AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

铭记红色岁月 传承长征精神——记四川省旺苍县老红军侯跃寿

在革命老区旺苍有个红军城,当年这里曾聚集了两万多名红军将士,为创建、巩固和发展川陕革命根据地作出了卓越的贡献,谱写了一曲壮丽的血与火之歌!当年英气勃勃的红军战士今安在？一个初春的午后,我们前往旺苍县老城区拜访老红军侯跃寿。走进一个近年新建的小区,我们来到侯老家。侯老正坐在沙发上看战争片,身穿深黑色外套,带着白黑色棉帽,双腿上搭着一条毯子。

午睡

在很多幼儿园幼儿的一日生活日程中,虽然也安排了充足的午睡时间,但却忽视了幼儿的睡眠卫生和睡眠习惯的培养。如: 1、幼儿睡眠的地方阴暗、潮湿、不见阳光、通风条件差、空气不新鲜。

彩叶灌木新品种朝阳醉鱼草

朝阳醉鱼草是河南名品彩叶苗木股份有限公司培育的一个彩叶灌木新品种,2014年12月获得国家植物新品种权证书。朝阳醉鱼草属落叶速生灌木,枝条长、斜生,高1~2.5米,树皮茶褐色,多分枝,小枝四棱状、有窄翅、两面被短白柔毛,成熟枝柔毛脱落。叶大,单叶对生、有叶柄,柄上密生茸毛。叶椭圆状披针形,长5~25厘米,缘有细齿,叶背密被星状茸毛,叶片黄绿色,新叶叶缘金黄色,叶心绿色,

Impact of a single bout of high-intensity interval exercise and short-term interval training on interleukin-6, FNDC5, and METRNL mRNA expression in human skeletal muscle

Background: Exercise promotes numerous phenotypic adaptations in skeletal muscle that contribute to improved function and metabolic capacity. An emerging body of evidence suggests that skeletal muscle also releases a myriad of factors during exercise, termed "myokines". The purpose of this study was to examine the effects of high-intensity interval training(HIIT) on the acute regulation of the mRNA expression of several myokines, including the prototypical myokine interleukin-6(IL-6), and recently identified myokines fibronectin type III domain-containing protein 5(FNDC5)(irisin) and meteorin-like protein(METRNL).Methods: Both before and after a 20-day period of twice-daily high-volume HIIT, 9 healthy males(20.5 ± 1.5 years performed a standardized bout of high-intensity interval exercise(HIIE; 5 × 4 min at ~80% pretraining peak power output) with skeletal muscle biopsy samples(vastus lateralis) obtained at rest, immediately following exercise, and at 3 h recovery.Results: Before training, a single bout of HIIE increased IL-6(p < 0.05) and METRNL(p < 0.05) mRNA expression measured at 3 h recovery when compared to rest. Following 20 days of HIIT, IL-6 and FNDC5 mRNA were increased at 3 h recovery from the standardized HIIE bout when compared to rest(both p < 0.05). Resting METRNL and FNDC5 mRNA expression were higher following training(p < 0.05), and there was an overall increase in FNDC5 mRNA post-training(main effect of training, p < 0.05).Conclusion: In human skeletal muscle(1) an acute bout of HIIE can induce upregulation of skeletal muscle IL-6 mRNA both before and after a period of intensified HIIT;(2) Resting and overall FNDC5 mRNA expression is increased by 20 days of HIIT; and(3) METRNL mRNA expression is responsive to both acute HIIE and short-term intense HIIT. Future studies are needed to confirm these findings at the protein and secretion level in humans.

Short mucin 1 alleles are associated with low virulent H pylori strains infection

TO THE EDITORWe read with interest the article published by Nguyen et al in the World Journal of Gastroenterology, showing an association between short MUC6 alleles and H pjloff infection. These results, together with previous observations by Vinall et al, reinforce the role of mucin genes （MUC6 and MUC1） VNTR polymorphisms for the variability in individual susceptibility to H pylori infection that cannot be explained by differences in environmental factors.

