On-going generation of multiple forms of HIV-1 intersubtype recombinants in the Yunnan Province of China

Objectives: To investigate the molecular epidemiology of HIV in China's Yunnan Province, where the initial HIV-1 outbreak among injecting drug users (IDU) occurred in 1989, and to analyse the genesis and interrelationship of the epidemic with that in surrounding areas. Design: A molecular epidemiological investigation was conducted among IDU in three prefectures in Yunnan Province, including Wenshan (east), Honghe (southeast) and Dehong (west). Methods: Thirty-nine specimens were collected from consenting IDU in 2000-2001. The nucleotide sequences of 2.6 kb gag-RT and 340 base pair (bp) env (C2/V3) regions were determined. Phylogenetic tree and recombination breakpoint analyses were performed. Results: The circulating recombinant form (CRF), CRF08_BC, predominated in east Yunnan near Guangxi Province (89% in Wenshan and 81% in Honghe), whereas it was not detected in Dehong(0/14) in the west. In contrast, 71% (10/14) of the Dehong isolates were unique recombinant forms (URF), mostly between subtypes B' (Thailand variant of subtype B) and C, with distinct profiles of recombination breakpoints. The subtype B' accounts for the remaining 29% (4/14) of Dehong isolates. Interestingly, two Honghe isolates (2/16) shared some of the precise B'/C recombination breakpoints with CRF07_BC. Conclusion: New recombinant strains are arising continually in west Yunnan near the Myanmar border. Some appeared to be secondary recombinants derived from CRF07_BC that had further recombined with other strains. The uneven distribution of subtypes, CRF and URF, suggests the presence of independent transmission networks and clusters among IDU in Yunnan. (C) 2002 Lippincott Williams Wilkins.

Identification and characterization of a new class of human immunodeficiency virus type 1 recombinants comprised of two circulating recombinant forms, CRF07_BC and CRF08_BC, in China

We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences if 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new "second-generation" recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.

Using sub-word-level information for confidence estimation with conditional random field models

The task of word-level confidence estimation (CE) for automatic speech recognition (ASR) systems stands to benefit from the combination of suitably defined input features from multiple information sources. However, the information sources of interest may not necessarily operate at the same level of granularity as the underlying ASR system. The research described here builds on previous work on confidence estimation for ASR systems using features extracted from word-level recognition lattices, by incorporating information at the sub-word level. Furthermore, the use of Conditional Random Fields (CRFs) with hidden states is investigated as a technique to combine information for word-level CE. Performance improvements are shown using the sub-word-level information in linear-chain CRFs with appropriately engineered feature functions, as well as when applying the hidden-state CRF model at the word level.

A confidence-based approach for improving keyword hypothesis scores

The task in keyword spotting (KWS) is to hypothesise times at which any of a set of key terms occurs in audio. An important aspect of such systems are the scores assigned to these hypotheses, the accuracy of which have a significant impact on performance. Estimating these scores may be formulated as a confidence estimation problem, where a measure of confidence is assigned to each key term hypothesis. In this work, a set of discriminative features is defined, and combined using a conditional random field (CRF) model for improved confidence estimation. An extension to this model to directly address the problem of score normalisation across key terms is also introduced. The implicit score normalisation which results from applying this approach to separate systems in a hybrid configuration yields further benefits. Results are presented which show notable improvements in KWS performance using the techniques presented in this work. © 2013 IEEE.

Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 mu g L-1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HIPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), ioclothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TR alpha and TR beta), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 mu g L-1 PFOS. A significant increase in NIS and Diol gene expression was observed at 200 mu g L-1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 mu g L-1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TR alpha and TR beta gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T-4) content remained unchanged, whereas triiodothyronine (T-3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones. (C) 2009 Elsevier Ltd. All rights reserved.

