A low power integrated CMOS transmitter for BCI system

The prototype wafer of a low power integrated CMOS Transmitter for short-range biotelemetry application has been designed and fabricated, which is prospective to be implanted in the human brain to transfer the extracted neural information to the external computer. The transmitter consists of five parts, a bandgap current regulator, a ring oscillator, a buffer, a modulator and a power transistor. High integration and low power are the most distinct criteria for such an implantable integrated circuit. The post-simulation results show that under a 3.3 V power supply the transmitter provides 100.1 MHz half-wave sinusoid current signal to drive the off-chip antenna, the output peak current range is -0.155 mA similar to 1.250 mA, and on-chip static power dissipation is low to 0.374 mW. All the performances of the transmitter satisfy the demands of wireless real-time BCI system for neural signals recording and processing.

滇杨(Populus yunnanensis)对增强紫外辐射、干旱和喷施脱落酸的生态生理响应

目前，随着全球气候变化的加剧，水分短缺更加明显。在干旱与半干旱地区，水分胁迫是影响植物存活和生长的主要限制因子。同时，随着大气平流层中臭氧浓度的减少，过量的紫外辐射(UV-B)到达地球表面，一些地区的植物不可避免地受到增强UV-B 和水分胁迫的共同作用。文献表明在UV-B 增强的情况下，干旱表现为减弱或增强UV-B 对植物的影响，这与种、品种有一定的相关性。另外，脱落酸(ABA)是近年来研究报道最多的信息调控物质，与植物抗旱性途径有较大的关系，但其对植株抗UV-B 的影响还有待于研究。本论文以滇杨(Populus yunnanensis)为模式植物，从形态和生理方面研究了增强UV-B、干旱和脱落酸对它的影响，并探讨了UV-B 与干旱的互作效应以及喷施脱落酸对植株抗旱性和抗UV-B 能力的影响。主要研究结果如下：1. 增强的UV-B 和干旱胁迫都影响了滇杨的形态生长和生理生化反应。它们都导致了滇杨植株的株高、基茎、整株叶面积、平均叶面积、总生物量和净光合速率的显著降低，使得叶片增厚，过氧化物酶(GPX)活性升高，脯氨酸和花色素苷含量增加，膜脂过氧化程度增大。不同的是干旱显著降低了植株叶片数目，增大了根/冠比(Rs)、细根/总根比(Ft)、提高了内源ABA 含量、碳同位素(δ13C)以及紫外吸收物质含量和超氧化物歧化酶(SOD)的活性，而UV-B 对它们没有影响。干旱与UV-B 的复合作用加剧了任一单独胁迫对植株的抑制，表现为更小的株高、基茎、整株叶面积、平均叶面积、总生物量，更低的光合作用和更高的MDA 含量。而且UV-B 辐射降低了干旱胁迫下生物量分配的可塑性，表现为降低了干旱情况下的Rs 和Ft，ABA 的含量也显著下降，复合胁迫下脯氨酸含量和过氧化氢酶(CAT)的活性比任一单独胁迫时都要低。这些实验结果表明，增强的UV-B 与干旱的复合胁迫加剧了对植株的抑制作用。II2. 干旱情况下同时施加外源ABA 提高了植株的根/冠比、细根/总根比和单位面积叶重，即提高了干旱胁迫下植株对生物量分配的可塑性。而且外源ABA 使干旱胁迫下的长期用水效率、ABA 含量、脯氨酸含量、GPX 活性进一步增加，并有效调节了活性氧代谢的平衡，抑制了受旱植株MDA 的增加。结果表明，外源ABA 的喷施提高了滇杨植株的抗旱性。3. 在增强的UV-B 情况下，外源ABA 加剧了UV-B 对滇杨形态生长的抑制效果，表现为进一步降低了滇杨植株的整株叶面积、平均叶面积、单位面积叶重和总生物量，而且ABA 还降低了UV-B 胁迫下的净光合速率和脯氨酸的含量，增大了MDA 的含量。通过以上的数据我们可以看出，外源ABA 虽然提高了滇杨植株的抗旱性，但却加剧了UV-B 胁迫对植株的抑制作用。Currently, drought is one of the most serious environmental stresses. In arid and semi-aridregions, drought is a major constraint imposed on tree survival and growth. The decrease ofozone layer leads to a significant increase in ultraviolet-B (UV-B, 280-320 nm) radiationreaching the earth surface. In some places, plants suffer both UV-B and water stresssimultaneously. Their combination will increase or decrease the sensitivity of plants to UV-Bstress which lies on the species. On