Differentially Expressed Proteome of Microcystis under Short-time Ultrasonic Stress

This study was conducted to explore the regulation mechanism for key protein expression. The Microcystis treated by short-time ultrasonic wave was select-ed to analyze the total protein based on 2-DE. The results showed that there were 71 up-regulated protein spots, 56 down-regulated protein spots, 54 new protein spots and 21 protein spots disappeared under short-time ultrasonic stress. Eight dif-ferential proteins were chosen for further MALDI-TOFTOF/MS analysis, and the re-sults showed that 2 unknown proteins and 6 functional proteins were detected. These proteins were relevant to some physiological processes, such as antioxidation and anti-inflammatory process, phosphate synthesis and electron transfer, which is beneficial to the metabolic balance and self-protection under short-time ultrasonic stress.

Role of ADAM10 and ADAM17 in CD16b Shedding Mediated by Different Stimulators

Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 （ADAM10） or ADAM17, sup- pressed with short hairpin RNA of ADAM10 or ADAM I 7, and reconstituted with ADAM 10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate （PMA） for 12 hours. The soluble CD 16b released from cell membrane was detected by immuno- precipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD 16b in cell supernatant after stimulation. Results HEK293 cell line stably expressing CD16b was successfully established. When CDI6b ex- pressing cell line was overexpressed with ADAM 10, shedding of CD 16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM I7, shedding of CDI6b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD 16b shedding was decreased after stimulation with ionomycin; when ADAM 17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM 17 and stimulated by PMA. Conclusions Both ADAM10 and ADAM17 could shed CD16b, but they possess differed prefer- ences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.

A Clinical observation of the therapy for malignant hydrothorax by intra-thoracic injection using IL-2 combining with Cisplatin

Objective: The aim of our study was to explore the short-term effects and complication of interleukin-2 (IL-2) combining with Cisplatin in the treatment of malignant hydrothorax.Methods: Sixty-two cases patients with malignant hydrothorax were randomly divided into two groups.Observation group was 31 examples,thoracic cavity injection IL-2 and Cisplatin;31 cases in control group using Cisplatin alone intra-thoracic injection.The regime of every week for 1–4 weeks was used to observe short term effects and complications.Results: The total response rate in observe group was higher than that in control group (90.3% vs.68.1%),which had statistically significant difference (P < 0.05).The complications included gastrointestinal tract reaction,bone marrow inhibition,chest pain and fever.The incidence rates of chest pain and fever in observe group was slightly higher than that in control group,but there was no statistically significant difference (P > 0.05).Conclusion: The IL-2 combining with Cisplatin intra-thoracic injection for malignant hydrothorax has the features of good therapeutic effects and slight poisonous side effects,which is worth to be used in clinic.

Pin1 expression affects cell proliferation and apoptosis of SW620 cells in colorectal carcinoma

Objective:The aim of our study was to investigate the effects of Pin1 reduction on SW620 cell proliferation and apoptosis in human colorectal carcinoma.Methods:We constructed a plasmid of RNA interfering(shRNA) for Pin1 gene(pGenesl-1-Pin1),then the plasmid was transfected into colorectal carcinoma SW620 cells line by liposome mediation.The protein expression of Pin1 was tested by Western blotting.The proliferation rate was analyzed by MTT and the apoptotic rate of cells was tested by flow cytometry.In order to explain further the effect of Pin1 in SW620 cells,the protein level of Bcl-2 was analyzed by Western blotting.Results:pGenesil-1-Pin1 plasmid was successfully constructed and confirmed by sequencing.The protein relative levels of Pin1 were 0.06 ± 0.04 for the P-shRNA/SW620 cells,and 0.32 ± 0.09 for the P-Con/SW620 cells.The cell growth rate of SW620 cells was slower while the apoptotic rate was increased after transfection with pGenesil-Pin1 plasmid,and the apoptotic rate was 12.38% ± 1.55% for the P-shRNA/SW620 group.At the same time,we found that the protein expression of Bcl-2 was also reduced.The results were 0.13 ± 0.04 for the P-shRNA/SW620 cells,and 0.36 ± 0.08 for the P-Con/SW620 cells.Conclusion:Inhibited Pin1 expression may suppress the cell proliferation and promote apoptosis of colorectal carcinoma cells in vitro.

