Abstract:Caloric restriction(CR) can delay aging and the onset of aging-related diseases. Sirtuin plays a key role in the aging process regulated by CR because of its ability to sense the metabolic status and to integrate into adaptive transcriptional outputs. Sirtuin regulates the aging process by altering protein activity and stability through lysine acetylation. Moderate CR in yeast influences replicative lifespan and chronological lifespan mainly by increasing the NAD⁺/NADH ratio and regulating the level of nicotinamide. Similar mechanism also exist among Caenorhabditis elegans and Drosophila melanogasters. SIRT1 protein level increases in response to CR in mammals, leading to an increase in PNC1/Nampt expression, which favors the synthesis of NAD⁺ from NAM, potentially acting as a major mechanism to drop the leash of SIRT1 inhibition. NO up-regulates SIRT1 and mitochondrial biogenesis. Cellular and organism's senescence may be influenced through the deacetylation of histone, p53, NES1, FOXO by SIRT1, indicating sirtuin and its homologous analogues play important roles in aging process and lifespan extension under CR in different organisms.

Abstract:Epigenetics indicates the heritable changes in gene expression without nucleotide sequence variation. The process of epigenetic regulation is very complex, which mainly includes DNA methylations, histone modifications and miRNA. Diabetes is a chronic metabolic disease accompanied with macrovascular and microvascular complications. The development of diabetes not only depends on genetic factors, but also is regulated by epigenetic regulation. Therefore, further exploration into epigenetic regulation will provide new ideas and methods to the prevention and treatment for diabetes and its complications.

Abstract:Mutations in the mitochondrial tRNAs are one of the causes of sensorineural hearing loss. Some tRNA mutations such as tRNA^Leu(UUR) A3243G are associated with hearing impairment and other clinical symptoms, while other tRNA mutations including tRNA^Ser(UCN) T7511C only produce the phenotype of hearing loss. These tRNA mutations are the primary factors for the development of hearing loss. On the other hand, other tRNA mutations such as tRNA^Thr G15927A act in synergy with the primary tRNA mutations, modulating the phenotypic manifestation. The mutations alter the secondary structures of tRNAs, impair translation and decrease the ATP production. Consequently, mitochondrial dysfunctions caused by these tRNA mutations lead to hearing loss. It this review we summarize the deafness-associated mitochondrial tRNA mutations and discuss the pathophysiology of these mitochondrial tRNA mutations.

Abstract:Eukaryotic DNA ligases play vital roles in DNA replication, recombination and repair through catalyzing ligation of nick in double-stranded DNA with an ATP-dependent reaction. DNA ligase Ⅲ(Lig3) is a unique ligase which is located in both nucleus and mitochondrion. Lig3 plays important roles in base excision repair and other single-stranded break repairs with its DNA repair protein XRCC1. But Lig3 is more important in maintenance of mitochondrial DNA (mtDNA) integrity without XRCC1-dependent DNA repair. These researches provide new perspective for Lig3 function and DNA repair.

Abstract:Traditional approaches to drug discovery are generally regarded as protracted and costly. The application of established drug compounds to new therapeutic indications, known as drug repositioning, offers several advantages over traditional drug development. Gene expression microarrays are regularly and broadly applied in clinical studies of human diseases, providing genome-wide characterization of a disease state. Microarrays are also widely used to discover gene expression patterns that signify pharmacologic perturbation, allowing for the development of high-quality signatures of drug effect. A pair of papers from one group recently published in Science Translational Medicine provided a concrete example of how to using bioinformatics approach to reinterpret and compare genome-wide gene expression data, that allows us to effectively hypothesize which drugs from one disease-indication can be repurposed for another disease. They examined publicly available gene expression data and determined the genes affected in 100 diseases and 164 drugs. By pairing drugs that correct abnormal gene expression in diseases, they confirmed known effective drug-disease pairs and predicted new indications for already approved agents. Experimental validation that an antiulcer drug and an antiepileptic can be reused for lung cancer and inflammatory bowel disease reinforced the promise of this approach. These two drugs are therefore good candidates for repositioning to treat lung cancer and inflammatory bowel disease that in need of better therapies, and we now have a way to mine available data for fast routes to new disease therapies.

