21 resultados para HIV, primary resistance to antirretrovirales, virologic faulier, secondary resistance

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毛壳菌属很多种类具有重要生防价值,其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今,学界对毛壳菌的研究主要集中在毛壳菌的生防机理,毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合,从产抗生素毛壳菌菌株的筛选开始,进而对产抗生素的角毛壳菌进行诱变选育,最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选:不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验,结果显示,菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究,菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌,菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较,发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱,表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌(CH21)和球毛壳菌(CH08)原生质体制备和再生条件研究:考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝,以0.06 M磷酸缓冲液(pH6.0)配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液,30℃酶解1.5 h,原生质体释放量2.02×107个/g;以PDA为再生培养基,0.7 mol/L的蔗糖再生稳渗剂,再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝,以0.06 M磷酸缓冲液(pH6.0)配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液,30℃酶解1 h,原生质体释放量达1.57×108个/g;以PDA为再生培养基,0.7 mol/L的蔗糖为再生稳渗剂,再生率可达41.48%。 3、角毛壳菌(CH21)原生质体紫外诱变选育:以CH21为出发菌株,制备原生质体进行紫外诱变,诱变条件为:15 w紫外灯,距离30 cm,照射90 s,致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛,获得一株突变菌株CH21-I-402,其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得:菌株CH21-I-402和CH08抗生素药敏试验表明, CH21-I-402菌株对潮霉素有抗性、对G418(Geneticin)敏感,菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌,经PCR检测,新霉素磷酸转移酶基因成功转化进菌株CH08-GR70,CH08-GR120。转化子对G418抗性提高3~4倍,对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析:制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体,以35%的PEG6000为助融剂进行原生质体融合,以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记,获得46个再生菌株。再生菌株连续传代5代后,再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明,再生菌株PF1、PF26为融合菌株。抑菌活性测试表明,融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用,且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

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A critical review on the mechanism and models on the bubble-to-slug transition of two-phase gas-liquid flows are presented in the present paper. It is shown that the most possible mechanism controlled the bubble-to-slug transition is the bubble coalescence. Focusing on the bubble-to-slug transition for the low-Re two-phase flow, a simple Monte Carlo method is used to simulate the influence of the initial bubble size on the bubble-to-slug transition. Some secondary factors, such as the liquid viscosity, the surface tension, and the relative slip between the two phases, are ignored in the present study. It is found that the locus of the dimensionless rate of collision is a universal curve. Based on this curve, it is determined that the bubble initial size can affect the phase distribution and flow pattern when its dimensionless value is in the range from 0.03 to 0.4. A simple relationship between the critical void fraction and the initial bubble size is proposed, which agrees very well with the experimental data.

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AIM: To determine whether trichobitacin, a novel ribosome-inactivating protein purified from the root tubers of Trichosanthes kirilowii, possesses the anti-HIV activity. METHODS: The inhibition of syncytial cell formation induced by human immunodeficiency virus type 1 (HIV-1),was determined under microscope, reduction of HIV-1 p24 antigen expression level was measured by ELISA, and decrease in numbers of HIV-1 antigen positive cells in acutely and-chronically infected cultures were detected by indirect immunofluorescence assay. RESULTS: Trichobitacin Was-found to greatly suppress syncytial cell formation induced by HIV-1 and to markedly reduce both expression of HIV-1 p24 antigen and the number of HIV antigen positive cells in acutely but not chronically HIV-1 infected culture. The median inhibitory concentration (IC50) in inhibition of syncytial cell formation and HIV antigen positive cells were 5 mu g.L-1 (95 % confidence limits: 1.3 - 20 mu g.L-1) and 0.09 mg.L-1 (95 % confidence limits: 0.011 - 0.755 mg.L-1), respectively. CONCLUSION: Trichobitacin is a novel ribosome-inactivating protein with anti-HIV-l activity.

