RESUMEN
BACKGROUND: The Candida glabrata does not develop into a pathogenic hiphal form; however, it has become the second most common pathogen of fungal infections in humans, partly because of its adhesion ability and virulence. OBJECTIVES: The present study aimed to determine whether Flo8, a transcription factor that plays an important role in the virulence and drug resistance in Candida albicans, has a similar role in C. glabrata. METHODS: We constructed FLO8 null strains of a C. glabrata standard strain and eight clinical strains from different sources, and a FLO8 complemented strain. Real-time quantitative PCR, biofilm formation assays, hydrophobicity tests, adhesion tests, Caenorhabditis elegans survival assay, and drug-susceptibility were then performed. RESULTS: Compared with the wild-type strains, the biofilm formation, hydrophobicity, adhesion, and virulence of the FLO8-deficient strains decreased, accompanied by decreased expression of EPA1, EPA6, and EPA7. On the other hand, it showed no changes in antifungal drug resistance, although the expression levels of CDR1, CDR2, and SNQ2 increased after FLO8 deletion. CONCLUSIONS: These results indicated that Flo8 is involved in the adhesion and virulence of C. glabrata, with FLO8 deletion leading to decreased expression of EPA1, EPA6, and EPA7 and decreased biofilm formation, hydrophobicity, adhesion, and virulence.
Asunto(s)
Candida glabrata , Proteínas Fúngicas , Antifúngicos/farmacología , Biopelículas , Candida albicans/metabolismo , Candida glabrata/genética , Candida glabrata/metabolismo , Farmacorresistencia Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , VirulenciaRESUMEN
BACKGROUND: Neonatal necrotizing enterocolitis is a common and often fatal gastrointestinal disease, especially in premature infants. To study potential mechanisms underlying the protective effect of breast milk on neonatal necrotizing enterocolitis, we induced intestinal inflammation in a Caco-2 cell model of neonatal necrotizing enterocolitis by hypoxia/re-oxygenation to investigate whether breast milk supernatant fluid inhibited the expression of proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-α. METHODS: Caco-2 cells were divided into normal (control) and neonatal necrotizing enterocolitis groups. Neonatal necrotizing enterocolitis was mimicked by exposing Caco-2 cells to hypoxia/re-oxygenation. Cells were independently maintained in minimal essential medium alone, minimal essential medium containing 5% breast milk supernatant, or 5% boiled breast milk supernatant. Production of interleukin-1ß, interleukin-6, and tumor necrosis factor-α was investigated in cell culture supernatants by ELISA, reverse transcription polymerase chain reaction, and immunofluorescence. RESULTS: Hypoxia/re-oxygenation significantly increased the expression of interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In the normal group, breast milk supernatant and boiled breast milk supernatant markedly downregulated the expression of interleukin-1ß, interleukin-6, and tumor necrosis factor-α when compared with the minimal essential medium group, with the reduction in inter-leukin-1ß expression being more pronounced in the breast milk group. In Caco-2 cells undergoing hypoxia/re-oxygenation, both breast milk supernatant and boiled breast milk supernatant significantly reduced the expression of interleukin-1ß, interleukin-6, and tumor necrosis factor-α, where the decrease in interleukin-1ß expression was greater in the breast milk group. CONCLUSIONS: Breast milk supernatant fluid inhibited the expression of proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-α in Caco-2 cells, especially after hypoxia/re-oxygenation. This may be one of the mechanisms underlying the protective effect of breast milk on neonatal necrotizing enterocolitis.
