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Mutations in mitochondrial DNA (mtDNA) have been associated with deafness and their pathophysiology remains poorly understood. In this study, we investigated the pathogenic mechanism of deafness-associated 7505A > G variant in the mitochondrial tRNASer(UCN). The m.7505A > G variant affected the highly conserved adenine at position 11 (A11), disrupted the highly conserved A11-U24 base-pairing of DHU stem of tRNASer(UCN) and introduced a tertiary base pairing (G11-C56) with the C56 in the TΨC loop. We therefore hypothesized that the m.7505A > G variant altered both structure and function of tRNASer(UCN). We demonstrated that the m.7505A > G variant perturbed the conformation and stability of tRNASer(UCN), as indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wild type molecule. Using the cybrids constructed by transferring mitochondria from the Chinese family into mitochondrial DNA (mtDNA)-less cells, we demonstrated the m.7505A > G variant led to significantly decreased steady-state levels of tRNASer(UCN) in the mutant cybrids, as compared with those of control cybrids. The aberrant tRNASer(UCN) metabolism resulted in the variable decreases in mtDNA-encoded polypeptides in the mutant cybrids. Furthermore, we demonstrated that the m.7505A > G variant decreased the activities of mitochondrial respiratory complexes I, III and IV, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the mutant cybrids. These results demonstrated that the m.7505A > G variant affected both structure and function of tRNASer(UCN) and consequently altered mitochondrial function. Our findings highlighted critical insights into the pathophysiology of maternally inherited deafness, which is manifested by the aberrant tRNA metabolism.

Although posterior mediastinal (PM) route and retrosternal (RS) route have been used for reconstruction after minimally invasive esophagectomy (MIE), the optimal route remains controversial. This study reviewed our experiences with McKeown MIEs for esophageal cancer and aimed to investigate which route was better for esophageal reconstruction.From December 2011 to December 2013, 103 patients who underwent McKeown MIE and esophageal reconstruction by PM or RS routes were reviewed. The decision regarding which approach was appropriated mainly depended on the first surgeon's preference and experience. Baseline demographics, operative, and postoperative data of the patients were analyzed.Fifty-six and forty-seven patients receiving PM and RS route reconstruction were reviewed, respectively. Shorter operation time (P = 0.001), less blood loss (P = 0.029), and longer route length (P0.001) were observed in PM route compared with RS route. No difference was observed in the resection type, harvested lymph node, intensive care unit and hospital stay, postoperative complications, and in-hospital mortality between the two routes (all P0.05).Both RS route and PM route were safe and effective. PM route was associated with shorter operation time, less blood loss, but longer route length compared with RS route.

We have developed a novel strategy to probe the topology of membrane proteins in their native bilayer environment. Our technique relies on unnatural amino acid mutagenesis to incorporate p-azido-L-phenlyalanine at a specific site in the expressed target protein. The reactive azido moiety facilitates Staudinger-Bertozzi ligation chemistry to introduce a monoclonal antibody (mAb) epitope-tagged phosphine derivative. This site-specific labeling method allows the flexibility and precision of single codon scanning, and appears to be superior to current biochemical approaches that rely on chemical modifications and/or introduction of epitope tags by mutagenesis. Our approach can be used to identify gross topological determinants for transmembrane proteins of unknown topology as well as to elucidate secondary structural elements with chemical precision. We demonstrate the experimental feasibility of our technique on human C-C chemokine receptor 5 (CCR5), a heptahelical transmembrane G protein-coupled receptor (GPCR) of known topology. CCR5 is a major co-receptor for HIV-1 entry into host cells. We labeled CCR5 in membranes with FLAG peptide epitope-phosphine at various sites on the receptor's intra- and extracellular surfaces. The differential reactivity of the FLAG epitope-phosphine reagent for the azido group on the various CCR5 mutants correlated to the known topology of CCR5 and defined specific helical boundaries. We further applied the new label/probe technology to mammalian cells in culture in order to label extracellular sites on surface-expressed CCR5. Our new method appears to be satisfactory to probe membrane protein topology in polytopic membrane proteins and has potential applications in the study of receptor signaling mechanisms in live cells.