CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum.However,the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear,which greatly restricts design of molecular breeding in chrysanthemum.Here,a chrysanthemum SG7 R2R3 MYB transcription factor(TF),CmMYB3-like,was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage.Quantitative real time PCR(qRT-PCR)assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage.Genetic transformation of chrysanthemum and dual-luciferase assays in N.benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly.However,overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols.Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation,but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage.Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

拟南芥转录因子Ethylene-insensitive3(EIN3)抑制花青素的合成

Ethylene-insensitive3(EIN3)和EIN3-like1(EIL1)蛋白是乙烯信号转导途径中一类重要的核转录因子。花青素是植物体中的一类水溶性天然色素,在植物的许多生理过程中起重要作用。本研究以拟南芥双突变体ein3-1eil1-3为研究材料,通过RT-PCR技术确定了拟南芥双突变体ein3-1eil1-3中EIN3和EIL1基因均已被敲除,单突变体ein3-1中的EIN3基因被敲除。通过肉眼定性观察发现突变体ein3-1eil1-3的种子和叶片内均呈紫色。通过紫外分光光度计定量分析发现,花青素积累量也明显比突变体ein3-1和野生型多。通过GUS染色发现EIN3启动子主要在花、柱头、成熟花粉、种子胚和果荚等组织中有较强的表达。这与突变体ein3-1eil1-3的种子和叶片内均呈紫色并花青素含量增高一致。因此,拟南芥转录因子EIN3可能与EIL1共同参与抑制花青素的合成。

MdDGK3-like as a negative regulator participates in ALA-induced PP2AC to promote stomatal opening in apple leaves

5-Aminolevulinic acid(ALA)can inhibit abscisic acid(ABA)-induced stomatal closure.However,the molecular mechanism is unclear.In this study,we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple(Malus domestica Borkh.cv.‘Fuji’)leaves.With the promoter of MdPP2AC as bait,a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid(Y1H)from the cDNA library of the epidermis of apple leaves treated by exogenous ALA.Additional to binding the promoter of MdPP2AC,MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter,while ALA significantly eliminated the role of MdDGK3-like.In tobacco leaves,MdDGK3-like was localized in the nucleus of stomatal guard cells.Therefore,MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure.To further identify MdDGK3-like functions,several transiently transgenic apple leaves(including overexpression and interference)were established.The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca^(2+)and H_(2)O_(2)and decreasing flavonol levels in the guard cells.Conversely,MdDGK3-like(i)led the stomatal opening with lower levels of Ca^(2+)and H_(2)O_(2)but higher flavonols.Based on these,we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity,which improved the stomatal aperture.Since it was the first time that MdDGK3-like was showed to act as a transcription factor,the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.

Location-based prediction model for Crohn’s disease regarding a novel serological marker,anti-chitinase 3-like 1 autoantibodies

BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.

菊苣对高尿酸血症鹌鹑肾脏有机阴离子转运体OAT3-LIKE的影响

目的从肾脏转运蛋白角度探究中药菊苣降尿酸的药效机制。方法将40只雄性迪法克鹌鹑按体质量随机分为5组,正常组给予普通饲料,其余各组均给予含酵母饲料(15 g·kg-1·d-1)建立高尿酸血症模型,同时给药组分别给予菊苣5,7.5 g·kg-1·d-1,苯溴马隆20 mg·kg-1·d-1,实验第35天处死。检测鹌鹑血清尿酸(Uric Acid,UA)、尿素氮(Urea Nitrogen,BUN)水平,并用Real-time RT-PCR(Real-time Reverse TranscriptionPolymerase Chain Reaction)方法检测其肾脏有机阴离子转运体OAT3-LIKE(Organic Anion Transporter3-LIKE)的mRNA表达水平,同时测定肝脏尿酸代谢相关酶活性。结果与正常组比较,实验第21天、28天、35天模型组鹌鹑血尿酸升高,第35天肾脏OAT3-LIKE m RNA表达水平下降。与模型组比较,实验第35天,菊苣可以降低鹌鹑血尿酸水平,上调其肾脏OAT3-LIKE mRNA表达水平并降低肝脏腺苷脱氨酶(ADA)活性。结论高嘌呤食饵可以成功诱导鹌鹑高尿酸血症;菊苣具有抑制高尿酸血症鹌鹑尿酸生成和促进尿酸排泄的双重作用;菊苣药效机制可能与降低ADA活性及上调OAT3-LIKE表达水平相关。