The search for the IFN-gamma receptor in fish: Functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFN gamma R1 and IFN gamma R2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFN gamma Rs in fish. They are widely expressed in tissues, with IFN gamma R1 typically more highly expressed than IFN gamma R2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1 beta down regulating IFN gamma R1 expression but up regulating IFN gamma R2 expression. Overexpression of the two receptor chains in RTG-2 cells revealed that the level of IFN gamma R2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gamma IP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFN gamma R may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction. (c) 2009 Elsevier Ltd. All rights reserved.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Microcalorimetric study on the toxic effect of Pb2+ to Tetrahymena

The toxic effect of Pb2+ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P (m)) and the growth rate constant (k) were determined, which showed that values of P (m) and k were linked to the concentration (C) of Pb2+. The addition of Pb2+ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb2+, and Pb2+ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb2+, indicating that the toxic effect of Pb2+ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb2+.

Electrically controllable RKKY interaction in semiconductor quantum wires

We demonstrate in theory that it is possible to all-electrically manipulate the RKKY interaction in a quasi-one-dimensional electron gas embedded in a semiconductor heterostructure, in the presence of Rashba and Dresselhaus spin-orbit interaction. In an undoped semiconductor quantum wire where intermediate excitations are gapped, the interaction becomes the short-ranged Bloembergen-Rowland superexchange interaction. Owing to the interplay of different types of spin-orbit interaction, the interaction can be controlled to realize various spin models, e.g., isotropic and anisotropic Heisenberg-like models, Ising-like models with additional Dzyaloshinsky-Moriya terms, by tuning the external electric field and designing the crystallographic directions. Such controllable interaction forms a basis for quantum computing with localized spins and quantum matters in spin lattices.

基于单字提示特征的中文命名实体识别快速算法

近年来条件随机场(CRF)模型在自然语言处理中的应用越来越广泛。标准的线性链(Linear-chain)模型一般采用L—BFGS参数估计方法，收敛速度慢。本文在分析模型复杂度的基础上提出了一种改进的快速CRF算法。该算法通过引入小规模单字特征降低特征的规模，并通过在推理过程中引入任务相关的人工知识压缩Viterbi和Baum-Welch格搜索空间，提高了训练的速度。在中文863命名实体识别评测语料和SIGHAN06语料集上进行的实验表明，该算法在不影响中文命名实体识别精度的同时，有效地降低了模型的训练代价。

黄土旱塬不同水分管理模式对旱作春玉米土壤温度的影响

基于春玉米生长季土壤温度动态观测资料,研究黄土高原南部旱作区不同水分管理模式对春玉米(Zeamays)农田土壤温度动态变化和玉米生育期土壤积温(>10℃)的影响,其结果对理解不同水分管理模式对玉米生长影响具有一定参考价值。【方法】以沈单10为供试作物品种,水分管理模式2007年采用补充灌溉、雨养和地膜覆盖3种处理。在2007年基础上,2008年增加秸秆覆盖,试验采用完全方案,于2007年和2008年连续进行2年田间试验。【结果】地膜覆盖具有明显的增温效应,而秸秆覆盖具有明显降温效应;玉米不同生育阶段>10℃土壤积温,以出苗阶段(PT-VE)最低,生殖生长阶段(R1-R6)次之,营养生长阶段(VE-R1)最高。不同水分管理模式对不同生育期的土壤积温影响不同:地膜覆盖在PT-VE阶段较低,补充灌溉在VE-R1阶段较低,雨养处理在R1-R6阶段较低。地膜覆盖条件下土壤温度对大气温度变化的响应更为敏感,与大气温度变化最为紧密;而秸秆覆盖土壤温度对大气温度变化的反应最迟钝。补充灌溉和地膜覆盖处理籽粒产量显著高于雨养处理(P<0.05),与雨养相比,2007和2008年补充灌溉分别增产30.0%和25.7%,地膜...

