: Cytology fluid samples can be used for predictive biomarker testing for lung cancer patients to provide in-house results with all fluids providing good-quality DNA.

Unmasking the True Identity of Rapamycin's Minor Conformer.

Rapamycin, a well-known macrocyclic natural product with myriad biological activities, has been the subject of intense study since its first isolation and characterization over five decades ago. Rapamycin has been found to adopt a single conformation in the solid state (both when protein bound and uncomplexed) and exists as a mixture of two conformations in solution. Early work established that the major conformer in solution is the trans amide isomer but left the minor conformer mostly uncharacterized. Since that time, it has been widely accepted that the minor conformer of rapamycin is the cis amide, based solely on analogy to FK-506, another potent immunosuppressive compound with some shared key structural elements. To address this long-standing and unresolved question, the solution structure of the minor conformer of rapamycin was investigated using a combination of NMR techniques and computational methods and determined to be a trans amide species with rotation about the ester linkage.

Decisions with Confidence: Application to the Conformation Sampling of Molecules in the Solid State.

Accurate conformations of a molecule are critical for reliable prediction of its properties, so good predictive models require good conformations. Here, we present a method for conformer sampling based on distance geometry, implemented in our conformation generator OMEGA, which we apply to both macrocycles and druglike molecules. We validate it in the usual fashion, reproducing conformations from the solid state, and compare its performance in detail to other methods. We find that OMEGA performs well on three key criteria: accuracy, speed, and ensemble size. To support our conclusions quantitatively, particularly on accuracy, we developed a workflow for method comparison that uses parameter estimation, inference from confidence intervals, classical null hypothesis significance testing, Bayesian estimation, and effect size. The workflow is designed to be robust to the highly skewed performance data often found when validating tools in computational chemistry and to provide reliable, easy to interpret results. In this workflow, we emphasize the importance of confidently distinguishing between methods, with particular reference to a priori estimation of sample size and statistical power (false negative or Type II error rate), a topic almost completely ignored hitherto in computational chemistry.

Speech-Language Pathologists and Behavior Analysts: A Survey of Video Modeling Use and Perspectives.

Speech-language pathologists (SLPs) and board-certified behavior analysts (BCBAs) work with clients with autism spectrum disorder (ASD). In general, these practitioners support a variety of skills during treatment, including language, social skills, daily living, and academics. Video modeling (VM) is an intervention that is often used to improve skills (e.g., independent living, social, behavioral, academic) for clients with disabilities. It is unknown how SLPs and BCBAs are specifically using VM to support clients, including those with ASD. The primary purpose of this study was to use an existing perception scale to analyze SLPs' and BCBAs' perspectives on and use of VM. Results have indicated that while most practitioners have heard of VM and use it with clients, barriers related to training and video creation still exist. VM is still predominantly used in clinics and special education classrooms, indicating that this evidence-based strategy is being used, but more training and generalization support is needed.

Conformation Generation: The State of the Art.

The generation of conformations for small molecules is a problem of continuing interest in cheminformatics and computational drug discovery. This review will present an overview of methods used to sample conformational space, focusing on those methods designed for organic molecules commonly of interest in drug discovery. Different approaches to both the sampling of conformational space and the scoring of conformational stability will be compared and contrasted, with an emphasis on those methods suitable for conformer sampling of large numbers of drug-like molecules. Particular attention will be devoted to the appropriate utilization of information from experimental solid-state structures in validating and evaluating the performance of these tools. The review will conclude with some areas worthy of further investigation.

The application of statistical methods to cognate docking: a path forward?

Cognate docking has been used as a test for pose prediction quality in docking engines for decades. In this paper, we report a statistically rigorous analysis of cognate docking performance using tools in the OpenEye docking suite. We address a number of critically important aspects of the cognate docking problem that are often handled poorly: data set quality, methods of comparison of the predicted pose to the experimental pose, and data analysis. The focus of the paper lies in the third problem, extracting maximally predictive knowledge from comparison data. To this end, we present a multistage protocol for data analysis that by combining classical null-hypothesis significance testing with effect size estimation provides crucial information about quantitative differences in performance between methods as well as the probability of finding such differences in future experiments. We suggest that developers of software and users of software have different levels of interest in different parts of this protocol, with users being primarily interested in effect size estimation while developers may be most interested in statistical significance. This protocol is completely general and therefore will provide the basis for method comparisons of many different kinds.

Conformer generation with OMEGA: learning from the data set and the analysis of failures.

We recently published a high quality validation set for testing conformer generators, consisting of structures from both the PDB and the CSD (Hawkins, P. C. D. et al. J. Chem. Inf. Model. 2010, 50, 572.), and tested the performance of our conformer generator, OMEGA, on these sets. In the present publication, we focus on understanding the suitability of those data sets for validation and identifying and learning from OMEGA's failures. We compare, for the first time we are aware of, the coverage of the applicable property spaces between the validation data sets we used and the parent compound sets to determine if our data sets adequately sample these property spaces. We also introduce the concept of torsion fingerprinting and compare this method of dissimilation to the more traditional graph-centric diversification methods we used in our previous publication. To improve our ability to programmatically identify cases where the crystallographic conformation is not well reproduced computationally, we introduce a new metric to compare conformations, RMSTanimoto. This new metric is used alongside those from our previous publication to efficiently identify reproduction failures. We find RMSTanimoto to be particularly effective in identifying failures for the smallest molecules in our data sets. Analysis of the nature of these failures, particularly those for the CSD, sheds further light on the issue of strain in crystallographic structures. Some of the residual failure cases not resolved by simple changes in OMEGA's defaults present significant challenges to conformer generation engines like OMEGA and are a source of new avenues to further improve their performance, while others illustrate the pitfalls of validating against crystallographic ligand conformations, particularly those from the PDB.