ORM1-like3基因单核苷酸多态性与客家人群哮喘发病的关系

目的 探讨ORM1-like 3基因的单核苷酸多态性（SNPs）与客家人群哮喘发病的相关性.方法 选取确诊的客家人群哮喘患者194例（哮喘组）和同期健康查体者200例（对照组）为研究对象,利用MassARRAY-IPLEX技术和基质辅助激光解吸电离飞行时间质谱平台对ORM1-like 3基因的6个SNPs位点进行基因分型.结果 哮喘组与对照组的性别（χ^2＝0.013,P=0.909）和年龄（t=0.728,P=0.259）差异均无统计学意义.与对照组相比,哮喘组嗜酸性粒细胞计数（Z=-5.670,P=0.000）和IgE（Z=-9.158,P=0.000）明显升高,CRP水平（Z=-1.716,P=0.086）差异无统计学意义,吸入性过敏原（χ^2＝55.452,P=0.000）和食物过敏原（χ^2＝12.853,P=0.000）检出率明显升高.单纯哮喘组与哮喘合并过敏性鼻炎组比较,除rs3859192差异有统计学意义（χ^2＝6.659,P=0.036）外,其余5个位点差异均无统计学意义（P〉0.05）.总的基因型分型成功率为97.34%.与对照组相比,哮喘组rs7216389的等位基因分布（χ^2=7.103,P=0.008）和基因型分布（χ^2=7.695,P=0.021）差异均有统计学意义,但进行Bonferroni校正后基因型分布差异无统计学意义（Pc=0.126）.与对照组比较,哮喘组rs12603332、rs8069176和rs3859192的等位基因分布（χ^2=2.119,P=0.145;χ^2=2.167,P=0.141;χ^2=0.026,P=0.871）和基因型分布（χ^2=2.197,P=0.333;χ^2=5.686,P=0.058;χ^2=5.052,P=0.080）差异均无统计学意义.与对照组比较,哮喘组rs2305480和rs4795400的等位基因分布（χ^2=14.722,P=0.000;χ^2=10.417,P=0.001）和基因型分布（χ^2=12.351,P=0.002;χ^2=12.271,P=0.002）差异均有统计学意义,进行Bonferroni校正后两组间差异仍均有统计学意义（Pc=0.012;Pc=0.012）.与CC基因型相比,rs2305480的CT、TT基因型显著增加哮喘发生的危险性（P=0.033,P=0.003）,OR值（95% CI）分别为1.763（1.044～2.978）和5.681（1.600～20.171）,CT＋TT基因型增加哮喘的发生,OR值（95% CI）为2.130（1.306～3.475）.与TT基因型相比,rs4795400的CT基因型不增加哮喘发生的危险性（P=0.059）,而CC基因型显著增加哮喘发生的危险性（P=0.002）,OR值（95% CI）为4.440（1.600～12.316）.在82例初次确诊的哮喘患者中,与CC基因型相比,rs2305480的CT＋TT基因型的FVC占预计值百分比和FEV1占预计值百分比下降明显（均P〈0.05）.与TT＋CT基因型相比,rs4795400的CC基因型的FVC占预计值百分比和FEV1占预计值百分比下降亦明显（均P〈0.05）;rs2305480的基因型分布在初诊哮喘的病情严重程度不同分级之间差异无统计学意义（χ^2=2.130,P=0.144）,rs4795400差异有统计学意义（χ^2=9.522,P=0.002）.结论 ORM1-like 3基因的rs2305480和rs4795400位点与客家人群哮喘的发病有关,它们的基因型分布可能影响初诊哮喘患者的肺功能,而且rs4795400可能与病情严重程度有关.