Abstract:Acetohydroxyacid synthase (AHAS) as the first common enzyme in the brunched-chain amino acids biosynthesis is the target of several classes of commercial herbicide. AHAS is normally composed of a larger catalytic subunit with FAD (flavin adenine dinucleotide), ThDP (thiamine diphosphate) and Mg²⁺ as cofactors and a smaller regulatory subunit bound to the end-product feedback signals such as valine, leucine or isoleucine to down regulate the holoenzyme activities. The Escherichia coli ilvN gene that encodes the regulatory subunit of AHAS Ⅰ was cloned into pET28a and expressed in soluble form at high levels in E. coli strain BL21 (DE3). The protein was purified using Ni²⁺-chelating chromatography followed by size-exclusion chromatography. Crystals of IlvN protein were obtained and diffracted to 2.6 Å. To further study the regulatory mechanism, crystals of IlvN co-crystallized with its effector valine were also obtained and diffracted to 3.0 Å.

Abstract:Auxin response factors (ARFs) are important transcription factors involved in auxin signal transduction pathway. In order to elucidate the function of tomato ARF2, we isolated the SlARF2 gene and analyzed its molecular features, in addition, we observed the subcellular localization of ARF2 in transgenic tomato plants. Physicochemical properties and molecular features of ARF2 were predicted by bioinformatic approaches including physical and chemical properties analysis, hydrophobicity analysis, domain analysis, phylogenetic tree analysis and subcellular localization analysis. Moreover, the full-length of SlARF2 gene was amplified by RT-PCR, and a binary vector consisting of ARF2 fused with the yellow fluorescent protein (YFP) coding sequence was further constructed. Using the method of Agrobacterium-mediated transformation, the recombinant vector was transformed into wild-type tomato, and the transgenic tomato plants were confirmed by PCR. The roots of transgenic tomato seedlings were observed under a fluorescence microscope. Bioinformatic analysis showed that the SlARF2 is an unstable soluble protein, which is rich in Ser, Gly, Pro and Leu residues, and it has typical structure and functional domain of ARF family. Tomato ARF2 shares 70.08 %, 66.94 %, and 60.87 % similarity in amino acid sequences with grapes, cassava and Arabidopsis, respectively. Analyses of restriction enzyme digestion and sequencing confirmed the expression vector pBA-ARF2-YFP was successfully constructed. PCR results showed the fusion protein was expressed in transgenic tomato plants. The subcellular localization of SlARF2 was found mostly in the nucleus. SlARF2 has the key characteristics of transcription factor, which is localized in the nucleus, and it might play a pivotal role in tomato fruit development and ripening. This might provide references for studying the function of SlARF2 gene.

Abstract:Mechanical forces are widely involved in regulating basic cellular functions, including proliferation, differentiation, adhesion, and migration. However, few studies show how the mechanical forces impact cell division direction, a crucial process in development, differentiation, embryogenesis and recovery of tissues. It has been documented that cell division direction is guided by stress fibers, which are cortical cues perpendicular to exogenous cyclic uniaxial stretch. In our study, we used the murine 3T3 fibroblasts as a model, to investigate the effect of uniaxial stretch on stress fiber alignment and cell division direction with custom-made stretch devices. We found that cyclic uniaxial stretch induced stress fiber alignment and cell division direction perpendicularly to the stretch direction. The blockage of actin assembly and myosinⅡ, two basic components of stress fibers, resulted in a disoriented cell division under uniaxial stretch, which suggested that the cell division direction was secondary to the stress fiber alignment. Our data suggested that Rho/Rho-kinase/MLC (Myosin Light Chain) and MLCK/MLC pathways were involved in the process of stretch-induced stress fiber alignment and cell division direction. Taken together, the present work demonstrated an important effect of uniaxial stretch on cell division direction via affecting the stress fibers alignment.

