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1.
Nat Commun ; 7: 10486, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26781218

RESUMO

Germline mutations are the source of evolution and contribute substantially to many health-related processes. Here we use whole-genome deep sequencing data from 693 parents-offspring trios to examine the de novo point mutations (DNMs) in the offspring. Our estimate for the mutation rate per base pair per generation is 1.05 × 10(-8), well within the range of previous studies. We show that maternal age has a small but significant correlation with the total number of DNMs in the offspring after controlling for paternal age (0.51 additional mutations per year, 95% CI: 0.29, 0.73), which was not detectable in the smaller and younger parental cohorts of earlier studies. Furthermore, while the total number of DNMs increases at a constant rate for paternal age, the contribution from the mother increases at an accelerated rate with age.These observations have implications related to the incidence of de novo mutations relating to maternal age.


Assuntos
Mutação em Linhagem Germinativa , Idade Materna , Adolescente , Adulto , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Idade Paterna , Adulto Jovem
2.
Mol Genet Genomic Med ; 2(6): 530-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25614875

RESUMO

Whole-genome sequencing and whole-exome sequencing are becoming more widely applied in clinical medicine to help diagnose rare genetic diseases. Identification of the underlying causative mutations by genome-wide sequencing is greatly facilitated by concurrent analysis of multiple family members, most often the mother-father-proband trio, using bioinformatics pipelines that filter genetic variants by mode of inheritance. However, current pipelines are limited to Mendelian inheritance patterns and do not specifically address disorders caused by mutations in imprinted genes, such as forms of Angelman syndrome and Beckwith-Wiedemann syndrome. Using publicly available tools, we implemented a genetic inheritance search mode to identify imprinted-gene mutations. Application of this search mode to whole-genome sequences from a family trio led to a diagnosis for a proband for whom extensive clinical testing and Mendelian inheritance-based sequence analysis were nondiagnostic. The condition in this patient, IMAGe syndrome, is likely caused by the heterozygous mutation c.832A>G (p.Lys278Glu) in the imprinted gene CDKN1C. The genotypes and disease status of six members of the family are consistent with maternal expression of the gene, and allele-biased expression was confirmed by RNA-Seq for the heterozygotes. This analysis demonstrates that an imprinted-gene search mode is a valuable addition to genome sequence analysis pipelines for identifying disease-causative variants.

3.
J Biol Chem ; 288(15): 10780-91, 2013 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-23322778

RESUMO

Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2-6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell.


Assuntos
Vacinas Bacterianas/imunologia , Células Epiteliais/imunologia , Francisella tularensis/imunologia , Regulação da Expressão Gênica/imunologia , Pinocitose/imunologia , Alvéolos Pulmonares/imunologia , Transcriptoma/imunologia , Tularemia/imunologia , Vacinas Bacterianas/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Francisella tularensis/metabolismo , Humanos , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/microbiologia , Alvéolos Pulmonares/patologia , Fatores de Tempo , Tularemia/metabolismo
4.
J Virol ; 86(10): 5647-59, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22419812

RESUMO

Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3' noncoding region (3'NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3'NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.


Assuntos
Doenças do Sistema Nervoso Central/virologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Infecções por Flavivirus/virologia , MicroRNAs/genética , Liberação de Vírus , Animais , Sequência de Bases , Vírus da Dengue/química , Vírus da Dengue/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/química , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Marcação de Genes , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fenótipo , Virulência , Replicação Viral
5.
RNA ; 16(8): 1679-85, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20584898

RESUMO

We previously showed the feasibility of using locked nucleic acid (LNA) for flow cytometric-fluorescence in situ hybridization (LNA flow-FISH) detection of a target cellular mRNA. Here we demonstrate how the method can be used to monitor viral RNA in infected cells. We compared the results of the LNA flow-FISH with other methods of quantifying virus replication, including the use of an enhanced green fluorescent protein (EGFP) viral construct and quantitative reverse-transcription polymerase chain reaction. We found that an LNA probe complementary to Sindbis virus RNA is able to track the increase in viral RNA over time in early infection. In addition, this method is comparable to the EGFP construct in sensitivity, with both peaking around 3 h and at the same level of infected cells. Finally, we observed that the LNA flow-FISH method responds to the decrease in levels of viral RNA caused by antiviral medication. This technique represents a straightforward way to monitor viral infection in cells and is easily applicable to any virus.