泛素连接酶E3 Nedd4-like家族与癌症关系的研究进展

泛素蛋白酶体系统参与体内80%以上蛋白质的降解,调节体内细胞增殖,分化及存活,其功能紊乱可导致包括癌症在内的多种疾病。该系统中泛素连接酶E3 Nedd4-like家族已被证实在多种癌症发生、发展中发挥重要作用。泛素连接酶E3 Nedd4-like家族成员能够调节多种蛋白的泛素化、溶酶体及蛋白酶体降解作用及细胞核转位作用,因而影响着肿瘤发生过程中多条信号转导通路,如TGFβ、EGF、IGF、VEGF、SDF-1及TNFα通路。此外,该家族还可直接调节Smads、p53、KLF、RUNX及Jun等多种癌相关转录因子。由此可见,充分了解E3 Nedd4-like家族对癌症的潜在影响将有利于对生物标志物及药物靶点的认知与开发。现将该家族的主要特点、作用机制及在多种癌症中的表达情况作一综述。

Chitinase 3-like 1基因-329 G/A多态性与冠心病相关性的研究

目的:探讨Chitinase 3-like 1基因-329 G/A多态性(rs10399931)与中国汉族人群冠心病的相关性。方法:收集189例冠心病患者的临床资料,应用连接酶检测反应(LDR)分析rs10399931各基因型,并与230例非冠心病者(对照组)进行比较。冠心病组按照发病年龄分为早发冠心病组(男性<55岁,女性<65岁)与非早发冠心病组,按照冠状动脉狭窄≥50%的支数分为1、2、3支血管病变亚组进行分析。结果:冠心病组与对照组rs10399931均存在CC、CT和TT 3种基因型;冠心病组与对照组CC、CT和TT基因型频率分别为39.7%、46.0%、16.3%及43.0%、43.9%、13.0%,C、T等位基因频率分别为62.7%、37.3%和65.0%、35.0%,两组间各基因型及等位基因频率差异无统计学意义(均P>0.05)。早发冠心病组、非早发冠心病组及对照组间rs10399931各基因型及等位基因频率差异无统计学意义(均P>0.05)。1、2、3支冠状动脉病变亚组间rs10399931各基因型分布频率差异无统计学意义(P>0.05)。结论:Chitinase 3-like 1基因-329 G/A多态性与汉族冠心病发病无相关性。

马铃薯Y病毒辅助成分蛋白酶与加工番茄OPA3-like蛋白结合及其定位研究

为了研究加工番茄OPA3-like蛋白与马铃薯Y病毒(Potato virus Y,PVY)的辅助成分蛋白酶(helper component proteinase,HC-Pro)的相互作用,进行了2种蛋白的结合及其定位研究。通过PCR的方法从加工番茄中克隆OPA3-like基因,同时利用Blast方法对其分析,进行了OPA3-like蛋白基因氨基酸序列同源性分析。通过构建双分子荧光互补载体pSPYCE-35S-OPA3-like和pSPYNE-35S-HC-Pro,利用农杆菌介导法共侵染洋葱表皮细胞60 h后,在共聚焦显微镜下观察洋葱表皮细胞中的黄色荧光。结果显示:加工番茄OPA3-like基因ORF长510 bp,编码169个氨基酸,编码蛋白属OPA3蛋白家族。激光共聚焦显微镜下可观察到沿细胞膜呈小泡状分布黄色荧光,说明PVY的HC-Pro蛋白与加工番茄OPA3-like蛋白结合并且共定位在细胞膜附近。这为进一步研究PVY的HC-Pro蛋白与加工番茄OPA3-like蛋白的相互作用机制奠定了一定的基础。

