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1.
Bioorg Med Chem Lett ; 20(8): 2493-6, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20304642

ABSTRACT

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure-activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.


Subject(s)
Ethanolamines/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Animals , Dose-Response Relationship, Drug , Drug Discovery , Drug Evaluation, Preclinical , Ethanolamines/chemistry , Humans , Potassium Channel Blockers/chemistry , Structure-Activity Relationship
2.
Nat Biotechnol ; 27(11): 1025-31, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19881494

ABSTRACT

Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across approximately 90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r(2) = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing.


Subject(s)
Microfluidics/methods , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Base Sequence , Humans , Mutation/genetics , Reproducibility of Results , Sequence Analysis, DNA/instrumentation
3.
PLoS One ; 4(7): e6342, 2009 Jul 22.
Article in English | MEDLINE | ID: mdl-19623263

ABSTRACT

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.


Subject(s)
Mass Spectrometry/methods , Orthopoxvirus/isolation & purification , Polymerase Chain Reaction/methods , Animals , Base Sequence , DNA Primers , DNA, Viral/genetics , Humans , Molecular Sequence Data , Orthopoxvirus/genetics , Rabbits , Sequence Homology, Nucleic Acid
4.
J Clin Microbiol ; 46(2): 644-51, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18094138

ABSTRACT

We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.


Subject(s)
Adenoviridae Infections/diagnosis , Adenoviridae Infections/virology , Adenoviridae/classification , Adenoviridae/isolation & purification , Environmental Microbiology , Polymerase Chain Reaction/methods , Spectrometry, Mass, Electrospray Ionization/methods , Adenoviridae/genetics , Chlamydiales , DNA Primers/genetics , Electronic Data Processing , Humans , Sensitivity and Specificity , Serotyping/methods
6.
PLoS One ; 2(5): e489, 2007 May 30.
Article in English | MEDLINE | ID: mdl-17534439

ABSTRACT

BACKGROUND: Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. METHODS AND PRINCIPAL FINDINGS: Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. CONCLUSION/SIGNIFICANCE: Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.


Subject(s)
Influenza A virus/genetics , Population Surveillance , Spectrometry, Mass, Electrospray Ionization/methods , Genotype , Influenza A virus/classification , Reverse Transcriptase Polymerase Chain Reaction
7.
J Med Chem ; 50(4): 807-19, 2007 Feb 22.
Article in English | MEDLINE | ID: mdl-17249648

ABSTRACT

The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease.


Subject(s)
Analgesics/chemical synthesis , Brain/metabolism , Pyrimidines/chemical synthesis , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Administration, Oral , Analgesics/pharmacokinetics , Analgesics/pharmacology , Animals , Antiparkinson Agents/chemical synthesis , Antiparkinson Agents/pharmacokinetics , Antiparkinson Agents/pharmacology , Biological Availability , Cell Line , Dogs , Female , Frontal Lobe/metabolism , Humans , Male , Pain Measurement , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Radioligand Assay , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship
8.
Proc Natl Acad Sci U S A ; 102(22): 8012-7, 2005 May 31.
Article in English | MEDLINE | ID: mdl-15911764

ABSTRACT

Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.


Subject(s)
Bacteria/genetics , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/microbiology , Sentinel Surveillance , Virulence Factors/genetics , Base Composition , California/epidemiology , Conserved Sequence/genetics , DNA Primers , Genetic Techniques , Genotype , Humans , Military Personnel , Species Specificity , Spectrometry, Mass, Electrospray Ionization
9.
J Biol Chem ; 279(29): 30440-8, 2004 Jul 16.
Article in English | MEDLINE | ID: mdl-15123614

ABSTRACT

During the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a nucleotide sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant.


Subject(s)
Bacterial Adhesion , Biological Transport , Carbohydrate Metabolism , Membrane Transport Proteins/physiology , Amino Acid Sequence , Animals , Caenorhabditis elegans , Cell Membrane/metabolism , Cloning, Molecular , DNA, Complementary/metabolism , Dogs , Dose-Response Relationship, Drug , Exons , Golgi Apparatus/metabolism , Green Fluorescent Proteins , Introns , Luminescent Proteins/metabolism , Membrane Transport Proteins/chemistry , Microscopy, Fluorescence , Models, Genetic , Molecular Sequence Data , Mutation , Phenotype , Plasmids/metabolism , Protein Structure, Tertiary , Ricin/pharmacology , Sequence Homology, Amino Acid , Transfection , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolism , Yersinia pseudotuberculosis
10.
Proc Natl Acad Sci U S A ; 100(26): 15706-11, 2003 Dec 23.
Article in English | MEDLINE | ID: mdl-14668445

ABSTRACT

The mouse meiotic mutant Mei1 was isolated in a screen for infertile mice descended from chemically mutagenized embryonic stem cells. Homozygotes of both sexes are sterile due to meiotic arrest caused by defects in chromosome synapsis. Notably, RAD51 protein does not load onto Mei1 mutant meiotic chromosomes, suggesting that there is a defect in either recombinational repair or the production of double-strand breaks (DSBs) that require such repair. Here, we show that treatment of mutant males with cisplatin restores RAD51 loading, suggesting that mutant spermatocytes have intact recombinational repair mechanisms. Levels of histone H2AX phosphorylation (gammaH2AX) at leptonema are significantly reduced compared with wild-type controls but comparable to that seen in animals deficient for SPO11, the molecule required for catalyzing DSB formation during meiosis. These observations provide evidence that genetically programmed DSB induction is defective in Mei1 leptotene spermatocytes. We also report the positional cloning of Mei1, which encodes a product without significant homology to any known protein. Expressed almost exclusively in gonads, Mei1 has no apparent homologs in yeast, worms, or flies. However, Mei1 orthologs are present in the genomes of mammals, chickens, and zebrafish. Thus, Mei1 is required for vertebrate meiosis. To our knowledge, Mei1 is the first meiosis-specific mutation identified by forward genetic approaches in mammals.


