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1.
J Stud Alcohol Drugs ; 79(4): 553-560, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30079870

RESUMO

OBJECTIVE: Infants with neonatal abstinence syndrome have significant morbidity, but the association with birth defects is poorly understood. This study aimed to determine how neonatal abstinence syndrome is related to birth defects, including the joint impact of neonatal abstinence syndrome and birth defects on infant morbidity. METHOD: A population-based cohort of 1,944,804 neonates born in the hospitals of Quebec, Canada (1989-2013), was compiled with data on maternal exposures and infant outcomes after delivery. The prevalence of neonatal abstinence syndrome and birth defects was estimated, including the association with neonatal morbidity and mortality after adjustment for maternal age, parity, morbidity, socioeconomic deprivation, and time period. Joint effects of neonatal abstinence syndrome and birth defects on infant outcomes were assessed. RESULTS: Infants with neonatal abtinence syndrome had a higher prevalence of birth defects (705.7 per 10,000) than infants with no drug exposure (568.9 per 10,000). Compared with no exposure, infants with neonatal abstinence syndrome had 1.35 times the chance of having birth defects (95% confidence interval [1.14, 1.59]). Associations were strongest for central nervous system defects (risk ratio = 6.06, 95% confidence interval [3.93, 9.35]). Neonatal abstinence syndrome combined with birth defects was associated with significantly more infant morbidity and mortality. CONCLUSIONS: Infants with neonatal abstinence syndrome have a greater prevalence of birth defects, particularly defects of the central nervous system. Neonatal abstinence syndrome with birth defects may be an underappreciated contributor to infant morbidity.


Assuntos
Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/epidemiologia , Síndrome de Abstinência Neonatal/diagnóstico , Síndrome de Abstinência Neonatal/epidemiologia , Estudos de Coortes , Feminino , Humanos , Lactente , Mortalidade Infantil/tendências , Recém-Nascido , Morbidade/tendências , Gravidez , Prevalência , Quebeque/epidemiologia , Fatores de Risco
2.
Cell Rep ; 12(5): 850-63, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26212330

RESUMO

Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs) at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.


Assuntos
Vírus da Dengue/fisiologia , Endorribonucleases/metabolismo , Vírus da Influenza A/fisiologia , Rhinovirus/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Replicação Viral/fisiologia , Endocitose/fisiologia , Endorribonucleases/genética , Células HeLa , Humanos , ATPases Vacuolares Próton-Translocadoras/genética
3.
Virology ; 444(1-2): 140-7, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23849792

RESUMO

Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/imunologia , Anticorpos Monoclonais/imunologia , Células Epiteliais/virologia , Feminino , Humanos , Gravidez , Ensaio de Placa Viral , Internalização do Vírus/efeitos dos fármacos
4.
Bioorg Med Chem Lett ; 23(13): 3841-7, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23726345

RESUMO

We describe here the design, synthesis and biological evaluation of antiviral compounds acting against human rhinovirus (HRV). A series of aminothiazoles demonstrated pan-activity against the HRV genotypes screened and productive structure-activity relationships. A comprehensive investigational library was designed and performed allowing the identification of potent compounds with lower molecular weight and improved ADME profile. 31d-1, 31d-2, 31f showed good exposures in CD-1 mice. The mechanism of action was discovered to be a host target: the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). The identification of the pan-HRV active compound 31f combined with a structurally distinct literature compound T-00127-HEV1 allowed the assessment of target related tolerability of inhibiting this kinase for a short period of time in order to prevent HRV replication.


Assuntos
Antivirais/farmacologia , Desenho de Fármacos , Rhinovirus/efeitos dos fármacos , Tiazóis/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química
5.
Antimicrob Agents Chemother ; 57(7): 3358-68, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23650168

RESUMO

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). A good correlation between PI4KIIIß activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIß inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIß inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIß were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIß is deleterious.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Cefalosporinas/farmacologia , Rhinovirus/efeitos dos fármacos , Rhinovirus/enzimologia , Tiazóis/farmacologia , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Antivirais/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Resfriado Comum/tratamento farmacológico , Resfriado Comum/virologia , Feminino , Células HeLa , Humanos , Camundongos , Oximas , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA Interferente Pequeno , Rhinovirus/crescimento & desenvolvimento , Sulfonamidas , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
6.
J Biomol Screen ; 16(3): 363-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21343600