Abstract:Gastric cancer (GC) seriously impacts on human health with high incidence and mortality rate in the world. Raf kinase inhibitor protein (RKIP) that was discovered in our previous studies is down-regulated in GC, which is associated with the occurrence, differentiation, invasion, and metastasis of GC. In order to investigate the molecular mechanisms and biological functions of RKIP in the occurrence and metastasis of GC, the fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transiently transfected into SGC7901 cells with liposome-mediated approach, the RKIP fusion proteins were purified with 3xFLAG tag affinity chromatography, and the RKIP-interacted proteins were identified with electrospray ionization-Quadrupole-time of flight (ESI-Q-TOF) tandem mass spectrometry (MS/MS). A total of 66 RKIP-interacted proteins were MS/MS-identified. The MS/MS-characterized components of the existing interaction complex (RKIP and 14-3-3) were confirmed with Western blotting in combination with co-immunoprecipitation. It is the first time to discover the interaction of RKIP with 14-3-3. This preliminary study on the molecular mechanisms that how RKIP inhibits the occurrence and development of GC provides novel clues for the early-stage diagnosis, prognosis monitoring, and targeted gene therapy of GC.

Abstract:Endometriosis is a common gynecological disease often resulting in chronic pelvic pain and infertility. The pathogenesis of endometriosis is likely multifactorial and several hypotheses have been suggested to explain the presence of ectopic endometrial tissue and stroma. There is a growing body of evidence indicating that miRNA is involved in the etiology of endometriosis, so this study explored the possible roles of Dicer and Drosha in endometriosis formation. Paired eutopic and ectopic endometrium in ovarian endometriosis was collected and cultured. Quantitative RT-PCR and Western blot of pairs of eutopic and ectopic endometrium demonstrated mRNA and protein levels for Dicer and Drosha in ectopic endometrium (EC) were decreased significantly compared with eutopic endometrium (EU). When transfected with Dicer or Drosha small hairpin RNA (shRNA) in EU, a significant increase in cell proliferation and decreased in cell apoptosis was detected. Furthermore, the Bcl2 was increased and Bax was deceased. In comparison, a scramble siRNA transfection did not affect protein levels for these molecules. Results demonstrate the low expression of Dicer and Drosha significantly correlates with EC, and shDicer or shDrosha can affect cell proliferation and apoptosis, manipulating Dicer and Drosha in EC may result in novel treatment strategies for EMs.

Abstract:The rich classes of secondary metabolites from the genus Sorangium have been an important source of new drugs. The proteome analysis is an effective method to study the regulation of metabolism. However, the genus Sorangium contains a large amount of exopolysaccharides or slime that interferes with protein solubility, resolution, and repeatability in proteome analysis. To perform high-throughput screening of the specific proteins expressed by Sorangium cellulosum So0157-2, we optimized the two-dimensional electrophoresis (2-DE) protocol. Firstly, the proteins have better solubility in lysis buffer. The pH 3～10 NL strip is appropriate for the first-dimensional isoelectric focusing, improving the resolution of protein spots. 1 mg of protein was used in the isoelectric focusing, improving the expression of low-accumulation proteins. 15% SDS-PAGE improved the resolution and repeatability for separation of these proteins. Based on the optimal 2-DE protocol, the protein patterns of S. cellulosum So0157-2 cultured in M26 medium for three days were acquired, and 552 protein spots were detected. Further, the expressed proteins (85.9%) were identified by MALDI-TOF-MS. The identified proteins included components of cell structure and function, and cell metabolic enzymes. Worthy to be mentioned, 8 proteins were related to the transformation and metabolism of carbohydrate, which were contributed to the in-depth study of epothiloneoside A. This optimal protocol laid the foundation for the further construction of proteome expression database of S. cellulosum So0157-2 in various industrial culture conditions.