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Aim of the study: Previously, we reported that the petroleum ether fraction, RC-1, and EtOAc fraction, RC-2, of the medicinal plant Rhus chinensis showed potent anti-HIV-1 activities. To address anti-HIV-1 constituents of RC-1 and RC-2, 17 compounds were

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Camptothecin (CPT), a traditional anti-tumor drug, has been shown to possess anti-HIV-1 activity. To increase the antiviral potency, the anti-HIV activities of two CPT derivatives, 10-hydroxy-CPT and 7-hydroxymethyl-CPT, were evaluated in vitro. The therapy index (TI) of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-1(IIIB) in C8166 were 24.2, 4.2 and 198.1, and against clinical isolated strain HIV-1(KM018) in PBMC were 10.3, 3.5 and 66.0, respectively. While the TI of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-2(CBL-20) were 34.5, 10.7 and 317.0, respectively, and the TI of the three compounds against HIV-2(ROD) showed the similar values. However, when the antiviral mechanisms were considered, we found there was no inhibition of 7-hydroxymethyl-CPT on viral cell-to-cell transmission, and was no inhibition on reverse transcriptase, protease or integrase in cell-free systems. 7-Hydroxymethyl-CPT showed no selective killing of chronically infected cells after 3 days of incubation. In conclusion, 7-hydroxymethyl-CPT showed more potent anti-HIV activity, while 10-hydroxy-CPT had less efficient activity, compared with the parent CPT. Though the antiviral mechanisms remain to be further elucidated; the modification of -OH residues at C-7 of CPT could enhance the antiviral activity, while of -OH residues at C-10 of CPT had decreased the antiviral activity, which provides the preliminary modification strategy for anti-viral activities enhancement of this compound.

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高效抗逆转录病毒治疗(HAART)的应用,极大的降低了AIDS发病率和死亡 率,延长了HIV感染者的生命。但HIV耐药在很大程度上影响了HAART的疗效, 耐药株的产生成为影响抗病毒治疗效果的主要因素。欧洲、美国的耐药监测技术 规范均推荐在新感染未经抗病毒药物治疗的患者中进行原发耐药检测。我国政府 于2003年底出台了艾滋病治疗的“四免一关怀”政策,陆续在全国范围内开展了大 规模的免费抗病毒治疗,监测我国未经抗病毒药物治疗HIV-1感染者中的耐药情 况可以为制定合理的用药方案和减少耐药毒株出现提供科学依据。 根据世界卫生组织(WHO)的“HIV 耐药监测指南”,无偿献血者中的HIV-1 感染者,可以认定为HIV 新诊断未治疗人群。分析了云南无偿献血者的血浆和 外周血单核细胞(PBMC),研究云南无偿献血人群的耐药状况。 已有实验室血清学方法识别HIV-1 新近感染和长期感染,用BED-CEIA 方 法,在河南、安徽、山西自愿咨询检测(VCT)人群中检出新近感染人群,进行耐 药基因研究, 对照研究了部分长期感染人群。 样品提取核酸后,巢式聚合酶链反应(nested-PCR)扩增pol 基因区(含蛋白酶 区1~99 氨基酸全长和逆转录酶区1~242 氨基酸)。PCR 产物双脱氧法测序,所 得序列与洛斯阿拉莫斯HIV 核酸序列库(Los Alamos HIV Database)标准株构建系 统进化树分析亚型;用斯坦福大学耐药数据库(Standford HIV Drug Resistance Database)分析耐药。 研究发现,云南省2005~2006 年无偿献血者中,有52 例为HIV-1 阳性,其 中49 例血浆和相应的PBMC 样品病毒基因扩增成功。序列分析表明,HIV 病毒 的亚型分布为CRF08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%)和B (4.1%);所有样品均未发现蛋白酶抑制剂(PI)耐药基因位点主要突变,只在6 例(11.7%)样品中发现7 例次PI 次要耐药位点突变;另外,在9 例(18.4%)样品中发现10 例次核苷类逆转录酶抑制剂(NRTI) 耐药突变,1 例(2.0%)发生非核苷类 逆转录酶抑制剂(NNRTI) 耐药突变;针对具体药物PI/NRTI/NNRTI 均只有1 例 有潜在的低度耐药,临床仍对药物敏感。PBMC 和血浆的病毒耐药没有显著差异。 从河南、安徽、山西27 个VCT 检测点2006~2007 年采集的10310 例样品 中,通过WB 和BED-CEIA 检测出新近感染人群63 例,分析成功50 例血浆样 品;河南VCT 长期感染样品中随机抽样,分析成功19 例样品。分析成功的69 例VCT 样品中,HIV 病毒株的亚型分布分别为B’ (95.7%),CRF01_AE(2.9%)和 C(1.4%)。上诉样品均未检出PI 主要耐药相关突变,只在26 例(37.7%)样品中存 在27 例次PI 次要耐药相关突变;3 例(4.3%)样品出现6 例次NRTI 耐药相关突 变,7 例(10.1%)样品出现8 例次NNRTI 耐药相关突变。通过与斯坦福大学耐药 数据库比对,没有发现针对PI 类药物的临床耐药;但有2 例(2.8%)针对NRTI 类 药物耐药,1 例有M184V 突变导致对拉米夫定(3TC)和氟代拉米夫定(FTC)高度 耐药;1 例样品存在T215Y、M41L、L210W 三重突变位点,对阿巴卡韦(ABC)、 去羟肌苷(ddI)和坦那夫韦(TDF)中度耐药,对齐多夫定(AZT)和司他夫定(d4T)高 度耐药;针对NNRTI 类药物,有3 例(4.3%)毒株有耐药,1 例有K103N 突变导 致对奈韦拉平(NVP)、地拉韦啶(DLV)和依菲韦伦(EFV)的高度耐药;1 例有Y188L 突变导致对NVP 和EFV 的高度耐药;1 例存在K101E 和G190A 双重突变,导 致对NVP 的高度耐药,对DLV、EFV 和依曲韦林(ETR)中度耐药。 比较长期感染和新近感染者之间的亚型和耐药,未发现显著差异。 研究结果表明,云南、河南和安徽未经治疗HIV-1 感染者中耐药处于低流行 状态。亚型分布云南无偿献血者以CRF_BC 为主,河南、安徽VCT 人群以B’ 为主。应持续在未经治疗人群中进行耐药监测。