Assuntos
Citometria de Fluxo/métodos , Hibridização de Ácido Nucleico/métodos , RNA Viral/análise , Animais , Antivirais , Linhagem Celular , Células/química , Cricetinae , Hibridização de Ácido Nucleico/genética , Ácidos Nucleicos/genética , Fenômenos Físicos , RNA/genética , RNA Mensageiro/genética , RNA Viral/genética , Transcrição Gênica , Replicação Viral/genética
6.
J Inorg Biochem ; 104(5): 592-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20189656

RESUMO

Transition metal complexes [Co(cyclen)(NH(3))(2)](ClO(4))(3)H(2)O (cyclen=1,4,7,10-tetraazacyclododecane) (2), [Co(NH(3))(5)(OH(2))](CF(3)SO(3))(3) (3) [Ni(NH(3))(6)]Br(2) (4) and [Ru(NH(3))(6)]Cl(3) (5) were tested against Sindbis infected baby hamster kidney (BHK) cells and show differential effects from the previously reported anti-viral complex [Co(NH(3))(6)]Cl(3) (1). The macrocyclic complex 2 and labile aqua complex 3 show either no or little effect on the survival on Sindbis virus-infected cells as compared to that for 1, which show a monotonic increase in % BHK cell survival. Nickel and ruthenium ammine complexes 4 and 5 had a moderate influence of cell survival. While the results showed some anti-viral activity for some of the structural variations, it appears that 1, with its potential to be a broad-spectrum anti-viral compound, occupies a unique position in its ability to both significantly enhance cell survival and to decrease viral expression of infected cells. We also show that 1 also shows anti-viral activity against Adenovirus lending support to the broad-spectrum potential of this complex.


Assuntos
Aminas , Cobalto , Complexos de Coordenação , Níquel , Rutênio , Sindbis virus/efeitos dos fármacos , Aminas/química , Aminas/farmacologia , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/virologia , Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cricetinae , Cricetulus , Estrutura Molecular , Níquel/química , Níquel/farmacologia , Oxirredução , Rutênio/química , Rutênio/farmacologia , Elementos de Transição/química , Elementos de Transição/farmacologia
7.
Anal Biochem ; 390(2): 109-14, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19393610

RESUMO

We present a novel method using flow cytometry-fluorescence in situ hybridization (flow-FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. beta-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to beta-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the beta-actin LNA probe and a biotinylated, nonspecific control LNA were used to determine optimal conditions for this type of flow-FISH. Multiple conditions for permeabilization and hybridization were tested, and it was found that conditions using 3 microg/ml of proteinase K for permeabilization and 90 min hybridization at 60 degrees C with buffer containing 50% formamide allow cells containing the LNA-bound mRNA to be detected and differentiated from the control LNA with high confidence (< 14% overlap between curves). This combined method, called LNA flow-FISH, can be used for detection and quantification of other RNA species as well as for telomerase measurement and detection.


Assuntos
Citometria de Fluxo/métodos , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos/análise , RNA Mensageiro/análise , Actinas/genética , Linhagem Celular , Permeabilidade da Membrana Celular , Fluorescência , Formamidas , Humanos , Oligonucleotídeos/metabolismo , Sensibilidade e Especificidade , Temperatura , Tempo
8.
BMC Genomics ; 9: 577, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19046445

RESUMO

BACKGROUND: Febrile respiratory illness (FRI) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. A particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (HRV) and enteroviruses (HEV) which are frequent causes of FRI. Resequencing Pathogen Microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking. RESULTS: Using HRV and HEV as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. A minimal number of probe sequences (26 for HRV and 13 for HEV), which were potentially capable of detecting all serotypes of HRV and HEV, were determined and implemented on the Resequencing Pathogen Microarray RPM-Flu v.30/31 (Tessarae RPM-Flu). The specificities of designed probes were validated using 34 HRV and 28 HEV strains. All strains were successfully detected and identified at least to species level. 33 HRV strains and 16 HEV strains could be further differentiated to serotype level. CONCLUSION: This study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse RNA viruses with a minimal number of prototype sequences. The results demonstrated that the newly designed RPM-Flu v.30/31 can provide comprehensive and specific analysis of HRV and HEV samples which implicates that this design strategy will be applicable for other genetically diverse viruses.


Assuntos
Enterovirus/isolamento & purificação , Infecções Respiratórias/diagnóstico , Rhinovirus/isolamento & purificação , Sequência de Bases , Diagnóstico Diferencial , Enterovirus/genética , Humanos , Sondas RNA , Vírus de RNA , Infecções Respiratórias/virologia , Rhinovirus/genética
9.
Bioorg Med Chem ; 16(2): 830-7, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17967542