牙鲆PIK3R3-like基因的表达分析及重组表达

本文从牙鲆(Paralichthys olivaceus)中鉴定得到了PI3K家族的成员之一——PIK3R3-like,并分析其可能为PIK3R3的亚型。由于其部分外显子在转录或修饰时被删除,导致该基因会产生一种较短的mRNA(PoPIK3R3-like-Ⅱ),与完整的转录组相比缺失21 nt。对PoPIK3R3-like的可变剪接长链(PoPIK3R3-like-Ⅰ)进行序列分析发现该基因包含一个1425 bp的开放性阅读框,共编码474个氨基酸,编码的蛋白共包含三个结构域,分别是一个nSH2结构域、一个iSH2结构域和一个cSH2结构域。同源性分析结果显示PoPIK3R3-like-Ⅰ、PoPIK3R3-like-Ⅱ均与其它硬骨鱼的PIK3R3-like相似度较高,系统进化分析显示PoPIK3R3-like与硬骨鱼聚为一支,与两栖类、鸟类和哺乳类相距较远。作者通过qRT-PCR检测了PoPIK3R3-like在各组织中的表达分布,发现其在牙鲆的各组织中均有表达,但在鳃和脑中表达量较高,使用迟缓爱德华氏菌侵染牙鲆鳃细胞及单核-巨噬细胞后,PoPIK3R3-like表达量分别呈现先上升再下降和显著上升的趋势,提示该基因在牙鲆抵抗迟缓爱德华氏菌感染相关的免疫应答中可能发挥作用。利用原核表达的方法在大肠杆菌中异源表达了PoPIK3R3-like-Ⅰ的重组蛋白,SDS-PAGE结果显示得到的重组蛋白与预测大小一致,蛋白纯化结果显示得到的蛋白纯度较高,可用于下一步PIK3R3-like基因功能的相关研究。

Caspase 3-like蛋白酶和TaeMCAⅡ基因对小麦胚乳细胞PCD的影响

对正常生长和淹水处理的华麦8号胚乳细胞发育过程中Caspase 3-like蛋白酶活性和TaeMCAⅡ基因表达量进行了研究。结果显示,正常生长条件下,在胚乳细胞发生PCD早期(花后12d),Caspase 3-like蛋白酶活性较强,且该酶主要定位于胚乳细胞中的淀粉质体上;整个胚乳细胞PCD过程中TaeMCAⅡ基因表达量变化不大。在淹水胁迫处理下,胚乳TaeMCAⅡ基因表达量和Caspase 3-like蛋白酶活性比同期正常处理高。上述研究结果表明,Caspase 3-like蛋白酶参与了正常生长的胚乳细胞PCD过程;淹水胁迫会导致TaeMCAⅡ基因超表达和Caspase 3-like蛋白酶活性升高,这可能是胚乳细胞PCD进程提前的原因之一。

Serum chitinase-3-like protein 1 is a biomarker of liver fibrosis in patients with chronic hepatitis B in China

Background:Serum chitinase-3-like protein 1(CHI3L1)is a potential biomarker for fibrosis assessment.We aimed to evaluate serum CHI3L1 as a noninvasive diagnostic marker for chronic hepatitis B virusrelated fibrosis.Methods:Serum CHI3L1 levels were measured by ELISA in 134 chronic hepatitis B(CHB)patients.Significant fibrosis was defined as a liver stiffness>9.7 kPa.The performance of CHI3L1 was assessed and compared to that of other noninvasive tests by receiver operating characteristic(ROC)analysis.Results:Serum CHI3L1 levels were significantly higher in CHB patients with significant hepatic fibrosis(≥F2)than in those without significant hepatic fibrosis(<F2)(56.5 ng/mL vs.81.9 ng/mL,P<0.001).In CHB patients,the specificity and sensitivity of CHI3L1 for predicting significant fibrosis were 75.6%and 59.1%,respectively,with a cut-off of 76.0 ng/mL and an area under the ROC curve of 0.728(95%CI:0.637–0.820).Conclusions:Serum CHI3L1 levels could be an effective new serological biomarker for the diagnosis of liver fibrosis.Moreover,CHI3L1 is feasible in monitoring disease progression.