Use of the Idylla EGFR Mutation Test for Variant Detection in Non-Small Cell Lung Cancer Samples.

OBJECTIVES: Stratified management of patients with non-small cell lung carcinoma (NSCLC) through epithelial growth factor receptor (EGFR) variant analysis has become standard clinical practice. The Idylla system is a fully compliant European in vitro diagnostics device, a fully automated platform designed to rapidly genotype formalin-fixed paraffin-embedded tissue samples. This retrospective study aims to validate the Idylla EGFR Mutation Test for use with extracted DNA from known NSCLC samples. METHODS: In this study, 20 µL of archival extracted DNA was placed directly inside the Idylla EGFR assay single-use cartridge. Idylla results were compared with the original Sanger sequencing reference method. RESULTS: The Idylla EGFR Mutation Test yielded valid results for all samples tested, confirming the variants identified by the reference method that lay within the Idylla target range. No false-positive cases were noted with the Idylla assay. Variant genotype reports were obtained within 150 minutes. CONCLUSIONS: The Idylla EGFR assay is sensitive for extracted DNA and can be reliably applied to cytologic specimens, enabling its implementation as an ancillary first-line test for patients with NSCLC.

Conformer generation with OMEGA: algorithm and validation using high quality structures from the Protein Databank and Cambridge Structural Database.

Here, we present the algorithm and validation for OMEGA, a systematic, knowledge-based conformer generator. The algorithm consists of three phases: assembly of an initial 3D structure from a library of fragments; exhaustive enumeration of all rotatable torsions using values drawn from a knowledge-based list of angles, thereby generating a large set of conformations; and sampling of this set by geometric and energy criteria. Validation of conformer generators like OMEGA has often been undertaken by comparing computed conformer sets to experimental molecular conformations from crystallography, usually from the Protein Databank (PDB). Such an approach is fraught with difficulty due to the systematic problems with small molecule structures in the PDB. Methods are presented to identify a diverse set of small molecule structures from cocomplexes in the PDB that has maximal reliability. A challenging set of 197 high quality, carefully selected ligand structures from well-solved models was obtained using these methods. This set will provide a sound basis for comparison and validation of conformer generators in the future. Validation results from this set are compared to the results using structures of a set of druglike molecules extracted from the Cambridge Structural Database (CSD). OMEGA is found to perform very well in reproducing the crystallographic conformations from both these data sets using two complementary metrics of success.

Using an Interactive Framework to Assess a Cultural Simulation for Learning: Tag Team Patient Safety Simulation.

BACKGROUND: The purpose of this pilot study, a replication of a study that originated in Australia for large classroom sizes, was to assess the effect of a culturally sensitive simulation-based pedagogical strategy on student learning. METHOD: This quasi-experimental approach, using a pretest-posttest design with a nonequivalent simulation (n = 43) and nonsimulation (n = 73) group, mimics an approach called Tag Team Patient Safety Simulation. A scenario was enacted focusing on the cultural needs of Nasifah, a 67-year-old woman requiring home-based palliative care. Challenging conversations were navigated by participants and observers through the engagement of antagonist and cue cards. RESULTS: The simulation-based strategy improved student understanding of working with culturally sensitive populations. CONCLUSION: This improvement had no greater learning effect than with the traditional based pedagogical strategy. However, student engagement may have been greater. The need to measure retention, the implications of the findings, the limitations of the study, and directions for future research are discussed. [J Nurs Educ. 2019;58(10):591-594.].

Ligand-Based Methods in GPCR Computer-Aided Drug Design.

This chapter describes two powerful 3D ligand-based shape similarity and scoring methods called ROCS and EON, their basic operation and selected validation data. The steps required to prepare a database of molecules for successful use with ROCS and EON are described and selected examples of their application in prospective lead discovery experiments are summarized.

Conformational Sampling of Macrocyclic Drugs in Different Environments: Can We Find the Relevant Conformations?

Conformational flexibility is a major determinant of the properties of macrocycles and other drugs in beyond rule of 5 (bRo5) space. Prediction of conformations is essential for design of drugs in this space, and we have evaluated three tools for conformational sampling of a set of 10 bRo5 drugs and clinical candidates in polar and apolar environments. The distance-geometry based OMEGA was found to yield ensembles spanning larger structure and property spaces than the ensembles obtained by MOE-LowModeMD (MOE) and MacroModel (MC). Both MC and OMEGA but not MOE generated different ensembles for polar and apolar environments. All three conformational search methods generated conformers similar to the crystal structure conformers for 9 of the 10 compounds, with OMEGA performing somewhat better than MOE and MC. MOE and OMEGA found all six conformers of roxithromycin that were identified by NMR in aqueous solutions, whereas only OMEGA sampled the three conformers observed in chloroform. We suggest that characterization of conformers using molecular descriptors, e.g., the radius of gyration and polar surface area, is preferred to energy- or root-mean-square deviation-based methods for selection of biologically relevant conformers in drug discovery in bRo5 space.

