Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长:一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs).我们发现MESFs、CMESFs、MOFs和MFGs,而不足MFGEs支持猕猴胚胎干细胞(rESCs,rhesus embryonic stem cells)的生长.通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达.在本研究中,我们运用Affymetrix公司的GeneChip Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用.结果表明,除MFGE外,包括GREM2、bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1和LTBF1等八个基因的mRNA在支持性的饲养层细胞中高表达.本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用.

Decay of vibrations along deepwater mooring lines

Model tests for global design verification of deepwater floating structures cannot be made at reasonable scales. An overview of recent research efforts to tackle this challenge is given first, introducing the concept of line truncation techniques. In such a method the upper sections of each line are modelled in detail, capturing the wave action zone and all coupling effects with the vessel. These terminate to an approximate analytical model, that aims to simulate the remainder of the line. The rationale for this is that in deep water the transverse elastic waves of a line are likely to decay before they are reflected at the seabed. The focus of this paper is the verification of this rationale and the ongoing work, which is considering ways to produce a truncation model. Transverse dynamics of a mooring line are modelled using the equations of motion of an inextensible taut string, submerged in still water, one end fixed at the bottom the other assumed to follow the vessel response, which can be harmonic or random. Nonlinear hydrodynamic damping is included; bending and VIV effects are neglected. A dimensional analysis, supported by exact benchmark numerical solutions, has shown that it is possible to produce a universal curve for the decay of transverse vibrations along the line, which is suitable for any kind of line with any top motion. This has a significant engineering benefit, allowing for a rapid assessment of line dynamics - it is very useful in deciding whether a truncated line model is appropriate, and if so, at which point truncation might be applied. Initial efforts in developing a truncated model show that a linearized numerical solution in the frequency domain matches very closely the exact benchmark. Copyright © 2011 by ASME.

Efficient truncation scheme for modeling deepwater mooring lines

This paper is aimed at enabling the confident use of existing model test facilities for ultra deepwater application without having to compromise on the widely accepted range of scales currently used by the floating production industry. Passive line truncation has traditionally been the preferred method of creating an equivalent numerical model at reduced depth; however, these techniques tend to suffer in capturing accurately line dynamic response and so reproducing peak tensions. In an attempt to improve credibility of model test data the proposed truncation procedure sets up the truncated model, based on line dynamic response rather than quasi-static system stiffness. The upper sections of each line are modeled in detail, capturing the wave action zone and all coupling effects with the vessel. These terminate to an approximate analytical model that aims to simulate the remainder of the line. Stages 1 & 2 are used to derive a water depth truncation ratio. Here vibration decay of transverse elastic waves is assessed and it is found that below a certain length criterion, the transverse vibrational characteristics for each line are inertia driven, hence with respect to these motions the truncated model can assume a linear damper whose coefficient depends on the local line properties and vibration frequency. Stage 3 endeavors to match the individual line stiffness between the full depth and truncated models. In deepwater it is likely that taut polyester moorings will be used which are predominantly straight and have high axial stiffness that provides the principal restoring force to static and low frequency vessel motions. Consequently, it means that the natural frequencies of axial vibrations are above the typical wave frequency range allowing for a quasi-static solution. In cases of exceptionally large wave frequency vessel motions, localized curvature at the chain seabed segment and tangential skin drag on the polyester rope can increase dynamic peak tensions considerably. The focus of this paper is to develop an efficient scheme based on analytic formulation, for replicating these forces at the truncation. The paper will close with an example case study of a single mooring under extreme conditions that replicates exactly the static and dynamic characteristics of the full depth line. Copyright © 2012 by the International Society of Offshore and Polar Engineers (ISOPE).

Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Background: Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results: A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 x 10(5) and 1.7 x 10(5) per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion: The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Induction of hepatic cytochrome P4501A1/2B activity and disruption of thyroglobulin synthesis/secretion by mono-ortho polychlorinated biphenyl and its hydroxylated metabolites in rat cell lines