刺五加活性成分及其相关非共价复合物的质谱研究

中药化学成分，尤其是活性成分，是中药发挥药效作用的物质基础，它的深入研究是中药现代化的关键和核心。而中药成分的复杂性和药效的整体性，一般的分析及研究方法无法简捷快速地解决中药有效成分鉴定的难题，直接影响到中药研究开发的进程。质谱技术，尤其是软电离质谱及其联用技术，由于其具有灵敏、快速、分辨率高的优点，使得质谱日益成为研究复杂生物分子和中药体系中的应用问题的强有力手段。本论文以刺五加新药的开发为背景，采用有效部分的研究方法，运用先进的质谱技术，以药理活性筛选为指导，全面地研究了刺五加的活性成分。首先，采用电喷雾多极串联质谱技术，研究了正负离子条件下传统中药刺五加中的有效成分刺五加营B和刺五加营E的特征质谱行为，并提出了质谱碎裂机理，可以此鉴定传统刺五加有效成分，对传统刺五加药材及相关药品的快速鉴别和质量控制具有重要意义。以人参皂营R1为例，定量地研究四种碱金属离子浓度对皂普结构分析的影响，为合理添加碱金属离子以提高皂着分析的灵敏度提供了依据。在此基础上，用电喷雾多极串联质谱技术简便而又快捷地区分了两对生理活性差别较大的人参皂营的同分异构体：Rg1和PF11，Rg2和Rg3，为深入研究人参皂普的药效成分和作用机制奠定了一定的基础。系统地研究了β-香树”脂醇型的三种五环三菇皂营在不同模式下的多级串联质谱特征，为从多角度阐明皂昔类化合物的结构提供了新的思路。结构相近的皂普类化合物在电喷雾多级串联质谱中都表现出了与其自身结构相符的特征质谱行为，为不同皂营混合物的快速鉴定提供了一种快速有效的分析方法。在此基础上，研究了两种典型的刺五加仆卜皂营的质谱特征，并用多种质谱手段从刺五加叶总皂普中检测到18种皂普成分，其中有8种皂营成分在刺五加中而未见有文献报道，并用质谱首次发现了刺五加叶皂营的结构和其在植物体内代谢的相关性，这一结果对研究刺五加皂营的体内代谢有着十分重要的意义。以药理目标活性成分为指标，采用对比实验和一系列正交实验，对刺五加活性成分的提取方法，提取工艺和纯化工艺进行了优化，首次采用联合树脂法一次性分离纯化刺五加中两类有效成分总皂昔和总黄酮，经工艺优化最终得到总有效成分达85％以上的刺五加制剂，为刺五加新药的开发提供了大量的工艺参数。基于环糊精包合物（CDs）在中药剂型改造及提高某些药物稳定性和生物利用度方面广阔的应用前景和刺五加新药开发中的实际需要，系统到研究了三种环糊精（a-CD，β-CD和γ-CD）与刺五加皂昔元齐墩果酸（OA）形成的复合物。纬果表明，三种环糊精都能与OA形成稳定的l:1复合物，而β-CD和γ-CD还能与以形成2:1“三明治”状的复合物。复合物气液两相性质具有良好的相关性，为质谱快速筛选和表征这类非共价复合物提供了必要的条件。采用同样的方法对三种环糊精与刺五加中黄酮类化合物（芦丁和懈皮素）的复合物进行了系统研究，结果发观，三种环糊精都与芦丁（R）和棚皮素（Q）形成1:1复合物。但复合物稳定性有差异，对环糊精与芦丁的1:1复合物，以β-CD复合物最稳定，而对于环糊精与槲皮素1:1复合物则以α-CD复合物最稳定，这与芦丁含有糖链有关。刺五加活性成分黄酮类化合物生物利用度和活性的另一有效途径是制备黄酮-金属络合物。黄酮-金属络合物作为潜在的药物开发正日益受到重视，本文利用电喷雾质谱系统地研究了第四周期五种二价金属离子与棚皮素形成的络合物，并采用多级串联质谱部分表征了这些络合物的结构和稳定性之间的关系，为建立黄酮金属络合物筛选体系和表征提供了一种较好的方法，为这类新药的研制开发提供了一定的理论基础。