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Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule. (C) 2002 Elsevier Science B.V. All rights reserved.

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A dense clad overlay with chemical inertness was achieved on top of the plasma-sprayed YSZ thermal barrier coatings by laser in order to protect them from hot-corrosion attack. The Al2O3-clad YSZ coating exhibited good hot-corrosion behavior in contact with salt mixture of vanadium pentoxide (V2O5) and sodium sulfate (Na2SO4) for a longtime of 100 h at 1173 K. The LaPO4-clad YSZ coating showed corrosion resistance inferior to the Al2O3-clad one. Yttria was leached from YSZ by reaction between Y2O3 and V2O5, which caused progressive destabilization transformation of YSZ from tetragonal (t) to monoclinic (m) phase. The chemical inertness of the clad layers and the restrained infiltration of the molten corrosive salts by the dense clad layers were primary contributions to improvement of the hot-corrosion resistances.

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This study deals with the formulation, mathematical property and physical meaning of the simplified Navier-Stokes (SNS) equations. The tensorial SNS equations proposed is the simplest in form and is applicable to flow fields with arbitrary body boundaries. The zones of influence and dependence of the SNS equations, which are of primary importance to numerical solutions, are expounded for the first time from the viewpoint of subcharacteristics. Besides, a detailed analysis of the diffusion process in flow fields shows that the diffusion effect has an influence zone globally windward and an upwind propagation greatly depressed by convection. The maximum upwind influential distance of the viscous effect and the relative importance of the viscous effect in the flow direction to that in the direction normal to the flow are represented by the Reynolds number, which illustrates the conversion of the complete Navier-Stokes (NS) equations to the SNS equations for flows with large Reynolds number.