Plasma chitinase 3-like 1 is persistently elevated during first month after minimally invasive colorectal cancer resection

AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plasma bank who underwent elective MICR for whom preoperative(PreO p), early postoperative(PostO p), and 1 or more late PostO p samples [postoperative day(POD) 7-27] available were included. Plasma CHi3L1 levels(ng/m L) were determined in duplicate by enzyme linked immunosorbent assay. RESULTS: PreOp and PostOp plasma sample were available for 80 MICR cancer patients for the study. The median PreOp CHi3L1 level was 56.8 CI: 41.9-78.6 ng/mL(n = 80). Significantly elevated(P < 0.001) median plasma levels(ng/mL) over PreOp levels were detected on POD1(667.7 CI: 495.7, 771.7; n = 79), POD 3(132.6 CI: 95.5, 173.7; n = 76), POD7-13(96.4 CI: 67.7, 136.9; n = 62), POD14-20(101.4 CI: 80.7, 287.4; n = 22), and POD 21-27(98.1 CI: 66.8, 137.4; n = 20, P = 0.001). No significant difference in plasma levels were noted on POD27-41. CONCLUSION: Plasma CHi3L1 levels were significantly elevated for one month after MICR. Persistently elevated plasma CHi3L1 may support the growth of residual tumor and metastasis.

Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 exhibits tumor-suppressing effects in hepatocellular carcinoma cells

BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.

AmphiSox1/2/3-like基因的系统进化学分析和在文昌鱼胚胎发育中的表达

为了研究文昌鱼胚胎发育中神经管发育的分子机制和脊椎动物中枢神经系统的起源,我们筛选和分析了与佛罗里达文昌鱼AmphiSox1/2/3基因同源的青岛文昌鱼AmphiSox1/2/3-like 基因,对其推测的氨基酸序列与17个脊椎动物和无脊椎动物的同源基因进行了系统的进化学分析,并利用胚胎整体原位杂交技术结合系列组织学切片技术,对该基因在青岛文昌鱼胚胎发育中的时空表达模式进行了系统的研究.AmphiSox1/2/3-like在原肠胚早中期开始在背部上胚层和预定神经外胚层中明显表达.在神经胚早期其表达定位于神经板.此后,表达区域位于形成中的神经管和胚胎中、后部分化中的神经管的两侧,其表达的水平从前向后逐渐下调和终止.此外,该基因在神经胚晚期和幼虫期的消化道壁中也有明显表达.研究结果显示,该基因与文昌鱼的神经分化和消化道分化密切相关.

Chitinase 3-like 1 secreted by peritumoral macrophages in esophageal squamous cell carcinoma is a favorable prognostic factor for survival

