Explore the vast range of titles available.

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.

Christopher Gordon, Cecilia Lo, Patrice Bouvagnet and colleagues report loss-of-function mutations in the MMP21 gene (encoding matrix metallopeptidase 21) that cause human heterotaxy with associated complex congenital heart defects. The authors confirm the role of MMP21 in heterotaxy and left-right patterning in zebrafish and mouse models. Heterotaxy results from a failure to establish normal left-right asymmetry early in embryonic development. By whole-exome sequencing, whole-genome sequencing and high-throughput cohort resequencing, we identified recessive mutations in MMP21 (encoding matrix metallopeptidase 21) in nine index cases with heterotaxy. In addition, Mmp21 -mutant mice and mmp21 -morphant zebrafish displayed heterotaxy and abnormal cardiac looping, respectively, suggesting a new role for extracellular matrix remodeling in the establishment of laterality in vertebrates.

Modeling is coming to the fore as it is now widely accepted and indeed expected during drug discovery and development. Modeling integrates knowledge, increases understanding and provides the ability to predict an outcome either before it occurs or when it is not possible to measure. This makes modeling an attractive option for inhaled drugs as it is not possible to routinely measure what is occurring to the drug (pharmacokinetics) and what effect the drug is having (pharmacodynamics) at local microscopic sites of such a diverse and complex organ as the lung. Many pieces of information (data and knowledge) exist like the pieces of a jigsaw puzzle and modeling brings the pieces together in a scientific and mechanistically coherent manner to increase understanding of both the efficacy and safety of inhaled drugs.

Physiologically based pharmacokinetic modelling is well established in the pharmaceutical industry and is accepted by regulatory agencies for the prediction of drug–drug interactions. However, physiologically based pharmacokinetic modelling is valuable to address a much wider range of pharmaceutical applications, and new regulatory impact is expected as its full power is leveraged. As one example, physiologically based pharmacokinetic modelling is already routinely used during drug discovery for in-vitro to in-vivo translation and pharmacokinetic modelling in preclinical species, and this leads to the application of verified models for first-in-human pharmacokinetic predictions. A consistent cross-industry strategy in this application area would increase confidence in the approach and facilitate further learning. With this in mind, this article aims to enhance a previously published first-in-human physiologically based pharmacokinetic model-building strategy. Based on the experience of scientists from multiple companies participating in the GastroPlus™ User Group Steering Committee, new Absorption, Distribution, Metabolism and Excretion knowledge is integrated and decision trees proposed for each essential component of a first-in-human prediction. We have reviewed many relevant scientific publications to identify new findings and highlight gaps that need to be addressed. Finally, four industry case studies for more challenging compounds illustrate and highlight key components of the strategy.

The gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation, or structural rearrangements. It was proposed that rs5758550, located 116 kb downstream of the gene locus, increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e., experimentally linking rs5758550, also referred to as the \"enhancer\" single-nucleotide polymorphism (SNP), to haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the \"enhancer\" SNP to interindividual variability in CYP2D6 activity. A large ethnically mixed population sample (n=3,162) was computationally phased to determine linkage between the \"enhancer\" SNP and haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept. Phasing predicted that the \"enhancer\" SNP can occur on numerous haplotypes including , and and suggested that linkage is incomplete, i.e., a portion of these alleles do not have the \"enhancer\" SNP. Phasing also revealed differences among the European and African ancestry data sets regarding the proportion of alleles with and without the \"enhancer\" SNP. DropPhase2D6 was utilized to confirm or refute the predicted \"enhancer\" SNP location for individual samples, e.g., of n=3 samples genotyped as , rs5758550 was on the allele of two samples and on the allele of one sample. Our findings highlight that the location of the \"enhancer\" SNP must not be assigned by \"default.\" Furthermore, linkage between the \"enhancer\" SNP and star allele haplotypes was confirmed with 10X Genomics technology. Since the \"enhancer\" SNP can be present on a portion of normal, decreased, or no function alleles, the phase of the \"enhancer\" SNP must be considered when investigating the impact of the \"enhancer\" SNP on CYP2D6 activity.

