Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

The “Sino-India Monitor on INDCs Adequacy and Necessity”(SIMIAN) initiative

The papers in this special topic of Sino-India Monitor on NDCs,authored by a select group of researchers from both China and India,provide a perspective on areas of common interest for societies in both countries as well as a focus on common objectives defining global action.

Severer nodular lesion in white matter than in gray matter in simian immunodeficiency virus-infected monkey, but not closely correlated with viral infection

Immune cell accumulation and white matter anomaly are common features of HIV(human immunodeficiency virus)-infected patients in combination antiretroviral therapy(cART) era.Neuroimaging tests on cART treated patients displayed prominent diffuse white matter lesions.Notably,immune cell nodular lesion(NL) was a conspicuous type of pathological change in HIV/SIV(simian immunodeficiency virus) infected brain before cART.Therefore,we used SIV infected brain to investigate the distribution of those NLs in gray and white matters.We found a significant higher number of NLs in white matter than that in gray matter.However,virus infection correlated with macrophage NLs but not with microglia NLs,especially in white matter.In addition,NLs interrupted white matter integrity more severely,since even tiny nodules could disconnect nerve fibers in white matter tracts.In the gray matter with dense myelinated axons,NLs obviously encroached those fibers;in the area of few myelinated axons,small nodules well co-localized with extracellular matrix between neurons.

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus （SIV） infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction （RT-PCR）. The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEMx174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA （b-DNA） assays or real-time PCR assays based on TaqMan probes.

The default mode network is affected in the early stage of simian immunodeficiency virus infection:a longitudinal study

Acquired immune deficiency syndrome infection can lead to cognitive dysfunction represented by changes in the default mode network.Most recent studies have been cross-sectional and thus have not revealed dynamic changes in the default mode network following acquired immune deficiency syndrome infection and antiretroviral therapy.Specifically,when brain imaging data at only one time point are analyzed,determining the duration at which the default mode network is the most effective following antiretroviral therapy after the occurrence of acquired immune deficiency syndrome.However,because infection times and other factors are often uncertain,longitudinal studies cannot be conducted directly in the clinic.Therefore,in this study,we performed a longitudinal study on the dynamic changes in the default mode network over time in a rhesus monkey model of simian immunodeficiency virus infection.We found marked changes in default mode network connectivity in 11 pairs of regions of interest at baseline and 10 days and 4 weeks after virus inoculation.Significant interactions between treatment and time were observed in the default mode network connectivity of regions of interest pairs area 31/V6.R and area 8/frontal eye field(FEF).L,area 8/FEF.L and caudal temporal parietal occipital area(TPOC).R,and area 31/V6.R and TPOC.L.ART administered 4 weeks after infection not only interrupted the progress of simian immunodeficiency virus infection but also preserved brain function to a large extent.These findings suggest that the default mode network is affected in the early stage of simian immunodeficiency virus infection and that it may serve as a potential biomarker for early changes in brain function and an objective indicator for making early clinical intervention decisions.

ELECTRON MICROSCOPIC OBSERVATION OF SIMIAN SARCOMA VIRUS TRANSFORMED NIH 3T3 CELLS IN VITRO

For electronic microscopic observation, we found SSV-transformed NIH 3T3 cells were different from non-transformed cells. In SSV-transformed NIH 3T3 cells nuclei cytoplasma ratio was increased and in cytoplasma the ribosomes (polyribosomes were attached to the swollen rough endoplasmic reticulum. It was likely that ribosomes were lined together functionally and structionally to produce specific protein (PDGF-like protein).

Purification and identification of simian parvovirus protein Vp2 expressed in E.coli

To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGE gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in E. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.