AIM To identify whether chitinase 3-like 1(CHI3 L1) serves as a suitable biomarker for the prognosis of esophageal squamous cell carcinoma(ESCC) and to analyze this protein's cellular source.METHODS An ELISA was conducted to detect the concentration of CHI3 L1 in the serum of 150 ESCC patients diagnosed between January 2001 and February 2005. The prognostic relevance of CHI3 L1 was evaluated by a Kaplan-Meier and Cox regression analysis. The immunohistochemistry was reanalyzed,and fluorescent staining was utilized to explore the cellular origins of CHI3 L1. We stimulated monocyte-derived macrophages(MDMs) with either IL-6 or the supernatant of the ESCC cell line Eca-109 and later investigated the level of CHI3 L1 by q PCR and ELISA.RESULTS The level of serum CHI3 L1 was higher in older patients(≥ 60) than in patients under the age of 60(P = 0.001). The patients with higher levels of CHI3 L1 had a significantly shorter overall survival,whereas the traditional markers,carcinoembryonic antigen and squamous cell carcinoma antigen,were less effective(P > 0.05). A multivariate Cox analysis(P = 0.001) indicated that CHI3 L1 was an independent prognostic factor for ESCC patients. Peritumoral macrophages in ESCC exhibited high levels of CHI3 L1. Interleukin-6(IL-6) and the supernatant of Eca-109 containing IL-6 stimulated MDMs to secrete CHI3 L1. The serum concentration of CHI3 L1 in the ESCC patients showed a weak correlation with the laboratory inflammatory parameters neutrophil(NEU,P = 0.045),neutrophil/lymphocyte rate(NLR,P = 0.016),and C-reactive protein(CRP,P < 0.001).CONCLUSION Our study first established a connection between the pretreated CHI3 L1 and patients with ESCC,and the serum CHI3 L1 was primarily secreted by ESCC-surrounded macrophages.

Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase

AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.

sp^(3)-like defect structure of hetero graphene-carbon nanotubes for promoting carrier transfer and stability

Three-dimensional(3 D) hybrid of nanocarbons is a very promising way to the high-performance design of electrocatalysis materials.However,sp^(3)-like defect structure,a combination of high strength and conduction of graphene and carbon nanotubes(CNTs) is rarely reported.Herein,3 D neural-like hybrids of graphene(from reduced graphene oxide) and carbon nanotubes(CNTs) have been integrated via sp^(3)-like defect structure by a hydrothermal approach.The sp^(3)-like defect structure endows 3 D nanocarbon hybrids with an enhanced carrier transfer,high structural stability,and electrocatalytic durability.The neural-like structure is shown to demonstrate a cascade effect of charges and significant performances regarding bio-electrocatalysis and lithium-sulfur energy storage.The concept and mechanism of "sp^(3)-like defect structure" are proposed at an atomic/nanoscale to clarify the generation of rational structure as well as the cascade electron transfer.

