The New Role of Sirtuin1 in Human Osteoarthritis Chondrocytes by Regulating Autophagy.

OBJECTIVE: The aim of this study is to investigate the role of Sirtuin1 (Sirt1) in the regulation of autophagy for human osteoarthritis (OA) chondrocytes. DESIGN: All cartilage samples were collected from human donors, including young group, aged group, and OA group. Primary chondrocytes were isolated and cultured with Sirt1 activator or inhibitor. Sirt1 expression in cartilage tissue and chondrocytes was evaluated, and the deacetylation activity of Sirt1 was determined. The alteration of autophagy activity after upregulating or downregulating Sirt1 was detected. Chondrocytes were treated with autophagy activator and inhibitor, and then the protein level of Sirt1 was examined. The interactions between Sirt1 and autophagy-related proteins Atg7, microtubule associated protein 1 light chain 3 (LC3), and Beclin-1 were determined by using immunoprecipitation. RESULTS: The assay of articular cartilage revealed that the expression of Sirt1 might be age-related: highly expressed in of younger people, and respectively decreased in the elderly people and OA patients. In vitro study was also validated this result. Further study confirmed that higher levels of Sirt1 significantly increased autophagy in aged chondrocytes, while the lower expression of Sirt1 reduced autophagy in young chondrocytes. Of note, the high levels of Sirt1 reduced autophagy in OA chondrocytes. When the chondrocytes were treated with autophagy activator or inhibitor, we found the expression of Sirt1 was not affected. In addition, we found that Sirt1 could interact with Atg7. CONCLUSION: These results suggest that Sirt1 in human chondrocytes regulates autophagy by interacting with autophagy related Atg7, and Sirt1 may become a more important target in OA treatment.

Codelivery of Anticancer Drug and Photosensitizer by PEGylated Graphene Oxide and Cell Penetrating Peptide Enhanced Tumor-Suppressing Effect on Osteosarcoma.

Objective: Graphene oxide (GO) has been widely used for various biological and biomedical applications due to its unique physiochemical properties. This study aimed to investigate the effects of cell penetrating peptide (CPP) modified and polyethylene-glycol- (PEG-) grafted GO (pGO) loaded with photosensitive agent 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-alpha (HPPH) and Epirubicin (EPI) (HPPH/EPI/CPP-pGO) on tumor growth in osteosarcoma. Methods: The HPPH/EPI/CPP-pGO were prepared, and then in vitro drug release assay was conducted. The detection of singlet oxygen (1O2) and cellular uptake of HPPH was performed as well. Next, the effects of control (saline solution), CPP-pGO, EPI, HPPH, HPPH/CPP-pGO, EPI/CPP-pGO, HPPH/EPI/pGO, and HPPH/EPI/CPP-pGO were evaluated by MTT assay, colony-forming assay, and cell apoptosis assay in MG-63 cells. Furthermore, the antitumor effects of HPPH/EPI/CPP-pGO on osteosarcoma xenograft mice were unraveled. Results: The 1O2 generation and cellular uptake of HPPH were significantly increased after CPP and pGO modification compared with free HPPH. In addition, compared with control cells, CPP-pGO treatment had low cytotoxicity in MG-63 cells. Compared with free HPPH or EPI, HPPH/CPP-pGO or EPI/CPP-pGO treatment significantly inhibited cell viability and colony forming number, as well as inducing cell apoptosis. HPPH/EPI-pGO treatment showed stronger inhibition effects on MG-63 cells than HPPH/CPP-pGO or EPI/CPP-pGO, and HPPH/EPI/CPP-pGO was the most effective one. Similarly, in vivo experiments revealed that, compared with control group, the tumor size and weight of osteosarcoma xenograft mice were obviously decreased after free HPPH or EPI treatment, which were further reduced in other groups, especially in HPPH/EPI/CPP-pGO group. Conclusion: HPPH/EPI/CPP-pGO had superior tumor-inhibiting effects in vitro and in vivo on osteosarcoma.

Cryopreservation of virus: a novel biotechnology for long-term preservation of virus in shoot tips.

BACKGROUND: Preservation of plant virus is a fundamental requirement in all types of virus-related research and applied applications. Development of efficient, reliable strategies for long-term preservation of plant virus would largely assist these studies. RESULTS: The present study reported a novel biotechnology allowing cryopreservation of Apple stem grooving virus (ASGV) in living shoot tips. Following cryopreservation by droplet-vitrification or encapsulation-dehydration, about 62-67% of shoot regrowth and 100% of ASGV cryopreservation were obtained. Although shoot proliferation and virus concentration were reduced in cryopreserved diseased shoots after 8 weeks of shoot regeneration, continuous subculture for 4 times (16 weeks) increased shoot proliferation and virus concentration to comparative levels as those produced by shoot tip culture (as a control to shoot tip cryopreservation). Cryopreserved ASGV was efficiently transmitted to a woody plant by micrografting and to a herbaceous indicator by mechanical inoculation. Gene sequencing in three fragments of ASGV genome including coat protein and movement protein showed that cryopreserved ASGV shared 99.87% nucleotide identities with shoot tip culture-preserved virus, indicating cryopreserved virus is genetically stable. CONCLUSIONS: The present study demonstrates ASGV, a representative virus that can infect meristematic cells of shoot tips, can be efficiently cryopreserved in shoot tips. To the best of our knowledge, this is the first report on plant virus cryopreservation in living tissues, and has great potential applications to long-term preservation of plant viruses.