RESUMO

We have investigated the potential antiviral activity of three cobalt(III) compounds. Two compounds, Co(III)-cyclen-methylbenzoic acid and its methyl ester derivative, are based on the macrocyclic chelator, cyclen, and were synthesized in our laboratory. Both compounds have been shown to bind tightly to nucleic acids and to hydrolyze phosphodiester bonds. However, neither compound exhibited any significant antiviral activity in an in vitro model of Sindbis virus replication. In contrast, a third compound, Co(III)hexammine, significantly inhibited Sindbis virus replication in baby hamster kidney (BHK) cells in a dose- and time-dependent manner. In plaque assays, the incubation of Co(III)hexammine with Sindbis virus resulted in a dose-dependent decrease in virus replication when measured at both 24 and 48-h post-infection. Over the concentration range of 0-5mM Co(III)hexammine, the IC(50) for the inhibition of viral replication was determined to be 0.10+/-0.04mM at 48h. Additionally, when BHK cell monolayers were pretreated with Co(III)hexammine for 6h prior to Sindbis infection, optimal cellular morphology and plasma membrane integrity were observed at 0.6-1.2mM Co(III)hexammine. Analysis by flow cytometry confirmed that Co(III)hexammine mediated a concomitant dose-dependent increase in BHK cell viability and a decrease in the percentage of Sindbis virus-infected cells (IC(50)=0.13+/-0.04mM). Our findings demonstrate for the first time that Co(III)hexammine possesses potent antiviral activity. We discuss our findings within the context of the ability to further functionalize Co(III)hexammine to render it a highly specific antiviral therapeutic reagent.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Cobalto/química , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Sindbis virus/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/toxicidade , Cobalto/farmacologia , Cobalto/toxicidade , Cricetinae , Ciclamos , Relação Dose-Resposta a Droga , Compostos Heterocíclicos/química , Estrutura Molecular , Compostos Organometálicos/química , Compostos Organometálicos/toxicidade
10.
Mol Biosyst ; 3(9): 623-34, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17700863

RESUMO

In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Galpha protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media. The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed.


Assuntos
Carbono/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Glicólise , Hifas/citologia , Mutação/genética , Proteômica , Saccharomyces cerevisiae/genética , Análise por Conglomerados , Meios de Cultura , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Maltose/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo
11.
Genome Res ; 16(4): 527-35, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16481660

RESUMO

The exponential growth of pathogen nucleic acid sequences available in public domain databases has invited their direct use in pathogen detection, identification, and surveillance strategies. DNA microarray technology has offered the potential for the direct DNA sequence analysis of a broad spectrum of pathogens of interest. However, to achieve the practical attainment of this potential, numerous technical issues, especially nucleic acid amplification, probe specificity, and interpretation strategies of sequence detection, need to be addressed. In this report, we demonstrate an approach that combines the use of a custom-designed Affymetrix resequencing Respiratory Pathogen Microarray (RPM v.1) with methods for microbial nucleic acid enrichment, random nucleic acid amplification, and automated sequence similarity searching for broad-spectrum respiratory pathogen surveillance. Successful proof-of-concept experiments, utilizing clinical samples obtained from patients presenting adenovirus or influenza virus-induced febrile respiratory illness (FRI), demonstrate the ability of this approach for correct species- and strain-level identification with unambiguous statistical interpretation at clinically relevant sensitivity levels. Our results underscore the feasibility of using this approach to expedite the early surveillance of diseases, and provide new information on the incidence of multiple pathogens.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Infecções Respiratórias/genética , Análise de Sequência de DNA , Técnicas de Tipagem Bacteriana/métodos , Humanos , Técnicas de Tipagem Micológica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Infecções Respiratórias/diagnóstico , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
12.
RNA ; 11(5): 831-6, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15840822

RESUMO

Metal ion-chelator catalysts based on main-group, lanthanide, or transition metal complexes have been developed as nonenzymatic alternatives for the hydrolysis of the phosphodiester bonds in DNA and RNA. Cobalt (III), with its high-charge density, is known for its ability to hydrolyze phosphodiesters with rate constants as high as 2 x 10(-4) s(-1). We have developed a kinetically inert Co(III)-cyclen-based complex, Co(III)-cycmmb that is very potent in inhibiting the translation of RNA into protein. Contact time as short as 10 min is sufficient to achieve the complete inhibition of the translation of a concentrated luciferase RNA solution into the enzyme in a cell-free translation system. The inhibition appears to proceed through two pathways. The first pathway involves the kinetic or substitutional inertness of Co(III) for the RNA template at short contact times. This interaction is mediated through the kinetic inertness of Co(III) for the phosphate groups of the nucleotides, as well as coordination of Co(III) to the nitrogenous bases. The second pathway occurs at longer contact times and is mediated by the hydrolysis of the phosphodiester backbone. This report represents the first demonstrated use of a metal-chelate complex to achieve the inhibition of the translation of RNA into protein. This Co(III) system can be useful in its present nonsequence-specific form as a novel viral decontamination agent. When functionalized to recognize specific nucleic acid sequences, such a system could potentially be used in gene-silencing applications as an alternative to standard antisense or RNAi technologies.