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In this paper, multi-hole cooling is studied for an oxide/oxide ceramic specimen with normal injection holes and for a SiC/SiC ceramic specimen with oblique injection holes. A special purpose heat transfer tunnel was designed and built, which can provide a wide range of Reynolds numbers (10(5)similar to 10(7)) and a large temperature ratio of the primary flow to the coolant (up to 2.5). Cooling effectiveness determined by the measured surface temperature for the two types of ceramic specimens is investigated. It is found that the multi-hole cooling system for both specimens has a high cooling efficiency and it is higher for the SiC/SiC specimen than for the oxide/oxide specimen. Effects on the cooling effectiveness of parameters including blowing ratio, Reynolds number and temperature ratio, are studied. In addition, profiles of the mean velocity and temperature above the cooling surface are measured to provide further understanding of the cooling process. Duplication of the key parameters for multi-hole cooling, for a representative combustor flow condition (without radiation effects), is achieved with parameter scaling and the results show the high efficiency of multi-hole cooling for the oblique hole, SiC/SiC specimen. (C) 2008 Elsevier Ltd. All rights reserved.

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In this paper, we propose a novel three-dimensional imaging method by which the object is captured by a coded cameras array (CCA) and computationally reconstructed as a series of longitudinal layered surface images of the object. The distribution of cameras in array, named code pattern, is crucial for reconstructed images fidelity when the correlation decoding is used. We use DIRECT global optimization algorithm to design the code patterns that possess proper imaging property. We have conducted primary experiments to verify and test the performance of the proposed method with a simple discontinuous object and a small-scale CCA including nine cameras. After certain procedures such as capturing, photograph integrating, computational reconstructing and filtering, etc., we obtain reconstructed longitudinal layered surface images of the object with higher signal-to-noise ratio. The results of experiments show that the proposed method is feasible. It is a promising method to be used in fields such as remote sensing, machine vision, etc. (c) 2006 Elsevier GmbH. All rights reserved.

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Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

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Pen-based user interface has become a hot research field in recent years. Pen gesture plays an important role in Pen-based user interfaces. But it’s difficult for UI designers to design, and for users to learn and use. In this purpose, we performed a research on user-centered design and recognition pen gestures. We performed a survey of 100 pen gestures in twelve famous pen-bases systems to find problems of pen gestures currently used. And we conducted a questionnaire to evaluate the matching degree between commands and pen gestures to discover the characteristics that a good pen gestures should have. Then cognition theories were applied to analyze the advantages of those characteristics in helping improving the learnability of pen gestures. From these, we analyzed the pen gesture recognition effect and presented some improvements on features selection in recognition algorithm of pen gestures. Finally we used a couple of psychology experiments to evaluate twelve pen gestures designed based on the research. It shows those gestures is better for user to learn and use. Research results of this paper can be used for designer as a primary principle to design pen gestures in pen-based systems.

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化石燃料的燃烧是百余年来大气中二氧化碳(CO2)浓度增加的主要原因。CO2的收集和处置则是抑制这一趋势的有效途径。本文通过对现有收集利用和处置技术的分析,认为火电厂是收集CO2的重点考虑对象;CO2用于三次采油及天然气回收在技术上和经济上比较可行;蓄水层储气前景广阔值得研究;深海处置有待进一步探索;CO2用于置换开采天然气水合物也是很有前景的方案。


The burning of fossil fuel is the primary cause to have the concentration of carbon dioxide(CO2) in atmosphere increased during the past more than a hundred of years,and the capture and disposal of CO2 is an effective method to control its rising tendency.By analysis of the current capture and disposal technologies of CO2,it is concluded that firepower plants are the key targets to capture CO2.The paper also puts forth that tertiary oil recovery and natural gas recovery with CO2 are feasible both technologically and economically;storage of CO2 in saline aquifer is a method of nice foreground and deserves to be researched; disposal of CO2 in deep seafloor will be further investigated;and displacement of gas hydrate with CO2 is a tempting programme also.