the other hand, abscisic acid (ABA), as a plant homoneand growth regulator, is better for plants resistant to drought stress, but it is uncleared aboutthe relationship between exogenous ABA and supplemental UV-B. In the present study, weemployed Populus yunnanensis Dode as a model species to characterize the growth andecophysiological responses of woody plants to supplemental UV-B, drought and exogenous ABA. The results are as follows:1. Both supplemental UV-B and drought affected the morphological, physiological andbiochemical responses of P. yunnanensis. They decreased the plant height, basal diameter,total leaf area, average leaf area, biomass and photosynthesis, and increased specific leaf mass,the activity of guaiacol peroxidase (GPX), the content of proline, anthocyanins andmalondialdehyde (MDA). However, drought decreased the leaf number and increasedroot/shoot ratio, fine root/total ratio, the activity of superoxide dimutase (SOD) and thecontents of ABA, carbon isotope composition (δ13C), UV-absorbing compounds. Whilesupplemental UV-B had no effects on them. The combination of drought and UV-Baugmented the growth inhibtion acting as further lower plant height and smaller basaldiameter, leaf area, biomass and higher MDA content. And compared with drought stress,root/shoot ratio and fine root/total root ratio decreased under the combination stresses. The photosynthesis, proline content and Catalase (CAT) activity became lower under combinationstresses than that of either stress lonely. According to these results, we suggested that,compared with the effect of stress lonely, the combination of supplemental UV-B and droughtdid not mitigate the harmful effect, but augmented it.2. Under drought conditions, exogenous ABA increased root/shoot ratio, fine root/total rootratio and the specific leaf mass. That was to say exogenous ABA increased plant plasticityunder drought conditions. Also ABA content, proline content, activity of GPX and δ13C wereenhanced further. In addition the enhancement of MDA was restrained. So the resultssuggested that exogenous ABA increased the seedling capacity of resistance to drought.3. Under supplemental UV-B conditions, exogenous ABA augmented the growth restrain ofUV-B to seedlings, which acted as further decreased leaf area, specific leaf mass and biomass.Compared with UV-B stress alone, proline content and photosynthesis were decreased andMDA content was increased under the combination of UV-B and ABA. These resultssuggested that although exogenous ABA increased the seedling capacity of resistance todrought, it augmented the growth restrain of supplemental UV-B to P. yunnanensis.