Simian virus 40 large tumor antigen forms specific complexes with p53 and pRb in human brain tumors

目的 探讨SV4 0早期区域基因编码产物大T抗原 (Tag)的表达及其与抑癌蛋白p53、pRb的相互作用在人脑肿瘤发生、发展中的意义。方法 采用免疫共沉淀结合银染色及Western印迹法检测 6 5例人脑肿瘤及 8例正常人脑组织中Tag的表达 ,并对 18例和 15例Tag阳性瘤组织分别检测Tag p53和Tag pRb复合物的形成。结果 Tag在 8例室管膜瘤及 2例脉络丛乳头状瘤中全部表达 ,垂体腺瘤Tag阳性率为 90 % (9/ 10 ) ,星形细胞瘤为 73% (11/ 15) ,脑膜瘤为 70 % (7/ 10 ) ,多形性胶质母细胞瘤为 50 % (4 / 8) ,髓母细胞瘤为 33% (2 / 6 ) ;5例少枝胶质细胞瘤、1例松果体细胞瘤及 8例正常人脑组织无Tag表达 ;检测 18例和 15例Tag阳性瘤组织 ,均发现Tag可与p53、pRb形成特异性复合物。结论 在人脑肿瘤组织中Tag不仅可以表达 ,而且还可与p53、pRb形成特异性复合物。Tag p53及Tag pRb特异性复合物的形成导致p53和pRb失活 ,这可能是SV4 0导致人脑肿瘤发生的一个重要机理。

Traditional Chinese Medicine etiology and pathogenesis of acquired immune deficiency syndrome in simian immunodeficiency virus-infected Chinese rhesus macaques

OBJECTIVE： To investigate the traditional Chinese Medicine （TCM） etiology and pathogenesis of ac- quired immune deficiency syndrome （AIDS） by 18-month observation of Chinese rhesus macaques infected with simian immunodeficiency virus （SIV） mac239. METHODS： Thirty-five healthy Chinese rhesus ma- caques were divided into a model group （n=30） and a control group （n=5）. The model was estab- lished by inoculating monkeys intravenously with SIVmac239. Changes in TCM symptoms after SIV in- fection within 18 months were then observed and recorded. Routine blood tests, SIV viral load, T-lym-phocyte subsets, plasma triiodothyronine （T3）, tet- raiodothyronine （14）, adrenocorticotropic hormone （ACTH） and cortisol （Cor） were tested periodically during the experiment. RESULTS： During the acute infection period of SIV, model monkeys temporarily showed clinical symp- toms such as diarrhea, dysphoria and slight weight loss. Decrease percentages of CD4~ T-lymphocytes were observed but levels of T3, 14, Cot, and ACTH were relatively unchanged. Monkeys in the model group during the early and middle periods of infec- tion showed no obvious symptoms, except few monkeys exhibited transient diarrhea and reduced food intake. All variables at this stage showed nor- mal fluctuations. In the middle period model group monkeys showed chronic and persistent diarrhea, weight loss, reduced food intake and low levels of 13 and Cot. In the late period, symptoms including emaciation, weight loss, listlessness, crouching in corners and low levels of T3appeared. CONCLUSION： The results suggest that the rhesus monkey SIWSAIDS model can be applied to re- search on TCM etiology and pathogenesis of AIDS. According to this model, the etiology of disease is the SIV virus. The pathogenesis manifests as the in- vasion of SIV virus, incubation of the virus, balance between virus and healthy ＂Qi＂, damage to spleen and kidney as the disease progressed, exhaustion of vitality and finally the failure of five zang and six fu organs.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain

Background The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07 BC. A highly replication-competent simian/human immunodeficiency viruses （SHIV） construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell （PBMC）, establish infection in Chinese rhesus macaque. Methods A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques （Macaca mulatta） was infected with one SHIV via intravenous infusion. Results One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection. Conclusions We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF 07BC recombinant strain.

Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40（SV40） is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine-homopurine duplex DNA（dsDNA） of SV40（4424-4440, gp6） was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5＇-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3＇（HP） containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structttre to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.

