RESUMO
Implementation of point-of-care tests may facilitate the health management of infectious diseases by reducing the timeframe on pathogen identification and host response measurements, allowing for immediate diagnosis and guided clinical intervention. In this feasibility study, a novel totally integrated and fully automated real-time polymerase chain reaction (PCR) platform (Idylla™, Biocartis) was assessed to determine the mRNA expression levels of multiple genes from 1 mL of whole blood. To this purpose, a sample-in result-out assay, including mRNA extraction and RT-qPCR-based detection, was ported to the platform. The genes used (matrix metallopeptidase 9, olfactomedin 4, NB1 glycoprotein and lipocalin 2) were previously identified as predictive for severity of disease caused by infection with respiratory syncytial virus (RSV). The reproducibility and robustness of the prototype assay was determined using the blood samples of 21 healthy donors. The data showed that the Idylla™ platform allows for a fast and user-friendly determination of the relative expression levels of the four selected mRNA markers.
Assuntos
Análise Química do Sangue/métodos , Perfilação da Expressão Gênica/métodos , Técnicas de Diagnóstico Molecular/métodos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Adulto , Automação Laboratorial/métodos , Estudos de Viabilidade , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Infecções por Vírus Respiratório Sincicial/patologia , Fatores de Tempo , Adulto JovemRESUMO
Linear regression modeling on a database of HIV-1 genotypes and phenotypes was applied to predict the HIV-1 resistance phenotype from the viral genotype. In this approach, the phenotypic measurement is estimated as the weighted sum of the effects of individual mutations. Higher order interaction terms (mutation pairs) were included to account for synergistic and antagonistic effects between mutations. The most significant mutations and interactions identified by the linear regression models for 17 approved antiretroviral drugs are reported. Although linear regression modeling is a statistical data-driven technique focused on obtaining the best possible prediction, many of these mutations are also known resistance-associated mutations, indicating that the statistical models largely reflect well characterized biological phenomena. The performance of the models in predicting in vitro susceptibility phenotype and virologic response in treated patients is described. In addition to a high concordance with in vitro measured fold change, which was the primary aim of model design, the models per drug show good predictivity of therapy response for regimens including that drug, even in the absence of other clinically relevant factors such as background regimen.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Modelos Lineares , Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral Múltipla , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Mutação , FenótipoRESUMO
The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.
Assuntos
Alcaligenes/classificação , Pseudomonas/classificação , Alcaligenes/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , Ácidos Graxos/análise , Genes Bacterianos/genética , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Hibridização de Ácido Nucleico , Fases de Leitura Aberta/genética , Oryza/microbiologia , Fenótipo , Filogenia , Pseudomonas/química , Pseudomonas/genética , RNA Ribossômico 16S/análiseRESUMO
Our approach to the isolation of plant-inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE-like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D-galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D-galactose, L-arabinose and D-fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.