Reciprocal antagonism between the netrin-1 receptor uncoordinated-phenotype-5A (UNC5A) and the hepatitis C virus.

Hepatitis C virus (HCV) infection is a leading cause of hepatocellular carcinoma (HCC), mainly through cirrhosis induction, spurring research for a deeper understanding of HCV versus host interactions in cirrhosis. The present study investigated crosstalks between HCV infection and UNC5A, a netrin-1 dependence receptor that is inactivated in cancer. UNC5A and HCV parameters were monitored in patients samples (n=550) as well as in in vitro. In patients, UNC5A mRNA expression is significantly decreased in clinical HCV(+) specimens irrespective of the viral genotype, but not in (HBV)(+) liver biopsies, as compared to uninfected samples. UNC5A mRNA is downregulated in F2 (3-fold; P=0.009), in F3 (10-fold, P=0.0004) and more dramatically so in F4/cirrhosis (44-fold; P<0.0001) histological stages of HCV(+) hepatic lesions compared to histologically matched HCV(-) tissues. UNC5A transcript was found strongly downregulated in HCC samples (33-fold; P<0.0001) as compared with non-HCC samples. In vivo, association of UNC5A transcripts with polyribosomes is decreased by 50% in HCV(+) livers. Consistent results were obtained in vitro showing HCV-dependent depletion of UNC5A in HCV-infected hepatocyte-like cells and in primary human hepatocytes. Using luciferase reporter constructs, HCV cumulatively decreased UNC5A transcription from the UNC5 promoter and translation in a UNC5A 5'UTR-dependent manner. Proximity ligation assays, kinase assays, as well as knockdown and forced expression experiments identified UNC5A as capable of impeding autophagy and promoting HCV restriction through specific impact on virion infectivity, in a cell death-independent and DAPK-related manner. In conclusion, while the UNC5A dependence receptor counteracts HCV persistence through regulation of autophagy in a DAPK-dependent manner, it is dramatically decreased in all instances in HCC samples, and specifically by HCV in cirrhosis. Such data argue for the evaluation of the implication of UNC5A in liver carcinogenesis.

RNA aptamer-mediated interference of HCV replication by targeting the CRE-5BSL3.2 domain.

The RNA genome of hepatitis C virus (HCV) contains multiple conserved structural RNA domains that play key roles in essential viral processes. A conserved structural component within the 3' end of the region coding for viral RNA-dependent RNA polymerase (NS5B) has been characterized as a functional cis-acting replication element (CRE). This study reports the ability of two RNA aptamers, P-58 and P-78, to interfere with HCV replication by targeting the essential 5BSL3.2 domain within this CRE. Structure-probing assays showed the binding of the aptamers to the CRE results in a structural reorganization of the apical portion of the 5BSL3.2 stem-loop domain. This interfered with the binding of the NS5B protein to the CRE and induced a significant reduction in HCV replication (≈50%) in an autonomous subgenomic HCV replication system. These results highlight the potential of this CRE as a target for the development of anti-HCV therapies and underscore the potential of antiviral agents based on RNA aptamer molecules.

Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain.

Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363-10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function. Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363-10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of either of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363-10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles. The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants.

Targets and tools: recent advances in the development of anti-HCV nucleic acids.

Hepatitis C virus (HCV), the major etiological agent of transfusion-associated non-A, non-B hepatitis, is a severe health problem affecting up to 3% of the world population. Since its identification in 1989, enormous efforts have been made to characterize the viral cycle. However, many details regarding the virus' penetration of hepatocytes, its replication and translation, and the assembling of virions remain unknown, mostly because of a lack of an efficient culture system. This has also hampered the development of fully effective antiviral drugs. Current treatments based on the combination of interferon and ribavirin trigger a sustained virological response in only 40% of infected individuals, thus the development of alternative therapeutic strategies is a major research goal. Nucleic acid based therapeutic agents may be of some potential in hepatitis C treatment. In recent years, much effort has gone into the improvement of DNA and RNA molecules as specific gene silencing tools. This review summarizes the state of the art in the development of new HCV therapies, paying special attention to those involving antisense oligonucleotides, aptamers, ribozymes, decoys and siRNA inhibitors. The identification of potential viral targets is also discussed.

Comparison of urease gene primer sequences for PCR-based amplification assays in identifying the gastric pathogen Helicobacter pylori.

Different genomic DNA samples and primer sequences were evaluated in urease (ure) gene-based PCR assays for rapid identification of Helicobacter pylori. Purified DNA and heated (boiled) cell lysates of bacterial cultures from gastric biopsies were tested with three primer sets for unique internal ureA, ureA+B and ureC sequences. The heated-lysates of H. pylori were quick to prepare but more frequently gave unexpected variable or negative PCR results than assays performed on purified DNA, which were highly specific and reproducible for all three primer sets. Results indicated that sensitivity of the assay was linked to the size of the amplified target region rather than any particular strain feature, with the small 294 bp ureC product providing more accurate assays with heated-lysates of H. pylori. We strongly recommend that negative results in any PCR assay should be checked on purified DNA to exclude the possibility of a false-negative result.

[Validity of a scale of vital prognosis in multiorgan failure syndrome: experience at a pediatric ICU]. / Validez de una escala de pronóstico vital en el síndrome de falla multiorgánica: experiencia de una UCI pediátrica.

In order to evaluate the utility of modified score index for multiple organ failure syndrome (MOFS) detection, we studied 31 critically ill pediatric patients at University Hospital of Puebla. They had a critical illness and they were successfully animated and developed two or more organ failure other than initial lesion. Were calculated sensibility, specificity, positive predictive value and negative predictive value of clinical and biochemical criterion of modified score index and results were few consistent. Were assessed that scale for know its predictive value and results were significant: sensitivity 81%, specificity 30% and accuracy, 54.5%. Finally we recommended caution to use anyone score system in evaluating MOFS.