Subject(s)
Chromosomes/genetics , Meiosis/genetics , Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Cycle Proteins , Chickens , Chromosomes/ultrastructure , Cisplatin/pharmacology , Cloning, Molecular , Consensus Sequence , DNA Primers , Humans , Meiosis/drug effects , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Zebrafish
11.
Biol Reprod ; 69(5): 1615-25, 2003 Nov.
Article in English | MEDLINE | ID: mdl-12855593

ABSTRACT

The genetic control of mammalian gametogenesis is inadequately characterized because of a lack of mutations causing infertility. To further the discovery of genes required for mammalian gametogenesis, phenotype-driven screens were performed in mice using random chemical mutagenesis of whole animals and embryonic stem cells. Eleven initial mutations are reported here that affect proliferation of germ cells, meiosis, spermiogenesis, and spermiation. Nine of the mutations have been mapped genetically. These preliminary studies provide baselines for estimating the number of genes required for gametogenesis and offer guidance in conducting new genetic screens that will accelerate and optimize mutant discovery. This report demonstrates the efficacy and expediency of mutagenesis to identify new genes required for mammalian gamete development.


Subject(s)
Gametogenesis/genetics , Mutation/genetics , Reproduction/genetics , Animals , Chromosome Mapping , Ethylnitrosourea/pharmacology , Female , Genotype , Infertility/genetics , Male , Meiosis/genetics , Mice , Mutagenesis/drug effects , Mutagenicity Tests , Mutagens/pharmacology , Oocytes/physiology , Phenotype , Pregnancy
13.
Dev Biol ; 242(2): 174-87, 2002 Feb 15.
Article in English | MEDLINE | ID: mdl-11820814

ABSTRACT

mei1 (meiosis defective 1) is the first meiotic mutation in mice derived by phenotype-driven mutagenesis. It was isolated by using a novel technology in which embryonic stem (ES) cells were chemically mutagenized and used to generate families of mice that were screened for infertility. We report here that mei1/mei1 spermatocytes arrest at the zygotene stage of meiosis I, exhibiting failure of homologous chromosomes to properly synapse. Notably, RAD51 failed to associate with meiotic chromosomes in mutant spermatocytes, despite evidence for the presence of chromosomal breaks. Transcription of genes that are markers for the leptotene and zygotene stages, but not genes that are markers for the pachytene stage, was observed. mei1/mei1 females are sterile, and their oocytes also show severe synapsis defects. Nevertheless, unlike arrested spermatocytes, a small number of mutant oocytes proved capable of progressing to metaphase I and attempting the first meiotic division. However, their chromosomes were unpaired and were not organized properly at the metaphase plate or along the spindle fibers during segregation. mei1 was genetically mapped to chromosome (Chr) 15 in an interval that is syntenic to human Chr 22q13. This region, which has been completely sequenced, contains no known homologs of genes specifically required for meiosis in model organisms. Thus, mei1 may be a novel meiotic gene.


Subject(s)
Chromosome Pairing , Meiosis/genetics , Mutation , Sex Characteristics , Animals , Blotting, Northern , Fluorescent Antibody Technique , Male , Mice , Microscopy, Electron , Oocytes/cytology , Reverse Transcriptase Polymerase Chain Reaction , Spermatocytes/ultrastructure , Transcription, Genetic
14.
J Org Chem ; 61(14): 4725-4738, 1996 Jul 12.
Article in English | MEDLINE | ID: mdl-11667404

ABSTRACT

An investigation of the scope and mechanism of a new synthesis of cyclopentenes from 3,6-dihydro-2H-thiopyrans is described. Alkyl halides substituted with an electron-withdrawing group in the alpha-position were reacted with sodium thiosulfate, yielding the corresponding Bunte salts, which could be transformed to reactive thiocarbonyl compounds by elimination of the elements of bisulfite with mild base treatment. In situ trapping by 1,3-dienes afforded in good yields a variety of 3,6-dihydro-2H-thiopyrans substituted with electron-withdrawing groups at the 2-position. Exposure of these cycloadducts to strong base at low temperature effected a novel ring contraction, affording 2-(methylthio)-3-cyclopentenes after quenching with methyl iodide. The level of diastereoselectivity exhibited during the generation of these cyclopentenes was found to be dependent on the nature of the electron-withdrawing group at the 2-position of the dihydrothiopyran as well as the substitution pattern originally present in the diene component. In some cases, reducing the temperature during the ring contraction resulted in the isolation of good yields of vinyl cyclopropanes of high isomeric purity. With one substrate, highly diastereoselective rearrangement of a vinyl cyclopropane to a cyclopentene was unambiguously demonstrated, suggesting that this might be a key feature of the overall ring contraction mechanism.

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