Improved survival in tumor-bearing SCID mice treated with interferon-γ-inducible protein 10 (IP-10/CXCL10)

Tumor growth requires angiogenesis, which in turn requires an imbalance in the presence of angiogenic and angiostatic factors. We have shown that the CXC chemokine family, consisting of members that are either angiogenic or angiostatic, is a major determinant of tumor-derived angiogenesis in non-small-cell lung cancer (NSCLC). Intratumor injection of interferon-inducible protein 10 (IP-10, or CXCL10), an angiostatic CXC chemokine, led to reduced tumor growth in a SCID mouse model of NSCLC. In this study, we hypothesized that treatment with CXCL10 would, by restoring the angiostatic balance, improve long-term survival in NSCLC-bearing SCID mice. To test this hypothesis, A549 NSCLC cells were injected in the subcutis of the flank, followed by intratumor injections with CXCL10 continuously (group I), or for ten weeks (group II), or a control group (human serum albumin). Median survival was 169, 130, and 86 days respectively (P<0.0001). We extended these studies to examine the mechanism of prolonged survival in CXCL10-treated mice. CXCL10 treatment inhibited lung metastases, but was dependent upon continued treatment, and was associated with an increased rate of apoptosis in the primary tumor, with no direct effect on the proliferation of the NSCLC cells. Furthermore, the inhibition of lung metastases was due to the angiostatic effect of CXCL10 on the primary tumor, since the rate of apoptosis within lung metastases was unaffected. These data suggest that anti-angiogenic therapy of human lung cancer should be continued indefinitely to realize persistent benefit, and confirms the anti-metastatic capacity of localized angiostatic therapy.     

m6A甲基转移酶家族Fiona/METT-10/METTL16的功能

RNA碱基上的化学修饰在其功能的精准调节中发挥关键作用,其中m⁶A是自然界中最普遍的RNA修饰之一,且该修饰在调控RNA稳定性、pre-mRNA剪接、翻译等方面具有重要功能。在真核生物中,m⁶A修饰主要由两种甲基转移酶完成,其在哺乳动物中分别命名为METTL3和METTL16。与METTL3相似,METTL16的底物多种多样,包括pre-mRNA、rRNA、snRNA和lncRNA等,因此似乎难以用一种分子机理解释METTL16对不同RNA底物进行m⁶A修饰的功能。此外,METTL16还在翻译调控中发挥重要作用,但此过程不依赖其甲基转移酶活性,这进一步增加了高度保守的METTL16的功能复杂性。本综述总结了METTL16及其同源蛋白质的结构域、甲基化底物以及它们的潜在功能,着重阐述了在不同物种中关于METTL16研究结果的矛盾之处,并推测METTL16调控S-腺苷基甲硫氨酸(SAM)代谢的功能是趋同进化的一个潜在案例。     

Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

Death receptors of TNFSF10/TRAIL (tumor necrosis factor superfamily member 10) contribute to immune surveillance against virus-infected or transformed cells by promoting apoptosis. Many viruses evade antiviral immunity by modulating TNFSF10 receptor signaling, leading to persistent infection. Here, we report that hepatitis B virus (HBV) X protein (HBx) restricts TNFSF10 receptor signaling via macroautophagy/autophagy-mediated degradation of TNFRSF10B/DR5, a TNFSF10 death receptor, and thus permits survival of virus-infected cells. We demonstrate that the expression of the TNFRSF10B protein is dramatically reduced both in liver tissues of chronic hepatitis B patients and in cell lines transfected with HBV or HBx. HBx-mediated downregulation of TNFRSF10B is caused by the lysosomal, but not proteasomal, degradation pathway. Immunoblotting analysis of LC3B and SQSTM1, and microscopy analysis of tandem-fluorescence-tagged LC3B revealed that HBx promotes complete autophagy. Inhibition of autophagy with a pharmacological inhibitor and LC3B knockdown revealed that HBx-induced autophagy is crucial for TNFRSF10B degradation. Immunoprecipitation and GST affinity isolation assays showed that HBx directly interacts with TNFRSF10B and recruits it to phagophores, the precursors to autophagosomes. We confirmed that autophagy activation is related to the downregulation of the TNFRSF10B protein in liver tissues of chronic hepatitis B patients. Inhibition of autophagy enhanced the susceptibility of HBx-infected hepatocytes to TNFSF10. These results identify the dual function of HBx in TNFRSF10B degradation: HBx plays a role as an autophagy receptor–like molecule, which promotes the association of TNFRSF10B with LC3B; HBx is also an autophagy inducer. Our data suggest a molecular mechanism for HBV evasion from TNFSF10-mediated antiviral immunity, which may contribute to chronic HBV infection.     