重庆四面山不同林分土壤抗蚀抗冲特征

为探讨重庆四面山地区不同林分土壤抗蚀抗冲特征,采用水浸试验和冲刷试验,计算土壤抗蚀指数与抗冲系数,对四面山4种林分类型(针叶林、阔叶林、针阔混交林和楠竹林),共9个不同植物组成的林地土壤抗蚀抗冲特征进行研究。结果表明:1)阔叶林的抗蚀指数最大,楠竹林抗蚀指数最小,随着土壤深度的增加,土壤抗蚀性能减弱,天然针阔混交林土壤抗蚀指数上下层差异最大(1.92倍)。2)二次多项式函数能高度拟合不同林分土壤抗蚀指数与水浸时间的关系(R2>0.95),随着水浸时间的增加,不同林分土壤抗蚀性能下降。3)土层越深,土壤抗冲系数越大,抗冲性能越强,坡面上层土壤抗冲系数为下层的1.05~5.79倍。阔叶林的抗冲性优于其他林分。4)≤1和>1~3 mm根径的根系总根长与根长密度与土壤抗蚀指数显著正相关(P<0.05),与土壤抗冲性呈显著负相关(P<0.05)。土壤抗蚀性和抗冲系数与总根质量、根质量密度呈显著负相关(P<0.05)。研究结果可为重庆四面山水土保持措施布设、选择合理的植被恢复模式及配置方式提供理论依据。

秭归丝绵茶鲜叶非挥发性成分及丝形态结构分析

为研究丝绵茶品质形成特点,以秭归当地生产丝绵茶的茶树种‘丝绵土茶’6个嫩度部位叶片为研究对象,通过超高效液相色谱飞行时间质谱、电感耦合等离子体发射光谱和扫描电子显微镜,分析其非挥发性代谢成分、矿质元素、“丝”结构及数量特点。结果表明,丝绵茶鲜叶中的非挥发性代谢成分在嫩度较高的叶位富集较多,其中一叶和二叶(L1、L2)中氨基酸、生物碱、儿茶素和香气糖苷物质含量较高;茶氨酸、有机酸和黄酮类在第三叶(L3)富集最多。而嫩度较低的五六叶中非挥发性成分含量相对较低。不同嫩度鲜叶原料积累的各种元素具有明显差异,氮、磷、钾、锌和铜元素在嫩度较高的一、二叶位(L1、L2)含量较高,L1分别为32.41 mg/g、4.53 mg/g、15.65 mg/g、45.45μg/g、10.75μg/g,L2分别为30.60 mg/g、3.70mg/g、14.12mg/g、35.82μg/g、9.02μg/g;而铁、锰和钙在成熟叶位含量较高。通过扫描电镜观察发现丝绵茶鲜叶中“丝”结构包括三股卷曲和单股卷曲两种形式,分布在主脉和侧脉的维管束木质部内螺纹或环纹导管;且二、三、四叶中“丝”的数量较芽头和一叶多。

论近代中国的汉武帝评价问题

汉武帝统治时期的一系列政策,不但对西汉朝局走向影响极大,而且很大程度上形塑着之后中国政治、经济与文化的格局。在近代变局下,人们也常从不同角度对之进行评价。近代民主思想在中国广泛传播之际,时人不仅从政治治理的实际效果来评价汉武帝的得失,还十分在意剖析其种种作为是否仅为了巩固个人统治、满足一己私欲。在这个意义上,近代关于汉武帝的评价,某种程度上也体现着人们对于现实当中的为政者的评判标准与期望值。此外,伴随着新思潮的迅速普及,社会主义成为绝大多数中国知识分子与政治人物思考历史与现实问题时的重要凭借,在此背景下,人们很容易基于当代立场把汉武帝时期的社会经济政策视为近代社会主义在中国古代的某种先声。当然,这种跨越古今的遥想,并不能替代对于汉武帝所作所为的具体分析。对于汉武帝的评价,堪称近代中国政治文化不容忽视的组成部分。

