As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.
Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non–small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8–3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.
Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.
Many Microbe Microarrays Database (M3D) is designed to facilitate the analysis and visualization of expression data in compendia compiled from multiple laboratories. M3D contains over a thousand Affymetrix microarrays for Escherichia coli, Saccharomyces cerevisiae and Shewanella oneidensis. The expression data is uniformly normalized to make the data generated by different laboratories and researchers more comparable. To facilitate computational analyses, M3D provides raw data (CEL file) and normalized data downloads of each compendium. In addition, web-based construction, visualization and download of custom datasets are provided to facilitate efficient interrogation of the compendium for more focused analyses. The experimental condition metadata in M3D is human curated with each chemical and growth attribute stored as a structured and computable set of experimental features with consistent naming conventions and units. All versions of the normalized compendia constructed for each species are maintained and accessible in perpetuity to facilitate the future interpretation and comparison of results published on M3D data. M3D is accessible at http://m3d.bu.edu/.
An anionic amphiphilic dendrimer is reported that possesses increased cytotoxicological potency against prokaryotic cells compared to eukaryotic cells. The half maximal effective concentration (EC 50 ) for the dendrimer against Bacillus subtilis, a Gram-positive bacterial strain, was measured to be 4.1×10 −5 M, while that against human umbilical vein endothelial cells (HUVEC) was more than 36x greater at a value of 1.5×10 −3 M. EC 50 ratios for two commercial amphiphiles, sodium dodecyl sulfate (SDS) and Triton X-100, in addition to a similar synthesized dendritic structure were at most only 3.8x greater. Furthermore, the observed EC 50 values appear to be correlated to the critical aggregation constant (CAC) in solution suggesting a mechanism of action for these anionic amphiphilic dendrimers related to their supramolecular structures.Dendritic macromolecules, due to their structure, unique properties, and precise compositions, are of significant interest 1 and are finding uses in an ever-increasing number of medical applications. 2 This is especially evident in the drug delivery area where the dendritic structure enables the attachment of a multitude of drugs or targeting moieties as well as the opportunity to control pharmacokinetics through alterations in generation number. 3 Our interest lies in the synthesis and evaluation of dendritic macromolecules composed of building blocks that are natural metabolites or known to be biocompatible for ocular tissue repair, 4 cartilage tissue engineering, 5 and drug delivery. 6 In an ongoing effort to expand the biomedical applications of dendrimers and our understandings of the resulting structure-activity relationships, we are investigating anionic dendritic macromolecules as antibacterial agents. Herein, we report the antibacterial activity of an anionic amphiphilic dendrimer and the striking selectivity in its E-mail: mgrin@bu.edu. There is a significant global need for new antibacterials and alternative mechanisms of action given the rise in resistance among bacteria. 7 Of the various known antibacterial agent classes, amphiphilic compounds act through perturbation and disruption of the prokaryotic membrane. 8 We hypothesized that amphiphilic anionic dendrimers may exhibit antibacterial activity with minimal eukaryotic cell cytotoxicity, since dendrimers with terminal anionic charges are generally non-cytotoxic and have low toxicity in zebrafish whole animal development studies. 9 On the other hand, cationic dendrimers, some of which have antibacterial properties if the positive charge is properly shielded, 10 have repeatedly shown cytotoxicity against a variety of eukaryotic cell lines. 3e,11 In addition, there are many reports of linear polycationic agents but only a few descriptions of linear polyanionic antibacterial agents (e.g., sulfonated polystyrene). 12 Consequently, we synthesized a series of surface-block anionic amphiphilic dendrimers composed of succinic acid, glycerol, and myristic acid possessing various numbers of acid and alkyl functionali...
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