TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Entecavir vs. lamivudine in chronic hepatitis B patients with severe acute exacerbation and hepatic decompensation.

BACKGROUND & AIMS: We compared the mortality and treatment response between lamivudine (LAM) and entecavir (ETV) in chronic hepatitis B (CHB) patients with severe acute exacerbation and hepatic decompensation. METHODS: From 2003 to 2010 (the LAM group) and 2008 to 2010 (the ETV group), 215 and 107 consecutive CHB naïve patients with severe acute exacerbation and hepatic decompensation treated with LAM and ETV respectively, were recruited. RESULTS: At baseline, the LAM group had higher AST levels and end-stage liver disease (MELD) scores, and lower albumin levels than the ETV group. Univariate analysis showed that the LAM group had a higher rate of overall (p=0.02) and liver-related mortality (p=0.052) at week 24 than the ETV group, including in patients with acute-on-chronic liver failure. Multivariate analysis showed that MELD scores, ascites, and hepatic encephalopathy were independent factors for overall and liver-related mortality at week 24. ETV or LAM treatment was not an independent factor for mortality in all patients or patients with acute-on-chronic liver failure. The best cut-off value of MELD scores were 24 for 24-week liver-related mortality. The ETV group achieved better virological response (HBV DNA <300 copies/ml) than the LAM group at week 24 (p=0.043) and 48 (p=0.007). The T1753C/A mutation was also an independent predictor associated with overall and liver-related mortality at week 24. CONCLUSIONS: The choice between ETV and LAM was not an independent factor for mortality in CHB patients with acute exacerbation and hepatic decompensation. Patients with ascites, hepatic encephalopathy, and MELD scores ⩾24 were associated with poor outcome and should be considered for liver transplantation.

A novel mutation in the connexin 29 gene may contribute to nonsyndromic hearing loss.

Connexins (Cxs) are homologous four-transmembrane domain proteins and constitute the major components of gap junctions. Among a cohort of patients with nonsyndromic hearing loss, we recently identified a novel missense mutation, E269D, in the GJC3 gene encoding connexin 29 (Cx29), as being causally related to hearing loss. The functional alteration of Cx29 caused by the mutant GJC3 gene, however, remains unknown. This study compared the intracellular distribution and assembly of mutant Cx29 (Cx29E269D) with that of the wild-type Cx29 (Cx29WT) in HeLa cells and the effect the mutant protein had on those cells. Cx29TW showed continuous staining along apposed cell membranes in the fluorescent localization assay. In contrast, the p.E269D missense mutation resulted in accumulation of the Cx29 mutant protein in the endoplasmic reticulum (ER) rather than in the cytoplasmic membrane. Co-expression of Cx29WT and Cx29E269D proteins by a bi-directional tet-on expression system demonstrated that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the formation of the gap junction. Based on these findings, we suggest that Cx29E269D has a dominant negative effect on the formation and function of the gap junction. These results provide a novel molecular explanation for the role Cx29 plays in the development of hearing loss.