An important component of precision medicine—the use of whole-genome sequencing (WGS) to guide lifelong healthcare—is electronic decision support to inform drug choice and dosing. To achieve this, automated identification of genetic variation in genes involved in drug absorption, distribution, metabolism, excretion and response (ADMER) is required. CYP2D6 is a major enzyme for drug bioactivation and elimination. CYP2D6 activity is predominantly governed by genetic variation; however, it is technically arduous to haplotype. Not only is the nucleotide sequence of CYP2D6 highly polymorphic, but the locus also features diverse structural variations, including gene deletion, duplication, multiplication events and rearrangements with the nonfunctional, neighbouring CYP2D7 and CYP2D8 genes. We developed Constellation, a probabilistic scoring system, enabling automated ascertainment of CYP2D6 activity scores from 2×100 paired-end WGS. The consensus reference method included TaqMan genotyping assays, quantitative copy-number variation determination and Sanger sequencing. When compared with the consensus reference Constellation had an analytic sensitivity of 97% (59 of 61 diplotypes) and analytic specificity of 95% (116 of 122 haplotypes). All extreme phenotypes, i.e., poor and ultrarapid metabolisers were accurately identified by Constellation. Constellation is anticipated to be extensible to functional variation in all ADMER genes, and to be performed at marginal incremental financial and computational costs in the setting of diagnostic WGS. Whole genome sequencing: Perfecting genetic search engines Software developed by US researchers can identify important genetic variants within whole genome sequencing (WGS) data. As WGS becomes faster and more affordable, it can help doctors to choose the safest and most effective treatments for individual patients. However, this requires electronic tools that can search the genome and identify variants of the so-called ADMER genes involved in absorbing, distributing, metabolizing, excreting and responding to drugs. A team including Andrea Gaedigk and Greyson Twist at Children’s Mercy-Kansas City and the University of Missouri in Kansas City developed a new probabilistic scoring system called Constellation, which successfully identified variations of CYP2D6–an important enzyme involved in metabolizing common drugs–in WGS data from 61 patients. The researchers hope that Constellation can identify variations in other ADMER genes, enabling future uses of WGS for personalized precision medicine.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

[This corrects the article DOI: 10.1371/journal.pgen.1007394.].

While the cost of whole genome sequencing (WGS) is approaching the realm of routine medical tests, it remains too tardy to help guide the management of many acute medical conditions. Rapid WGS is imperative in light of growing evidence of its utility in acute care, such as in diagnosis of genetic diseases in very ill infants, and genotype-guided choice of chemotherapy at cancer relapse. In such situations, delayed, empiric, or phenotype-based clinical decisions may meet with substantial morbidity or mortality. We previously described a rapid WGS method, STATseq, with a sensitivity of >96 % for nucleotide variants that allowed a provisional diagnosis of a genetic disease in 50 h. Here improvements in sequencing run time, read alignment, and variant calling are described that enable 26-h time to provisional molecular diagnosis with >99.5 % sensitivity and specificity of genotypes. STATseq appears to be an appropriate strategy for acutely ill patients with potentially actionable genetic diseases.

Background and Objectives Physiologically based pharmacokinetic (PBPK) modelling has evolved to accommodate different routes of drug administration and enables prediction of drug concentrations in tissues as well as plasma. The inhalation route of administration has proven successful in treating respiratory diseases but can also be used for rapid systemic delivery, holding great promise for treatment of diseases requiring systemic exposure. The objective of this work was to develop a PBPK model that predicts plasma and tissue concentrations following inhalation administration of the PI3Kδ inhibitor nemiralisib. Methods A PBPK model was built in GastroPlus ® that includes a complete mechanistic description of pulmonary absorption, systemic distribution and oral absorption following inhalation administration of nemiralisib. The availability of clinical data obtained after intravenous, oral and inhalation administration enabled validation of the model with observed data and accurate assessment of pulmonary drug absorption. The PBPK model described in this study incorporates novel use of key parameters such as lung systemic absorption rate constants derived from human physiological lung blood flows, and implementation of the specific permeability-surface area product per millilitre of tissue cell volume (SpecPStc) to predict tissue distribution. Results The inhaled PBPK model was verified using plasma and bronchoalveolar lavage fluid concentration data obtained in human subjects. Prediction of tissue concentrations using the permeability-limited systemic disposition tissue model was further validated using tissue concentration data obtained in the rat following intravenous infusion administration to steady state. Conclusions Fully mechanistic inhaled PBPK models such as the model described herein could be applied for cross molecule assessments with respect to lung retention and systemic exposure, both in terms of pharmacology and toxicology, and may facilitate clinical indication selection.