Comparative effectiveness of electro-acupuncture plus lifestyle modification treatment for patients with simple obesity and overweight: study protocol for a randomized controlled trial.

BACKGROUND: Acupuncture is considered to be an effective and safe treatment for obese and overweight patients, although high-quality evidence regarding the effects of acupuncture on obesity are not conclusive. The aim of the current study is to investigate the effectiveness of electro-acupuncture plus lifestyle modification for treating obese and overweight patients, in comparison with lifestyle modification alone in China. METHODS/DESIGN: To compare the effectiveness of acupuncture plus lifestyle modification, a 2-armed, controlled trial with randomization using minimization will be conducted on 150 simple obesity and overweight patients, aged 18-50 years, for a 36-week study duration. All patients will be randomly assigned to one of two groups and will receive either acupuncture plus lifestyle modification or lifestyle modification alone. Outcomes will be evaluated at baseline and at 4 weeks, 8 weeks, and 12 weeks during treatment as well as at 6-week, 12-week, and 24-week follow-up. The primary endpoint is change of body mass index (BMI) during the 12th week. Secondary endpoints are body weight; waist-to-hip ratio; biochemical tests including serum cholesterol (TC), triglyceride (TG) and high-density lipoprotein (HDL) levels; and answers to the Short Form 36 (SF-36) and the Impact of Weight on Quality of Life Questionnaire-Lite Version (IWQOL-Lite). Statistical analyses will be based on the intention-to-treat (ITT) principle. The main endpoint will be analyzed by analysis of covariance (ANCOVA), and the objective outcome results will be analyzed by logistic regression analysis. To avoid potential confounding factors, additional sensitivity analyses will be conducted following these statistical analyses. DISCUSSION: This trial is the first to compare the effectiveness of acupuncture plus lifestyle modification for treating obesity relative to lifestyle modification treatment alone by using a pragmatic study design. We hope that the results of this study will contribute to advancing the current methodology of acupuncture trials for obesity and will facilitate the application of useful acupuncture strategies in real-world clinical settings. TRIAL REGISTRATION: ChiCTR-TRC- 12002762 . The date of registration is 31 October 2012.

Tripartite interactions of Barley yellow dwarf virus, Sitobion avenae and wheat varieties.

The tripartite interactions in a pathosystem involving wheat (Triticum aestivum L.), the Barley yellow dwarf virus (BYDV), and the BYDV vector aphid Sitobion avenae were studied under field conditions to determine the impact of these interactions on aphid populations, virus pathology and grain yield. Wheat varietal resistance to BYDV and aphids varied among the three wheat varieties studied over two consecutive years. The results demonstrated that (1) aphid peak number (APN) in the aphid + BYDV (viruliferous aphid) treatment was greater and occurred earlier than that in the non-viruliferous aphid treatment. The APN and the area under the curve of population dynamics (AUC) on a S. avenae-resistant variety 98-10-30 was significantly lower than on two aphid-susceptible varieties Tam200(13)G and Xiaoyan6. (2) The production of alatae (PA) was greater on the variety 98-10-30 than on the other varieties, and PA was greater in the aphid + BYDV treatment on 98-10-30 than in the non-viruliferous aphid treatment, but this trend was reversed on Tam200(13)G and Xiaoyan6. (3) The BYDV disease incidence (DIC) on the variety 98-10-30 was greater than that on the other two varieties in 2012, and the disease index (DID) on Tam200(13)G was lower than on the other varieties in the aphid + BYDV and BYDV treatments in 2012, but not in 2011 when aphid vector numbers were generally lower. (4) Yield loss in the aphid + BYDV treatment tended to be greater than that in the aphid or BYDV alone treatments across varieties and years. We suggested that aphid population development and BYDV transmission tend to promote each other under field conditions. The aphids + BYDV treatment caused greater yield reductions than non-viruliferous aphids or virus treatment. Wheat varietal resistance in 98-10-30 affects the aphid dispersal, virus transmission and wheat yield loss though inhibits aphid populations from increasing.

Detection and identification of the phytoplasma associated with China ixeris (Ixeridium chinense) fasciation.

BACKGROUND: Phytoplasmas are always associated with symptoms in host plants such as stunting of stems, witches'-broom, yellowing of leaves, formation of sterile-deformed flowers, virescence and phyllody. Recently also symptom of fasciation was reported associated with phytoplasma presence. In the present work, China ixeris fasciation was observed associated with phytoplasmas in Guanzhong Area, Shaanxi, China. RESULTS: Phytoplasma-like bodies were observed under transmission electron microscope in stem tissues of symptomatic samples. The 16S rRNA operon and tuf genes from phytoplasmas were amplified by PCR assays. Phylogenetic trees were calculated respectively based on sequences data of these two genes. The pattern of restriction fragment length polymorphism (RFLP) was generated via digesting the PCR products of 16S rRNA gene with eight restriction enzymes. CONCLUSION: The presence of phytoplasma in China ixeris exhibiting fasciation symptom was confirmed by the results of TEM observation and PCR testing. Based on sequence data, phylogeny analysis and actual restriction fragment length polymorphism (RFLP) analysis, the associated phytoplasma was classified as related to 16SrI-C subgroup. This was the first record of phytoplasmas in China ixeris.