甜菜增产菌P10菌株的鉴定

在新疆甜菜块根皮层内分离并筛选到对甜菜具有增产增糖作用的优化菌株Ｐ_１０。通过形态培养征和生理生化特性研究,将该菌株鉴定为短小芽孢杆菌（Ｂａｃｉｌｕｓｐｕｍｉｌｕｓ）。     

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL-10 is a potent anti-inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could up-regulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could down-regulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was up-regulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL-10 alleviates LPS-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL-10 can alleviate the LPS-induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down-regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.     

沉默GmATG10导致大豆免疫反应的激活

自噬途径是真核生物中普遍存在的物质降解及循环利用的保守机制,在真核生物的生长发育以及免疫反应等方面起着至关重要的作用。而ATG10在自噬体(autophagosomes)的形成过程中起着非常重要的作用。为探讨大豆(Glycine max) ATG10在免疫防御反应中的功能,本研究采用大豆豆荚斑驳病毒(bean pod mottle virus,BPMV)诱导的基因沉默技术(virus-induced gene silencing,VIGS)成功地在大豆中同时沉默ATG10的两个同源基因(GmATG10a和GmATG10b);通过黑暗诱导的碳饥饿处理以及GmATG8积累水平的Western blotting分析证明,同时沉默GmATG10a/10b可导致大豆叶片出现自噬缺陷;抗病性鉴定与激酶分析证明沉默GmATG10a/10b可通过负调控Gm MPK3/6激活而参与免疫反应,是大豆免疫反应的负调控因子。     

红花玉兰MwZFP10基因的克隆及功能分析

该研究选取了湖北五峰地区红花玉兰新品种‘娇红1号’为研究材料,基于实验室前期对红花玉兰转录组测序差异显著表达基因中筛选到的645bp花发育相关的核心片段,采用RACE技术克隆得到1个SUPERMAN类基因MwZFP10(GenBank登录号为MH037314)。生物信息学分析显示,MwZFP10基因全长为1 117bp,开放阅读框726bp,编码241个氨基酸,蛋白分子式为C_(1128)H_(1760)N_(322)O_(383)S_(14),分子量26.4kD,理论等电点4.42,含有一个C2H2型锌指结构域。对比分析发现MwZFP10属于SUPERMAN类锌指蛋白,具有N端的QALGGH和C端富含亮氨酸的L(I)DLXLR(K)L保守结构域。α螺旋(Hh)、延伸链(Ee)和β转角(Bt)构成红花玉兰MwZFP10蛋白的基本结构,蛋白的三级结构具有典型的锌指空间构型。MwZFP10蛋白为亲水性蛋白,可能定位于细胞核中。半定量和实时荧光定量结果显示,MwZFP10在花器官、叶片和苞片中均有表达,且花器官的雌蕊中表达量较高。过表达MwZFP10转基因拟南芥表现为植株矮小且不育,叶片卷缩,花器官发育畸形等特征。研究认为,MwZFP10基因在植物花器官的生长和发育中起重要作用。     

利用ARIMA模型对木聚糖酶F/10和G/11的进化过程分析

目的:利用ARIMA模型对木聚糖酶(Xylanase)F/10和G/11家族的进化过程进行分析,并分析两家族中同时含量较高的氨基酸在进化中的特征.方法:计算出每条序列中均含量较高的几种氨基酸,利用SAS时间序列中的ARIMA模型,选取木聚糖酶F/10和G/11两个家族的各十条进化阶段不同的序列,设计一个时间序列实验进行分析.结果:F/10家族的进化是平稳的,甘氨酸含量逐渐降至7%左右,缬氨酸的含量稳定的保持在7%上下,而G/11家族的进化存在较大波动,甘氨酸含量升至14%左右,丙氨酸含量相对于F/10家族稳定.结论:F/10家族的进化比G/11家族稳定,甘氨酸在两家族进化存在明显差异,这对研究木聚糖酶分子的进化以及同义密码子的偏好性具有一定的指导意义.     