Assuntos
Compostos Organometálicos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Hidrólise/efeitos dos fármacos , Luciferases/biossíntese , Luciferases/genética , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , RNA Mensageiro/genética , Fatores de Tempo
13.
J Immunol Methods ; 283(1-2): 269-79, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14659918

RESUMO

Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges-one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR, but has not been assessed at the transcriptome level on Affymetrix microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at -20 degrees C for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/sangue , Humanos
14.
Biosens Bioelectron ; 18(8): 1065-72, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12782470

RESUMO

Viruses form a significant class of bio-threat agents. Currently, the only method to determine the bioactivity of viruses in vitro is to measure viral and cellular responses after co-incubation of cells with virus. Our goal is to find biomarkers for classification of agents, establishment of bioactivity, and/or prediction of disease outcomes. To begin development of a cell-based biosensor for detection of bioactive Sindbis virus (SV), our model analyte, we surveyed the outcomes of SV interaction with primary rat neural precursor cells (NPC) and human peripheral blood mononuclear cells (PBMC). Confocal fluorescence analysis of NPC treated with recombinant SV carrying green-fluorescent-protein (SV-GFP) showed that most cells were GFP positive by day 1 post inoculation. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus showed nuclear condensation and fragmentation, and the percentage of TUNEL positive cells were higher in virus-treated cells than in mock-treated control. Also, there were less BrdU positive cells in virus-treated cells compared to control. Thus, SV infects NPC, decreases cellular proliferation, and induces cell death via apoptosis. PBMC were treated with SV- or UV-inactivated SV. By day 5 post infection, there were fewer adherent cells in SV-treated PBMC compared to UV-inactivated SV treated PBMC. However, the percentage of viable cells remained the same, and virus growth curves showed only clearance of virus. Thus, SV induces detachment of a subpopulation of PBMC while not killing most of the cells. Together, these results indicate that NPC and PBMC respond to bioactive SV inoculation, suggesting potential use as detectors of SV in cell-based biosensor paradigm. These studies also provide the rationale, time-scale, and phenotypic correlates for further studies with gene expression arrays.


Assuntos
Técnicas Biossensoriais/métodos , Leucócitos Mononucleares/virologia , Neurônios/virologia , Sindbis virus/isolamento & purificação , Sindbis virus/fisiologia , Células-Tronco/virologia , Animais , Apoptose , Técnicas Biossensoriais/instrumentação , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Estudos de Viabilidade , Humanos , Leucócitos Mononucleares/patologia , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Sindbis virus/patogenicidade , Sindbis virus/ultraestrutura , Células-Tronco/patologia , Replicação Viral
15.
J Virol Methods ; 109(2): 153-60, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12711058

RESUMO

The effects of extracellular matrix (ECM) components on the outcomes of alphavirus interaction with cells are not known. Studies that address such interactions have to address several methodological difficulties, including: the survival of the cells within the matrix; the passage of the virus through the matrix to infect embedded cells; and the dissociation of cells and matrix into single-cell suspension, before and after virus infection, for quantitative analysis. Herein, these issues were addressed in the context of a model system of collagen as the ECM component, baby hamster kidney (BHK) cells, and Sindbis virus. The outcomes of Sindbis virus infection of BHK cells, grown in three-dimensional (3D) collagen gel versus on plastic, and on two-dimensional (2D) collagen versus bovine serum albumin (BSA)-coated surfaces were compared. Cell morphology was more slender in 3D and on 2D collagen than on plastic or BSA-coated surfaces. The cells were able to survive in the 3D environment. Using Sindbis virus carrying the green fluorescent protein gene, the virions were found to be capable of penetrating the 3D collagen matrix and infecting the cells. There was more infectious virus in cultures of cells in 3D and on 2D collagen than on plastic or BSA-coated surfaces, respectively. Higher virus titers from cells on 2D collagen compared to BSA-coated surfaces was not associated with uninfected cell number or viability but with increased cell survival after infection. Infected cells on BSA surfaces became detached, while those on 2D collagen remained attached. These experiments establish procedures for analysis of interaction of collagen, BHK cells, and Sindbis virus and suggest that collagen increases infectious Sindbis virus titers from BHK cells by enhancing post-infection cell survival.


Assuntos
Colágeno/farmacologia , Sindbis virus/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Rim/citologia , Rim/virologia , Camundongos , Sindbis virus/crescimento & desenvolvimento , Replicação Viral/efeitos dos fármacos
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