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Background. In situ hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

The α-subunit of G proteins of the G12/13 family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of Gα12/13 to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction be- tween Gα13 and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of Gαi proteins. Using Gαi2, which does not bind RGL domains, as the backbone in which Gα13 sequences were swapped or mutated, we observed that the switch region of Gα13 is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surpris- ingly, the switch region of Gα13 was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of Gα13 and chimeric Gαi2 proteins was also necessary for Rho activation. These findings revealed the structural features by which Gα13 interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs. [ABSTRACT FROM AUTHOR]