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Background The development of a fast and accurate, non-sputum-based point-of-care triage test for tuberculosis (TB) would have a major impact on combating the TB burden worldwide. A new fingerstick blood test has been developed by Cepheid (the Xpert MTB Host Response [MTB-HR] prototype), which generates a "TB score" based on messenger RNA (mRNA) expression of 3 genes. Here we describe the first prospective findings of the MTB-HR prototype. Methods Fingerstick blood from adults presenting with symptoms compatible with TB in South Africa, The Gambia, Uganda, and Vietnam was analyzed using the Cepheid GeneXpert MTB-HR prototype. Accuracy of the Xpert MTB-HR cartridge was determined in relation to GeneXpert Ultra results and a composite microbiological score (GeneXpert Ultra and liquid culture) with patients classified as having TB or other respiratory diseases (ORD). Results When data from all sites (n = 75 TB, 120 ORD) were analyzed, the TB score discriminated between TB and ORD with an area under the curve (AUC) of 0.94 (95% confidence interval [CI],.91–.97), sensitivity of 87% (95% CI, 77–93%) and specificity of 94% (88–97%). When sensitivity was set at 90% for a triage test, specificity was 86% (95% CI, 75–97%). These results were not influenced by human immunodeficiency virus (HIV) status or geographical location. When evaluated against a composite microbiological score (n = 80 TB, 111 ORD), the TB score was able to discriminate between TB and ORD with an AUC of 0.88 (95% CI,.83–.94), 80% sensitivity (95% CI, 76–85%) and 94% specificity (95% CI, 91–96%). Conclusions Our interim data indicate the Cepheid MTB-HR cartridge reaches the minimal target product profile for a point of care triage test for TB using fingerstick blood, regardless of geographic area or HIV infection status. [ABSTRACT FROM AUTHOR]

Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10 , which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens. [ABSTRACT FROM AUTHOR]

The quality control of mRNA maturation is a highly regulated process that surveys pre-mRNA integrity and eliminates improperly matured pre-mRNAs. In nature, certain viruses regulate the expression of their genes b! hijacking the endogenouv RNA quality control machinerv We demonstrate that the inclusion of 5' splice sites within the 3'-untranslated region of a reporter gene in plants alters the pre-mRNA cleavage and polvadenvlation process, resulting in pre-mRNA degradation, esemplifying a regulatory mechanism conserved hetn'een kingdoms. Altered pre-mRNA processing nas associated with an inhibition of homologous gene expression in trans and the preferential accumulation of 24-nucleotide (nt) short-interfering R'sAs (siRNAs) as opposed to 21-nt siRNA subspecies, suggesting that degradation of the aberrant pre-mRNA involves the silencing machinery. However, gene expression was not restored by coexpression of a silencing suppressor or in an RNA-dependent RNA potymerase )RDR6)-deficient background despite reduced 24-nt siRNA accumutation. Our data highlight a complex cross talk between the quality control RNA machinery, 3'-end pre-mRNA maturation, and RN A-silencing pathways capable of discriminating among different type' of aberrant RN As. [ABSTRACT FROM AUTHOR]

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained .VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless 1], and β-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRY helper protein 2b may have an essential role in the host regulatory mechanisms that control TRY invasion. [ABSTRACT FROM AUTHOR]

The quality control of mRNA maturation is a highly regulated process that surveys pre-mRNA integrity and eliminates improperly matured pre-mRNAs. In nature, certain viruses regulate the expression of their genes b! hijacking the endogenouv RNA quality control machinerv We demonstrate that the inclusion of 5' splice sites within the 3'-untranslated region of a reporter gene in plants alters the pre-mRNA cleavage and polvadenvlation process, resulting in pre-mRNA degradation, esemplifying a regulatory mechanism conserved hetn'een kingdoms. Altered pre-mRNA processing nas associated with an inhibition of homologous gene expression in trans and the preferential accumulation of 24-nucleotide (nt) short-interfering R'sAs (siRNAs) as opposed to 21-nt siRNA subspecies, suggesting that degradation of the aberrant pre-mRNA involves the silencing machinery. However, gene expression was not restored by coexpression of a silencing suppressor or in an RNA-dependent RNA potymerase )RDR6)-deficient background despite reduced 24-nt siRNA accumutation. Our data highlight a complex cross talk between the quality control RNA machinery, 3'-end pre-mRNA maturation, and RN A-silencing pathways capable of discriminating among different type' of aberrant RN As. [ABSTRACT FROM AUTHOR]