Comparison of shape-matching and docking as virtual screening tools.

Ligand docking is a widely used approach in virtual screening. In recent years a large number of publications have appeared in which docking tools are compared and evaluated for their effectiveness in virtual screening against a wide variety of protein targets. These studies have shown that the effectiveness of docking in virtual screening is highly variable due to a large number of possible confounding factors. Another class of method that has shown promise in virtual screening is the shape-based, ligand-centric approach. Several direct comparisons of docking with the shape-based tool ROCS have been conducted using data sets from some of these recent docking publications. The results show that a shape-based, ligand-centric approach is more consistent than, and often superior to, the protein-centric approach taken by docking.

Periconceptional undernutrition in sheep accelerates maturation of the fetal hypothalamic-pituitary-adrenal axis in late gestation.

We investigated the effects of moderate maternal periconceptional undernutrition from 60 d before to 30 d after mating on fetal hypothalamic-pituitary-adrenal axis function in late gestation. Ewes were sampled regularly during the period of undernutrition for circulating hormone levels. Vascular catheters were inserted into ewes and their singleton fetuses at 112 d gestation (term, 145 d), and fetal ACTH(1-24) and metyrapone challenge tests were performed at 127 and 128 d. Postmortems were performed at 132 d. Fetuses of undernourished ewes (UN, n = 12) had elevated baseline cortisol concentrations (P < 0.05), compared with fetuses of ad libitum-fed ewes (n = 10). There were no differences between groups in fetal responses to ACTH challenge, but only UN fetuses demonstrated ACTH and 11-deoxycortisol responses to metyrapone (P < 0.05). UN fetuses had increased mRNA levels for proopiomelanocortin and prohormone convertase-1, but not -2, in the pars intermedia of the pituitary gland (P < 0.05). Glucocorticoid receptor mRNA levels were not different between groups in pituitary or hypothalamus. Maternal cortisol and ACTH levels during undernutrition were profoundly suppressed (P < 0.001), rather than elevated, in UN ewes. Furthermore, the normal pregnancy rise in maternal serum progesterone concentrations was delayed in undernourished mothers. These data demonstrate that events around the time of conception have profound effects on fetal hypothalamic-pituitary-adrenal development in late gestation and that factors other than fetal exposure to excess glucocorticoids may be important.

How to do an evaluation: pitfalls and traps.

The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used.

Periconceptional undernutrition alters growth trajectory and metabolic and endocrine responses to fasting in late-gestation fetal sheep.

Maternal undernutrition during pregnancy can lead to impaired metabolic regulation in postnatal offspring. The extent to which such abnormalities are determined in early gestation, and may already be present in fetal life, is unknown. We studied the effect of periconceptional undernutrition on fetal growth and metabolism in late gestation. Ewes were either well fed throughout pregnancy (N) or undernourished to reduce ewe weight by 15% from 61 d before until 30 d after mating (UN). At 121 d, one cohort of ewes and their singleton fetuses (N, n=10; UN, n=10) were fasted for 72 h, given 25 g of glucose i.v. over 8 h, and refed. Fetal and placental weights were not different at 131 d. UN ewes had higher glucose, amino nitrogen, and Hb concentrations than N ewes. UN fetuses had higher glucose and lactate, and lower amino nitrogen and O2 concentrations and pH. UN ewes had higher insulin and lower IGF-I concentrations, and UN ewes and fetuses had higher placental lactogen concentrations. A second cohort (N, n=8; UN, n=10) were studied until term. Chest girth increment was reduced in UN fetuses until delivery, but birth weights were not significantly reduced. These findings suggest that fetal growth, metabolic and endocrine status, and placental function in late gestation are influenced by maternal nutrition in the periconceptional period, independent of fetal size. Metabolic and endocrine adaptations in the mother may mediate some of these effects. Such adaptations may allow continued fetal growth and survival in the face of a potentially adverse nutritional environment.

Versatile and facile synthesis of diverse semisynthetic tetracycline derivatives via Pd-catalyzed reactions.

A diverse collection of tetracycline derivatives has been synthesized utilizing Heck, Suzuki, and other palladium-coupling reactions via tetracycline arenediazonium and iodoarene salts. Large numbers of tetracyclines are now possible via these reactions, including numerous upper periphery derivatives of doxycycline, minocycline, sancycline, and methacycline modified at positions C7, C9, and C6-C13 on the tetracycline naphthacene ring. Application of palladium-coupling reactions to the tetracyclines has yielded new tetracycline classes with differing structural attributes, greatly increasing the structural diversity of this family of antibiotics, one of the last of the early antibiotic families to be expanded by organic and medicinal chemistry.