TMED10对骨关节炎中TGF-β/Smads信号通路的影响

摘要 目的：探究跨膜P24转运蛋白10（TMED10）对骨关节炎中转化生长因子-β（TGF-β）/Smads信号通路的影响。方法：将大鼠软骨细胞分为Control组、IL-1β组、NC-sh+IL-1β组、TMED10-sh+IL-1β组、NC-OE+IL-1β组和TMED10-OE+IL-1β组。Control组和IL-1β组细胞不进行转染处理,NC-sh+IL-1β组、TMED10-sh+IL-1β组、NC-OE+IL-1β组和TMED10-OE+IL-1β组细胞分别转染NC-sh、TMED10-sh、NC-OE和TMED10-OE。转染后,除Control组之外,其他组软骨细胞均用白介素-1β（IL-1β）（10 ng/mL）处理24 h模拟OA软骨细胞的病理微环境。通过MTT法检测细胞增殖,通过TUNEL法检测细胞凋亡。采用前交叉韧带切断加内侧半月板部分切除法建立OA大鼠模型,将OA大鼠分为OA组、OA+NC-sh组和OA+TMED10-sh组（n=12）,Sham组（n=12）大鼠进行假手术操作。Sham组和OA组大鼠关节腔内注射40 μL生理盐水,OA+NC-sh组和OA+TMED10-sh组大鼠关节腔内分别注射40 μL的NC-sh和TMED10-sh（滴度为1×10⁹ TU/mL）,每周注射1次,共4周。采用番红O/固绿染色法评价膝关节软骨形态,采用TUNEL法检测软骨细胞凋亡。采用RT-qPCR检测软骨细胞和大鼠关节软骨中TMED10的mRNA水平,采用Western blot检测软骨细胞和大鼠关节软骨中TMED10、TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3、Collagen II和MMP-13的蛋白表达水平。结果：与Control组比较,IL-1β组TUNEL阳性率、TMED10 mRNA和TMED10、MMP-13蛋白水平均升高（P<0.05）,相对细胞活力以及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II的蛋白水平均降低（P<0.05）。与IL-1β组和NC-sh+IL-1β组比较,TMED10-sh+IL-1β组TUNEL阳性率、TMED10 mRNA水平和TMED10、MMP-13的蛋白水平均降低（P<0.05）,相对细胞活力以及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II的蛋白水平均升高（P<0.05）。与IL-1β组和NC-OE+IL-1β组比较,TMED10-OE+IL-1β组TUNEL阳性率、TMED10 mRNA水平和TMED10、MMP-13的蛋白水平均升高（P<0.05）,相对细胞活力以及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II的蛋白水平均降低（P<0.05）。与Sham组比较,OA组大鼠的OARSI评分、TUNEL阳性率、TMED10 mRNA水平以及TMED10和MMP-13的蛋白水平均升高（P<0.05）,TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II蛋白水平均降低（P<0.05）。与OA组和OA+NC-sh组比较,OA+TMED10-sh组大鼠的OARSI评分、TUNEL阳性率、TMED10 mRNA水平以及TMED10和MMP-13的蛋白水平均降低（P<0.05）,TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和Collagen II蛋白水平均升高（P<0.05）。结论：本研究表明TMED10可能通过抑制TGF-β/Smads信号通路导致软骨细胞丢失和软骨退变,TMED10/TGF-β/Smads信号通路可能是治疗OA的新靶标。     

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.     

PKCγ过表达诱导C3H10T1/2细胞生长失控的初探

通过DNA重组构建蛋白激酶C_γ(PKC_γ)亚类的重组质粒并经基因转染技术和DNA印迹、蛋白质印迹与PKC活性分析,获得了过表达PKC_γ的C₃H₁₀T_1/2细胞——NCP4.NCP4细胞生长速率提高,流式细胞光度术检测表明,NCP4细胞G1期百分率下降,S期和G2+M期百分率升高,与对照组细胞相比,血清依赖性明显下降,贴壁依赖性降低,在软琼脂中形成小集落,出现部分转化表型.进一步检测,首次观察到NCP4细胞中癌基因c-sis表达明显增强,这可能是NCP4细胞血清依赖性下降的分子机理之一.实验表明,在正常C₃H₁₀T_1/2细胞中PKC_γ的过表达可直接导致细胞增殖加速并可诱导出现部分转化特征.     

穗醋栗MYB10基因克隆结构分析及功能验证

为探究花色苷合成相关转录因子MYB10在不同颜色穗醋栗果实着色差异的分子机理,通过cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)法从果实花青素含量有较大差异的黑穗醋栗(Ribes nigrum L.)、红穗醋栗(Ribes rubrum L.)和白穗醋栗(Ribes album L.)中分别克隆出MYB10基因,分别命名为RnMYB10 (KY786107)、RrMYB10 (KY786108)和RaMYB10(MW660848)。系统发育分析表明,RnMYB10和RrMYB10在进化上具有同源性。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)结果表明:黑穗醋栗各时期果实中MYB10表达量均高于红穗醋栗且远远高于白穗醋栗。随着果实直径加大颜色加深,RnMYB10和RrMYB10表达量呈现先上升后下降的趋势(在果实转色程度75%时达到最大值),RaMYB10表达量极低,几乎无表达。过表达RnMYB10和RrMYB10的拟南芥呈现紫色叶柄和叶片,过表达RaMYB10的拟南芥无明显变化。说明...     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.