Study on properties of residue-residue contacts in protein

Residue-residue contacts are very important in forming protein structure. In this work, we calculated the average probability of residue-residue contacts in 470 globular proteins and analyzed the distribution of contacts in the different interval of residues using Contacts of Structural Units (CSU) and Structural Classification (SCOP) software. It was found that the relationship between the average probability PL and the residue distance L for four structural classes of proteins could be expressed as lgPL=a+b×L, where a and b are coefficients. We also discussed the connection between two aspects of proteins which have equal array residue number and found that the distribution probability was stable (or un- stable) if the proteins had the same (or different) compact (for example synthase) in the same structural class.

Tentative Research of Short Process of Jute Degumming and Bleaching

The feasibility of combination process of jute degumming and bleaching with alkali-hydrogen peroxide in one-step-one-bath was discussed. The combination process basically has the similar function as the traditional two-step-two-bath method. The factors such as hydrogen peroxide concentration, CBI concentration, sodium hydroxide concentration, treatment time and temperature were studied respectively, and then an orthogonal experiment was designed to study the interactions among the hydrogen peroxide concentration, CBI concentration, sodium hydroxide concentration. After the designed experiments, the optimum treatment conditions were obtained as follows: hydrogen peroxide of 12g/L, sodium hydroxide of 4g/L, CBI of 4g/L, JFC of 1g/L, treatment time of 60min and temperature of 75℃.

Differential protein expression during colonic adaptation in ultra-short bowel rats

AIM： To investigate the proteins involved in colonic adaptation and molecular mechanisms of colonic adapration in rats with ultra-short bowel syndrome （USBS）. METHODS： Sprague Dawley rats were randomly as- signed to three groups： USBS group （10 rats） undergoing an approximately 90%-95% small bowel resection; sham-operation group （10 rats） undergoing small bowel transaction and anastomosis; and control group （ten normal rats）. Colon morphology and differential protein expression was analyzed after rats were given postsurgical enteral nutrition for 21 d. Protein expression in the colonic mucosa was analyzed by two-dimensional electrophoresis （2-DE） in all groups. Differential protein spots were detected by ImageMaster 2D Platinum soft-ware and were further analyzed with matrix-assisted laser desorption/ionization-time-of-flight/time-of-flightmass spectrometric （MALDI-TOF/TOF-MS） analysis. RESULTS： The colonic mucosal thickness significantly increased in the USBS group compared with the control group （302.1 ± 16.9 um vs 273.7 ± 16.0 um, P 〈 0.05）. There was no statistically significant difference between the sham-operation group and control group （P 〉 0.05）. The height of colon plica markedly improved in USBS group compared with the control group （998.4 ± 81.2 um vs 883.4 ± 39.0 um, P 〈 0.05）. There was no statistically significant difference between the shamoperation and control groups （P 〉 0.05）. A total of 141 differential protein spots were found in the USBS group. Forty-nine of these spots were down-regulated while 92 protein spots were up-regulated by over 2-folds. There were 133 differential protein spots in USBS group. Thirty of these spots were down-regulated and 103 were upregulated. There were 47 common differential protein spots among the three groups, including 17 down- regulated protein spots and 30 up-regulated spots. Among 47 differential spots, eight up-regulated proteins were identified by MALDI-TOF/TOF-MS. These proteins were previously reported to be involved in sugar and fat metabolism, protein synthesis and oxidation reduction, which are associated with colonic adaption. CONCLUSION： Eight proteins found in this study play important roles in colonic compensation and are associated with sugar and fat metabolism, protein synthesis, and molecular chaperoning

如果感到幸福你就跺跺脚

那一年,青年德皮勒完成了全部学业从州立六学毕业了,他做了一名教文 学的老师。所以,从那时开始,我们应该叫他德皮勒老师。 其实德皮勒非常想去做一个优秀的长跑运动员。四年前的他曾是那么单纯 而痴迷的一个运动青年。但是,他的梦想却在生活中成了幻想。 拿捏着自已从最新的教育学书籍上学来的方法,德皮勒在自己的学生们身 上试验着。书是麦尔教授推荐的,应该不会错。麦尔教授是他大学选修心理学 的主课教授,是一个有着短白胡子的小老头。