猴副流感病毒RT-PCR检测方法的建立及初步应用

为对易感动物以及相关生物材料和生物制品进行猴副流感病毒(SV5)的快速病原检测,根据SV5病毒SH基因序列,设计了1对引物,建立了检测SV5的RT-PCR方法,通过优化确定了RT-PCR体系的最佳工作条件。核酸序列分析显示,所扩增片段与GenBank数据库中报道的SV5病毒株WR-21005序列同源性达到97%,证明该方法特异。敏感性分析显示,该方法可检测的最小RNA浓度为2.1 ng/μL,能检出的最小病毒滴度为3.98CCID50/0.1 mL。初步将该方法运用于猴、地鼠、常用传代细胞和猴源生物制品的检测,在所检测的样品中均未检出猴副流感病毒核酸序列。结果表明,所建立的RT-PCR方法可用于猴副流感病毒(SV5)的诊断和分子流行病学调查。

猴免疫缺陷病毒（SIV）猴模型的建立

应用ＳＩＶｍａｃ毒株感染中国恒河猴１３只，感染剂量为病毒液的１０－１～２×１０－５；感染食蟹猴４只，感染剂量为１０－２。感染后２周出现各种症状和体征如皮疹，浅表淋巴结肿大，脾肿大，血象白细胞总数下降，出现异常淋巴细胞和中性白细胞。感染后期Ｔ４下降，Ｔ４、Ｔ８比例倒置等。从外周血淋巴细胞和血浆分离病毒阳性，血清抗体上升。淋巴结组织切片呈规律性改变，即淋巴滤泡增生－滤泡耗竭－淋巴组织耗竭或逐渐恢复。感染后２．５个月有急性死亡病例，以后呈散在死亡例，尸检还发现机遇性感染如肺寄生虫，肺、肝巨细胞病毒感染等。

重庆四面山常绿阔叶林建群种种子雨、种子库研究

对重庆四面山常绿阔叶林建群种种子雨、种子库的研究表明,建群种早期和晚期的种子雨无活力;种子偏早或偏晚成熟及大籽粒的树种,其种子雨被取食的比例大;种子雨、有活力种子雨、种子库三者的数量变化不一致;有活力种子雨量较大的栲、石栎、小叶青冈、扁刺栲、香桂等,其种子库密度在早期以近几何级数的方式增长,元江栲、银木荷种子库小,存在时间短,翌年无一年生萌发苗;种子库数量动态、消减率动态决定于种子被取食的强度、种子衰老的速度以及种子对病菌、逆境的抗性和种子萌发的整齐性.

中研2号治疗HIV/AIDS的临床和实验研究

目的:观察中研2号对HIV/AIDS患者和猴艾滋病毒SIVmac251感染猴的疗效,评价该方抗艾滋病毒和对机体的保护作用。方法:根据统一的诊断标准,对确诊的HIV/AIDS患者给予中研2号治疗,疗程为3个月,从免疫功能、病毒载量和临床症状体征等方面进行疗效判定;采用猴艾滋病模型,以病毒分离、酶联免疫法及流式细胞仪检测 SIV水平、CD_4^+、CD_8^+细胞及新喋呤水平,并进行病理学检查。结果:中研2号对治疗HIV/AIDS患者临床有效率为45％～55％;对猴艾滋病模型不仅能降低感染猴病毒血症及SIVp27抗原水平,同时能持续提高CD_4^+细胞数量,降低血清新喋呤含量,与叠氮胸苷阳性药对照结果近似,与生理盐水比较,差异有显著性。病理检查显示中研2号能激活并促进淋巴结中心细胞的增殖。结论:中研2号具有一定的临床效果,可抑制感染猴血浆病毒血症及保护机体免疫功能。

大豆皂甙复合物抑制猴免疫缺陷病毒活性的观察

以猴免疫缺陷病毒（ＳＩＶ）为靶病毒和相应的ＣＥＭｘ１７４细胞系统为模型，以ＡＺＴ为阳性对照药物，观察了从大豆提取的皂甙复合物及大豆胰蛋白酶抑制剂对细胞病变的影响．结果表明，大豆皂甙复合物具有明显的抗ＳＩＶ作用，而大豆胰蛋白酶抑制剂则无抗ＳＩＶ作用．