天花粉蛋白抗HIV-1构效关系研究及机制探讨

天花粉蛋白（Trichosanthin，TCS）是从葫芦科植物括楼（TrichosanthesKiriloii）球根中提取的一种由247个氨基酸组成的I型核糖体失活蛋白（RibosomeInactivatingProtein，RIP）。TCS具有广谱的生物学和药学活性，包括抗肿瘤、免疫抑制、中期引产以及抗病毒活性，TCS还具有抗白血病和淋巴瘤的作用。TCS能够抑制HI卜1在急性感染的T淋巴细胞和慢性感染的巨噬细胞中的复制。TCS抗HIV的机制还不清楚，一般认为与R1（RibosolneInactivating）活性有关。TCS的毒副作用限制了它在抗HIV／AIDS临床上的进一步应用。人们期望通过改造或修饰，在保留TCS抗HIV活性的同时，降低其神经毒副作用及所引起的变态反应。因此，研究TCS的抗HIV-1作用机制及构效关系，有利于拓宽TCS的临床应用适应症，也对RIP类化合物基础研究和应用研究具有重要的指导作用。本论文在实验室己有研究工作的基础上，对TCS抗HIV-1作用、机制及构效关系进一步研究。首先，采用MTT比色法检侧TcS对人T淋巴细胞系C8166、HIV-1慢性感染细胞系H9／HIV-1IIIB细胞毒性作用以及采用台盼蓝染色法检测了TCs对刺激转化的人外周血单个核细胞（PeriphejralBloodMononuclearCen，PBMC）的细胞毒性作用；以合胞体形成抑制实验检测了TCS对实验株HIV-1ms诱导C8166细胞致细胞病变的抑制作用；以捕捉HIV-1p24抗原ELISA方法检测TCS对实验．株HIV-1IIIB在急性感染C8166，细胞和慢性感染Hg细胞中复制的抑制作用、临床分离株HIV-1KMO18在PBMC中复制的抑制作用、耐药株HIV-174v在C8166细胞中复制的抑制作用。其次，利用荧光实时定量RT-PCR检测了TCS对HIV-IIIB吸附和融合宿主细胞的抑制作用；采用高效液相色谱（HighPerformanceLiquidChromatograPhy，HPLC）检测了TCS脱HIV-IRNA腺嘿呤作用；另外还检测了TCS对病毒颗粒的直接杀伤作用、TCS对HIV感染和未感染细胞融合的抑制以及对HIV重组逆转录酶活性的抑制作用。最后，利用以蛋白工程技术构建的14个TCD突变体研究其抗HIV的构效关系，其中活性中心突变体：结果表明，TCS不仅能够有效抑制实验株HIV-1mB在C8166细胞中的复制和HIV-1ms诱导宿主细胞的病变作用，还能抑制临床分离株HIV-1KM018在PMBC中的复制和耐药株HIV-174v在C8166细胞中的复制，但TCS对HIV-1在慢性感染H9细胞中的复制无直接抑制作用。TCS不能抑制HIV-1进入宿主细胞；对感染细胞和未感染细胞的融合没有抑制作用；TCS也不能抑制HIV-1重组逆转录酶活性；TCS对病毒颗粒的直接杀伤作用不大；但TCS能够使裸露的HIV-1RNA脱腺漂呤，可能TCS的脱缥岭活性或其它酶活性直接损伤病毒或者病毒感染细胞的核酸，而胞质腺嗦岭含量的增高则可以导致线粒体膜电位的降低、细胞色素C的释放、活性氧的增加和抗凋亡因子表达的下降，结果使感染细胞更多的凋亡，这可能是TCS抗HIV的一个机制。结果也表明，活性中心突变体TCSM（120-123）与TCSE160A/E189A，在失去绝大部分R工活"性的同时，也几乎完全失去抗HIV活性。、而另一个活性中心突变体TCSR122G，RI活性下降1的倍，却仍保留一定的抗HIV活性。TcSC末端删除突变体（TCSC2，TCSC4和TCSC14）抗HIV活性的下降（1.4-4.8倍）与其R1活性呈平行下降（1.2-3.3倍）。这些结果表明TCS抗HIV-1活性与其R1活性显著相关，但似乎又不是唯一的决定因素，因为我们发现二个分别在C末端加上末端19个氨基酸延伸肤或KDEL信号肤的突变体TCS饥触与TCSKDEL，虽然保留全部的RI活性，但却几乎完全失去抗HIV活性，表明有其它机制介入了TCS的抗HIV-1活性。TCS抗原决定簇位点突变后对TCS抗HIV-1活性没有显著影响，但当在抗原决定簇突变体所引入的Cys残基上加上PEG漱后，这些突变体则显著降低了抗HIV-1的活性。