猪Ⅰ型补体受体与C3b活性片段相互结合的体外检测

【目的】检测猪红细胞类补体受体Ⅰ型(Complement receptor 1-like,CR1-like)与C3b活性片段能否发生结合,以期为阐明猪红细胞发挥免疫粘附功能的分子机理提供科学数据。【方法】利用前期已构建的CR1-like_(3-6)、CR1-like_(8-11)功能域片段的重组质粒建立酵母双杂交检测体系,运用酵母共转化的方法将诱饵质粒(重组pGBKT7-CR1-like)与捕获质粒(重组pGADT7-C3b)共同转入Y2HGold酵母细胞中,分别利用一缺平板SD/-Leu、SD/-Trp和二缺平板SD/-Leu/-Trp(DD0)严格筛选共转化成功的酵母细胞,再根据报告因子是否表达来鉴别转化子在SD/-Leu/-Trp/X-α-Ga1 (DDO/X)、SD/-Leu/-Trp/X-α-Ga1/Aba (DDO/X/A)二缺培养板上的生长情况,并结合菌落的颜色变化现象综合判定CR1-like活性片段与补体C3b在酵母细胞中是否发生相互结合;然后运用免疫沉淀技术分离酵母细胞中CR1-like与C3b结合复合物,并对该复合物的特异性进行Western blot鉴定。【结果】试验成功将pGBKT7-CR1-like与pGADT7-C3b基因共转入Y2HGold酵母细胞。共转化的酵母克隆在SD/-Leu、SD/-Trp、DDO平板上能够正常生长,在DDO/X、DDO/X/A平板上正常生长且菌落呈现蓝色,由此表明,试验中酵母双杂交系统建立成功,并通过试验获得了阳性酵母克隆。共同转化了 pGBKT7-CR1-like和pGADT7-C3b质粒的酵母菌落PCR反向鉴定结果显示,在共转化的酵母菌中含有目的基因CR1-like_(3-6)和CR1-like_(8-11),共转化组的质粒酶切后出现C3b基因片段,与设计大小一致,说明重组质粒成功共转化入酵母细胞中。免疫沉淀试验中应用pGBKT7载体的标签抗体c-Myc沉淀酵母细胞中的融合蛋白,以c-Myc为一抗进行Western blot检测发现,单独转化了pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)的融合蛋白在 50 kD 处出现特异性条带;共转化 pGBKT7-CR1-like_(3-6)+pGADT7-C3b和共转化pGBKT7-CR1-like(8-11)+pGADT7-C3b的酵母融合蛋白在83kD处出现特异性条带;以HA单克隆抗体为一抗进行Western blot检测时,在pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)融合蛋白中没有出现特异性条带,只有3、4泳道中共转化的酵母融合蛋白在83kD处出现特异性条带,表明在Y2HGold酵母细胞中存在CR1-like与C3b识别结合的复合物。使用CR1-like单克隆抗体沉淀酵母细胞中的融合蛋白,以CR1-like单克隆抗体为一抗进行Western blot检测发现,单独转化了pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)的融合蛋白在50kD处出现特异性条带;共转化pGBKT7-CR1-like_(3-6)+pGADT7-C3b和共转化pGBKT7-CR1-like_(8-11)+pGADT7-C3b的酵母融合蛋白在83kD处出现特异性条带;以C3单克隆抗体为一抗进行Western blot检测发现,在pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)融合蛋白中没有出现特异性条带,泳道3、4所示只有共转化的酵母融合蛋白在83 kD处出现特异性条带,表明在Y2HGold酵母细胞中存在具有生物活性的CR1-like与C3b识别结合的复合物。通过多个单克隆抗体杂交结果,可看出诱饵质粒的表达产物CR1-like_(3-6)、CR1-like_(8-11)片段与捕获质粒的表达产物C3b片段可在酵母细胞内发生结合。【结论】猪红细胞CR1-like发挥免疫粘附功能的识别配体为C3b,为猪红细胞CR1-like功能域分子结构的进一步解析提供了重要数据依据。

RAVL1 Activates IDD3 to Negatively Regulate Rice Resistance to Sheath Blight Disease

Sheath blight disease (ShB) has a severe impact on the production of rice. ABI3/VP1-like 1(RAVL1) negatively regulated the rice defense mechanism against ShB, however, this regulatorymechanism is not clearly understood. In this study, we identified that indeterminate domain 3 (IDD3) waspositively regulated by RAVL1. Further, chromatin immunoprecipitation (ChIP) assay, yeast one-hybridassay and transient expression assay indicated a direct binding between RAVL1 and the IDD3 promoterregion. IDD3 was ubiquitously expressed in different tissues and at different stages, and its expressionwas significantly enhanced by Rhizoctonia solani infection. IDD3 exhibited transcription activation activityin yeast and IDD3-GFP was found to be localized in the nucleus. IDD3 mutants exhibited no significantdifferences in response to ShB, while IDD3 overexpressors were more susceptible to ShB compared withwild type (WT) plants. Furthermore, IDD3 repressors were less susceptible to R. solani than WT plants.Interestingly, the expression of brassinosteroid-related genes (D2, D11 and BRI1) was lower in IDD3repressors and higher in IDD3 overexpressors compared with WT. However, the ChIP assay revealedthat IDD3 did not directly bind to the D2 and D11 promoters. Overexpression of IDD3 in BRI1 mutantd61-1 inhibited the activity of IDD3, reducing its susceptibility to ShB compared with IDD3 overexpressorand WT plants, indicating that IDD3 negatively regulated the rice defense mechanism against ShB by activatingthe BR signaling pathway. Thus, our analyses provided information to enhance the understanding of therice defense mechanism against ShB.