Asunto(s)
Citocinas/metabolismo , Enterocolitis Necrotizante/prevención & control , Mediadores de Inflamación/metabolismo , Leche Humana/metabolismo , Células CACO-2 , Culinaria , Regulación hacia Abajo/genética , Enterocolitis Necrotizante/fisiopatología , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Hipoxia , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Oxígeno/administración & dosificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The analysis of molecular phylogenies estimated from the gene sequences of sampled viruses can provide important insights into epidemiological processes. METHODS: The demographic and migration histories of the prevalent hepatitis C virus (HCV) subtypes 1a and 1b were inferred from viral gene sequences sampled in 5 countries. Estimated viral phylogenies were analyzed by use of methods based on parsimony and coalescent theory. RESULTS: The parsimony migration analysis suggested that the global subtype 1a and 1b epidemics are geographically structured, with asymmetrical movement of HCV strains among the sampled countries. The coalescent analysis indicated that subtype 1a infections in the United States, Brazil, and Indonesia began to increase exponentially during the 1940s and 1950s, whereas in Vietnam the increase began after the 1970s. In contrast, subtype 1b infections in these 4 countries and in Japan began to increase exponentially between 1880 and 1920, with a possible recent decrease in infection rates in Indonesia and Japan. In the United States, Brazil, and Vietnam, the epidemic growth rates for subtype 1a strains were higher than those for subtype 1b strains, whereas the growth rates were similar in Indonesia. CONCLUSIONS: The estimated histories of migration and population growth indicated that patterns of HCV transmission differ among countries and viral subtypes.
Asunto(s)
Genes Virales , Hepacivirus/genética , Hepatitis C/epidemiología , Hepatitis C/virología , ARN Viral/genética , Brasil/epidemiología , Genotipo , Hepacivirus/clasificación , Hepacivirus/aislamiento & purificación , Humanos , Indonesia/epidemiología , Japón/epidemiología , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , Prevalencia , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ADN , Estados Unidos/epidemiología , Vietnam/epidemiologíaRESUMEN
A limited number of hepatitis A virus (HAV) isolates from South America have been characterised at the genomic level. IgM anti-HAV positive serum samples collected from patients with hepatitis A living in the five geographical regions of Brazil (North, Northeast, Central, South, and Southeast) were used to obtain HAV isolates and determine their genetic relatedness. Of the 232 case isolates, sequence data were obtained from the VP1/2A junction region of the HAV genome. All isolates were classified in genotype I; 231 belonged to subgenotype IA, and one to subgenotype IB. HAV isolates from four States formed distinct clusters of highly related sequences. However, isolates from other states did not cluster and the sequences from those states were intermingled with sequences found in the other states. The amino acid sequences of all but two isolates showed a Leu --> Ile substitution at position 42 in the 2A protein. This substitution appeared to be a characteristic geographic fingerprint of HAV sequences within Brazil.
Asunto(s)
Genoma Viral , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/aislamiento & purificación , Hepatitis A/virología , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos , Brasil , Niño , Preescolar , Femenino , Genotipo , Virus de la Hepatitis A/clasificación , Humanos , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , ARN Viral/sangre , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genéticaRESUMEN
The level of in vitro detection of viral genomes in mixes with two different hepatitis C virus (HCV) subtypes was investigated by artificially mixing previously measured subtype-specific HCV RNA genomes. The RNAs in these mixtures were reverse transcribed and then PCR amplified by using two sets of primers corresponding to the 5' untranslated region and digested with endonucleases to analyze the restriction fragment length polymorphism patterns. This approach facilitated detection of a wider range of type-specific HCV genomes than originally described, beyond equimolar concentrations of contributing HCV subtypes. Moreover, by using computerized image analysis, this study also demonstrated that the true contribution of each virus type-and consequently of mixed infections-may be underestimated when only visual observation is carried out. These results may be useful for comparing data obtained from this and other currently used methodologies.
Asunto(s)
Genoma Viral , Hepacivirus/clasificación , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/virología , Polimorfismo de Longitud del Fragmento de Restricción , ARN Viral/sangre , Genotipo , Hepacivirus/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The complete genome sequences of hepatitis B virus (HBV) from 12 HBV-infected Yucpa Indians of Venezuela, a group with highly endemic HBV, were amplified and sequenced. The 12 isolates were closely related to each other, with 98.6-100% nucleotide identity. A phylogenetic tree based on the complete genome indicated clearly that they were genotype F. Three individuals had evidence of infection with two different HBV deletion mutants. In two individuals, a three amino acid deletion was identified just prior to the 'a' determinant loop of the S region. A third individual was infected with virus that contained a complete core reading frame and a population that contained a deletion in the middle of the core region. These results indicate that genotype F HBV is present in the Venezuelan Yucpa Amerindians and the complete genome sequence allowed the identification of two unique deletion mutants in a limited set of samples.