RESUMO

The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Canais Iônicos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismo , Cromatografia Líquida , Humanos , Canais Iônicos/genética , Canais Iônicos/isolamento & purificação , Lipossomos/química , Lipossomos/metabolismo , Meliteno/metabolismo , Permeabilidade , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade , Bibliotecas de Moléculas Pequenas , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
7.
J Virol Methods ; 171(1): 169-75, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21034775

RESUMO

Many anti-HCV antibodies are available, but more are needed for research and clinical applications. This study examines whether ascitic fluid from cirrhotic patients could be a source of reagent-grade antibodies. Ascitic fluid from 29 HCV patients was screened by ELISA for anti-HCV antibodies against three viral proteins: core, NS4B, and NS5A. Significant patient-to-patient variability in anti-HCV antibody titers was observed. Total ascitic fluid IgG purified by Protein-A chromatography reacted with HCV proteins in immunoblots, cell extracts, and replicon-expressing cells. Affinity-purification using synthetic peptides as bait allowed the preparation of cross-genotypic antibodies directed against pre-selected regions of HCV core, NS4B, and NS5A proteins. The performance of the polyclonal antibodies was comparable to that of monoclonal antibodies. Anti-NS4B antibody preparations reacted with genotype 1a, 1b, and 2a NS4B proteins in immunoblots and allowed NS4B to be localized in replicon-expressing cells. Ascitic fluid is an abundant source of human polyclonal cross-genotypic antibodies that can be used as an alternative to blood. This study shows the utility of selectively purifying human polyclonal antibodies from ascitic fluid. Affinity purification allows antibodies to be selected that are comparable to monoclonal antibodies in their ability to react with targeted regions of viral proteins.


Assuntos
Líquido Ascítico/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/análise , Hepatite C/imunologia , Idoso , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Anticorpos Anti-Hepatite C/isolamento & purificação , Humanos , Imunoglobulina G/análise , Masculino , Pessoa de Meia-Idade , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/imunologia
8.
J Nat Prod ; 69(2): 173-7, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16499312

RESUMO

A screening program aimed at discovering inhibitors of the bacterial type III secretion system identified the MeOH extract of the Caribbean sponge Caminus sphaeroconia as an active hit in the initial assay. Bioassay-guided fractionation of the crude extract led to the isolation of caminosides A (1) to D (4), a family of antimicrobial glycolipids. The structures of the three new caminosides B (2) to D (4) have been elucidated by spectroscopic analysis.


Assuntos
Antibacterianos , Glicolipídeos , Poríferos/química , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Região do Caribe , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Glicolipídeos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Plantas/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
9.
Antimicrob Agents Chemother ; 49(10): 4101-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16189086

RESUMO

The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by enteropathogenic Escherichia coli (EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC.


Assuntos
Escherichia coli/genética , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Fatores de Virulência/antagonistas & inibidores , Movimento Celular/genética , Movimento Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Genes Bacterianos , Virulência/genética , Fatores de Virulência/genética
10.
J Bacteriol ; 185(23): 6747-55, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617638

RESUMO

Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones. It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus. The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system. We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC). Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN. We showed by affinity chromatography that EscN and Tir bind CesT specifically. Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT. Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC. These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Superfície Celular/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Ligação Proteica , Transporte Proteico , Especificidade da Espécie
12.
Infect Immun ; 71(6): 3310-9, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12761113

RESUMO

At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm. EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.


Assuntos
Proteínas de Bactérias/fisiologia , Escherichia coli/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Citoplasma/metabolismo , Proteínas de Escherichia coli/metabolismo , Transporte Proteico , Receptores de Superfície Celular/metabolismo
13.
Org Lett ; 4(23): 4089-92, 2002 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-12423093

RESUMO

Extracts of the marine sponge Caminus sphaeroconia showed potent activity in a screen for bacterial type III secretion inhibitors. Bioassay guided fractionation of the extract led to the isolation of the novel antimicrobial glycolipid caminoside A (1). The structure of caminoside A was elucidated by analysis of spectroscopic data and chemical degradation.[structure: see text]


Assuntos
Anti-Infecciosos/isolamento & purificação , Ciclobutanos/síntese química , Glicolipídeos/isolamento & purificação , Poríferos/química , Sulfonamidas/síntese química , Animais , Anti-Infecciosos/química , Configuração de Carboidratos , Ciclobutanos/química , Glucose/química , Glicolipídeos/química , Modelos Moleculares , Estrutura Molecular , Sulfonamidas/química
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