鞑靼荞麦（Fagopyrum tataricum Gaertn.）离体再生与遗传转化研究

鞑靼荞麦是我国特有的农业产品，具有抗寒耐旱特性和较高的营养保健功能。荞麦的开花习性及遗传特点导致其人工杂交授粉难以成功，这成为荞麦杂交育种难以获得突破的重要原因。因此利用转基因技术导入有益基因有可能成为荞麦遗传改良的新途径，而再生及转化体系的建立是开展转基因研究的基础。 本文研究了苗龄、外植体、几种激素配比对鞑靼荞麦（Fagopyrum tataricum Gaertn.）离体培养的影响，初步建立了鞑靼荞麦离体再生体系。结果表明，鞑靼荞麦离体再生的最佳取材时间为苗龄6-8d；诱导愈伤组织的最适培养基为MS＋2.0 mg/L 2,4-D＋1.5 mg/L 6-BA，子叶诱愈率达75％左右，下胚轴的可高达86.62％；愈伤组织分化的最适培养基为MS 0.1mg/L IAA＋2.0mg/L 6-BA＋1.0 mg/L KT＋0.5mg/L TDZ，下胚轴的分化率可达9.52%。下胚轴的诱愈率与分化率均高于子叶，更适于离体再生培养。培养基中加入AgNO3后，能有效降低褐化率。生根最适培养基为含有0.5mg/L NAA的1/2MS培养基，生根率在50％左右。TDZ在诱导鞑靼荞麦的愈伤组织分化出芽的过程中起到明显的促进作用，可提高分化率约20%。 在上述研究基础上，本文还对鞑靼荞麦的遗传转化体系进行了探索性研究。分别利用根癌农杆菌（Agrobacterium tumefaciens）介导法和微粒轰击法（基因枪法）对黑水苦荞下胚轴进行遗传转化。 在农杆菌介导的方法中，携带有质粒pCAMBIA2301的农杆菌菌株EHA105用于转化。载体质粒pCAMBIA2301包含有gus和npt-II 基因, 并受35s启动子驱动。研究结果表明，在侵染方式选择上，浸泡方式比吸打方式更有效，根癌农杆菌侵染的较适浓度为OD600＝0.5，共培养3天，恢复培养7天，能检测到gus基因的表达。 基因枪法使用质粒pBI121，同样包含有gus和npt-II基因, 并受CaMV35s 启动子驱动。轰击距离为9cm较合适，甘露醇前处理在本研究中未表现出明显优势。 两种转化方法比较，基因枪法比农杆菌介导法更快速有效。 本研究为进一步的遗传操作研究打下基础。 Tartary buckwheat (Fagopyrum tataricum Gaertn.), the traditional and unique agricultural product of China, is a kind of crop with strong drought and cold tolerance, abundant nutrition and high medical value. Artificial hybridization is hard in buckwheat because of its flowering habits and genetic characteristics, which leads to no breakthrough in tartary buckwheat breeding. However, biotechnological approaches, especially genetic transformation for the direct introduction of good genes into tartary buckwheat for quality improvement, hold great promise. In this study, we established tartary buckwheat regeneration system in vitro. It is the foundation for genetic manipulation of this crop. The effects of seedling age, hypocotyl and cotyledon as explants, and proportions of several growth regulators were tested in tissue culture of tartary buckwheat for establishing its in vitro regeneration system. The results showed that the best seedling age for callus induction was 6 to 8 days. On the MS medium containing 2.0mg/L 2, 4-D and 1.5mg/L 6-BA, the induction rate of callus from hypocotyls was up to 86.62%, while from cotyledons was about 75％. The suitable shooting medium was the MS medium+0.1mg/L IAA+2.0mg/L 6-BA+1.0 mg/L KT+0.5mg/L TDZ, and the shooting rate from hypocotyls was 9.52%. The callus induction and shooting rates were higher from hypocotyls than from cotyledons. Browning reduced when the medium mixed with AgNO3. Half strength MS supplemented with 0.5mg/L NAA was the best for rooting, the rate was around 50% after 30 days culture. TDZ can accelerate the shoot differentiation distinctively, and it could improve the shooting rate nearly 20%. On the base of above, the explorative research of the genetic transformation in tartary buckwheat was done. In the study, hypocotyls from Heishui tartary buckwheat were transformed by Agrobacterium-mediated method and microprojectile bombardment method (gene-gun), comparatively. In Agrobacterium-mediated method, a disarmed Agrobacterium tumefaciens strain EHA105 harboring plasmid pCAMBIA2301 was used. The vector pCAMBIA2301 contains gus and npt-II genes, driven by CaMV35s promoter. The results showed that the appropriate concentration of Agrobacterium tumefaciens for infecting was OD600＝0.5, and co-culture time was 3d. Seven days later after coculture, GUS expression could be tested. In particle bombardment transformation, plasmid pBI121 was used. pBI121 also contains gus and npt-II genes, driven by 35s promoter. Hypocotyls pretreated with mannitol, no effect was observed, and the suitable distance of bombardment is 9cm. Comparing with Agrobacterium-mediated method, gene-gun method is more convenient and effective. All above results could be a basic work for further study in tartary buckwheat transformation.

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

Quantitative sample injection for capillary electrophoresis

An apparatus including a rotary-type injector was designed for quantitative sample injection in capillary electrophoresis (CE), in which both pressurized flow and electroosmotic flow were used to drive the background electrolyte solution. A relative standard deviation of peak area of lower than 1% was achieved by using this apparatus. The effects of back-pressure regulator, restrictor, and applied voltage on separation efficiency and resolution were investigated. The utility of this apparatus in both micro-HPLC and pressurized capillary electrochromatography (pCEC) was also demonstrated.