Sp7/Osterix is involved in the up-regulation of the mouse pro-α1(V) collagen gene (Col5a1) in osteoblastic cells.

Sp7/Osterix, a transcription factor whose expression is restricted in osteoblasts, belongs to the Sp family of transcription factor that bind to G/C-rich sequences. Previous studies have identified a Sp1binding site in the proximal promoter region of the mouse Col5a1 gene, but it did not activate or repress this gene in a mouse fibroblast cell line and a human rhabdomyosarcoma cell line. The purpose of the present study was to clarify the involvement of Sp7/Osterix in the mouse Col5a1 gene. A functional analysis revealed that mutation of the Sp1 binding site specifically decreased the promoter activity in osteoblastic cells. An overexpression of Sp7/Osterix significantly increased the promoter activity and the endogenous mRNA levels of the Col5a1 gene in osteoblastic cells. Conversely, siRNA-mediated knockdown of Sp7/Osterix decreased the promoter activity and the endogenous mRNA levels of the Col5a1 gene. These effects on promoter activity were canceled when the mutant construct of Sp1 binding site was introduced. Consistent with these data, the experiments using an osteoblast differentiation model showed increased promoter activity and endogenous mRNA levels, along with increased Sp7/Osterix during differentiation. Therefore, type V collagen appears to be involved in bone formation.

The Sp1 and CBF/NF-Y transcription factors cooperatively regulate the mouse pro-alpha3(V) collagen gene (Col5a3) in osteoblastic cells.

The purpose of this study was to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Transient transfection into rat osteosarcoma ROS17/2.8 cells demonstrated that a region from nucleotides 337 to 1 was involved in the transcriptional activity of the Col5a3 gene. An electrophoretic mobility shift assay showed that Sp1/Sp3 and CBF/NF-Y bound to a GC-rich domain (194/186) and a CCAAT box (134/130) in the Col5a3 gene, respectively. Introduction of mutations or deletion into a GC-rich domain, the CCAAT box, or both elements decreased the transcription activity. Overexpression of Sp1 increases the transcription activity and interferes with Sp family binding to the GC-rich domain to decrease promoter activity. Therefore, the transcription of the mouse Col5a3 gene is cooperatively regulated by Sp1 and CBF/NF-Y in osteoblastic cells.

[The inactivating effect of chito-oligosaccharides on TMV particles in vitro].

To confirm the inactivating effect of chito-oligosaccharides on Tobacco mosaic virus (TMV) par ticles in vitro, the difference of TMV pathogenicity was evaluated according to the decrease of local lesion numbers after inoculating with TMV mixed with chito-oligosaccharides (DP3-10) in Nicotiana glutinosa, and the virion structural change was studied by transmission electron microscopy after mixed with chito-oligosaccharides. In the range of tested concentrations of chito-oligosaccharides (100-1000 microg /mL), the numbers of local lesions were strongly reduced with over 30% decrement, and the 88.4% reduction gained at the concentration of 600g /mL. It revealed that treatment with chito-oligosaccharide solution of 300-500 microg /mL directly broke TMV particles into tiny pieces of 50-150nm long, and that treatment with solutions of 600-1000 microg/mL caused virus particle agglomerated. The data presented here suggested that chito-oligosaccharides exerted strong inactivating effect on plant virus in vitro.

[Research on secretion expression in Pichia pastoris and function of the HC-pro gene of watermelon mosaic virus].

HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPI(9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease Sal I , the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut+ /His+ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE, the results showed that a specific protein with a molecular weight of about 66 kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to "bridge" hypothesis that HC-Pro help aphid transmission of non-persistent viruses.

[Activity and ecological distribution of actinomycetes from soil in the southeastern of Tibet].

The soil samples were collected from primeval forest, alpine meadow, marshy, grainfield and vegetable farmland ect. 9 different kinds of Vegetative type and from 2970 - 4590 altitude of southeastern of Tibet. The amount,composition, antimicrobial activity of mesophilic and psychrophilic actinomycetes and some mesophilic actinomycetes of physiological and biochemical characteristics were studied by normal methods. These actinomycetes were identified at genus level by their morphological properties. Results indicated that: (1) 9 genus of actinomycetes were isolated from soil and Actinosynnema was never reported in China. Grain field contains the largest quantity of actinomycetes. (2) Primeval forest contains the largest quantity of antimicrobal actinomycetes, so more and more antimicrobal actinomycetes information can be obtained from them. (3) The strains tested had stronger antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria, had stronger antimicrobial activity against fungi than bacteria. (4) Many strains of streptomyces have some enzyme activities such as gelatin utilization, Milk-clotting enzyme etc.