Capillary electrophoretic study on UDP-sugars in cells

Glucose is an important regulator of cell growth and metabolism. Uridine diphosphate sugars (UDP-sugars), as the intermediate products of metabolism, play pivotal roles as precursors in the synthesis of complex carbohydrates and glycolipids as well as lectose. It is very important to study their metabolism in cells in clinical biochemistry. A capillary electrophoretic method has been developed for the analysis of UDP-sugars and nucleotides, By using an uncoated capillary (70cm x 50 mu m) and 20 mmol/L borax buffer (pH 9), 4 important UDP-sugars can be analyzed in 15 min at 22 kV with satisfactory precision and sensitivity. The developed method has been applied to analyze UDP-sugars concentrations in lymphocytes, fibroblasts and mesangial cells, and the results show it not only is much better than HPLC method, but also can be used to measure the energy charge of cells.

Preparation and characterization of La2Zr2O7 coating with the addition of Y2O3 by EB-PVD

Thermal barrier coatings (TBCs) of La2Zr2O7 (LZ) with the addition of 3 wt.% Y2O3 (LZ3Y) were deposited by electron beam-physical vapor deposition (EB-PVD). The phase stabilities, thermophysical and mechanical properties, and chemical compositions of these ceramics and coatings were studied in detail. The phase stability and thermal expansion behavior of LZ3Y bulk material are identical to those of LZ bulk material, but the mechanical properties of the former are superior to those of the latter. Elemental analysis and X-ray diffraction indicate that compositional deviation of LZ coating can be optimized after doping by 3 wt.% Y2O3, Y2O3 acts as a dopant as well as a process regulator. The optimal composition of LZ3Y coating could be effectively achieved by the addition of excess Y2O3 into the ingot and by properly controlling the current of electron beam (i.e. similar to 650 mA).

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Effects of rare earth ions and calmodulin on the activity of lactate dehydrogenase

In this paper, the effects of rare earth ions (La3+, Eu3+, Dy3+, Yb3+) and their complexes with calmodulin on the activity of lactate dehydrogenase (LDH) were investigated. The results reveal that whether binding with calmodulin or not, rare earth ions show a minor activation effects on LDH when their concentrations are less than 3 mu mol (.) L-1, but indicate some strong inhibitory effects on LDH activity when the concentrations are above 5 mu mol (.) L-1. Calmodulin, which is a calcium-dependent regulator, can stimulate LDH activity and release the inhibitory effects of rare earth ion. Diethylenetriamine pentaacetic acid(DTPA) and its derivatives bisdimethylamide-diethylenetriamine pentaacetic acid (DTPA-BDMA), bisisonicotinyl-diethylenetriamine pentaacetic acid (DTPA-BIN), which are often used as ligands to metal ions, inhibit LDH activity when their concentrations are above 5 mu mol (.) L-1. Calmodulin can also release their inhibitory effects at the same time.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Molecular analysis of the copper-responsive CopRSCD of a pathogenic Pseudomonas fluorescens strain

CopRS/CopABCD is one of the known systems that control copper homeostasis in bacteria. Although CopRS/CopABCD homologues are found to exist in Pseudomonas fluorescens, the potential role of this system in P. fluorescens has not been investigated. In this study a genetic cluster, consisting of copR, S, C, and D but lacking copAB, was identified in a pathogenic P. fluorescens strain (TSS) isolated from diseased fish. The copRSCD cluster was demonstrated to be required for full copper resistance and regulated at the transcription level by Cu. Expression of copCD is regulated directly by the two-component response regulator CopR, which also regulates its own expression. Interruption of the regulated expression of copR affected bacterial growth, biofilm formation, and tissue dissemination and survival. A mutant CopR, which lacks the N-terminal signal receiver domain and is constitutively active, was found to have an attenuating effect on bacterial virulence when expressed in TSS. To our knowledge, this is the first report that suggests a link between CopR and bacterial pathogenicity in P. fluorescens.

Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35A degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25A degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.