As the active metabolites of polychlorinated biphenyl (PCBs), hydroxylated polychlorinated biphenyls (OH-PCBs) are found in wildlife and human tissues. They have been proposed as main contributors for endocrine disruption of PCBs in living organisms. In this study, mono-ortho PCB 156 and its hydroxylated metabolites 4'-OH-PCB 159, 4'-OH-PCB 121, and 4'-OH-PCB 72 were selected to investigate the toxic effects on rat hepatoma H4IIE cell line and rat thyroid follicle FRTL-5 cell line at concentrations of 1, 10(2), 10(4) nM. 7-Ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) activities were determined with micro-EROD/PROD to indicate cytochrome P4501 A1 (CYP1A1) and cytochrome P4502B (CYP2B) induction in the H4IIE cell after exposure for 72 h. To assess thyroid disruption of these compounds, thyroglobulin concentrations also were detected inside FRTL-5 cell with immunocellularchemistry and in its medium with radioimmunoassay after exposure for 24 It. Significant inductions of EROD activity by PCB 156 at 102 and 104 nM (p < 0.05) were observed, but no effects by the three OH-PCBs in H4IIE cell line. 7-Pentoxyresorufin-O-dealkylase activities were induced only by 10(4) nM of PCB156 and the three OH-PCBs (p < 0.05). Meanwhile, significant increases of thyroglobulin concentrations were observed in the medium of FRTL-5 cell exposed to 4'-OH-PCB 121 and 4'-OH-PCB 72 at all of the test concentrations (p < 0.05), but not to the other compounds. The results demonstrated that mono-ortho PCBs mainly could be metabolized to hydroxylated metabolites through CYP1A1 instead of CYP2B. Moreover, after being metabolized, OH-PCBs still sustained the ability to induce PROD activity and did exhibit the disruption on thyroglobulin synthesis/excretion in rat cells.

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method. All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp. Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group. In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group. The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively. The values of natural silver carp and bighead carp are 0.161 and 0.104. From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out. A new breeding process of establishing a pure line is developed.

Effects of doubled atmospheric CO2 concentration on the photosynthesis and growth of Chlorella pyrenoidosa cultured at varied levels of light

Chlorella pyrenoidosa was cultured with 350 and 700 p.p.m.v. CO2 at varied levels of light to see the impacts of doubled atmospheric CO2 concentration on its growth and photosynthesis. The CO2 enrichment did not affect the growth rate (mu), but significantly increased the cell density when light was sufficiently supplied. The CO2 enrichment significantly depressed light-saturated photosynthesis and dark respiration in the cells grown under a high-light regime, but not those under a low-light regime. The light-saturating point for photosynthesis and photosynthetic efficiency was not affected by the CO2 enrichment under either the high-light or low-light conditions.

High electrical-to-green efficiency high stability intracavity-frequency-doubled Nd:YAG-LBO QCW 532 nm laser with a straight cavity

An electrical-to-green efficiency of more than 10% was demonstrated by intracavity-frequency-doubling a Q-switched diode-side-pumped Nd:YAG laser with a type II lithium triborate (LBO) crystal in a straight plano-concave cavity. An average power of 69.2 W at 532 nm was generated when electrical input power was 666 W. The corresponding electrical-to-green conversion efficiency is 10.4%. To the best of our knowledge, this is the highest electrical-to-green efficiency of second harmonic generation laser systems with side-pumped laser modules, ever reported. At about 66 W of green output power, the power fluctuation over 4 hours was better than +/-0.86%.

An augmented method for free boundary problems with moving contact lines

An augmented immersed interface method (IIM) is proposed for simulating one-phase moving contact line problems in which a liquid drop spreads or recoils on a solid substrate. While the present two-dimensional mathematical model is a free boundary problem, in our new numerical method, the fluid domain enclosed by the free boundary is embedded into a rectangular one so that the problem can be solved by a regular Cartesian grid method. We introduce an augmented variable along the free boundary so that the stress balancing boundary condition is satisfied. A hybrid time discretization is used in the projection method for better stability. The resultant Helmholtz/Poisson equations with interfaces then are solved by the IIM in an efficient way. Several numerical tests including an accuracy check, and the spreading and recoiling processes of a liquid drop are presented in detail. (C) 2010 Elsevier Ltd. All rights reserved.

水稻同源四倍体三系法杂种优势利用研究

水稻是重要的粮食作物，其产量的增加和品质的改良都是关系国计民生的大事。就我国现阶段的国情而言，水稻产量在现有水平上稳步提升仍是未来十几年甚至几十年农业生产最重要的目标之一。尽管根据“超级杂交水稻育种”的战略设想和水稻育种实践，通过不断地改进育种技术可望在更高的产量水平上进行水稻杂种优势利用，在稻属植物内还具有很大的产量潜力可以挖掘。然而，仅仅从现有的种质基础出发，要更大幅度提高水稻单产，实现“超级杂交稻”的目标也存在一些困难：现有的推广品种是二倍体，尽管种类众多，但是其基因组的来源相对单一；同时，水稻基因组DNA含量也是作物中最少的，基因组内寻求开发潜力有一定困难；水稻作为C3植物，光合利用效率不高也是制约水稻产量提高的因素之一。因此，寻求常规手段以外的技术突破或者方法创新，是实现“超级杂交稻”的目标的迫切需求。本研究利用秋水仙素能抑制细胞分裂中纺锤丝的收缩、使细胞染色体加倍的作用，对水稻幼穗诱导的愈伤组织细胞进行加倍，并分化出再生植株；创制出水稻同源四倍体新的种质材料，在此基础上选育水稻同源四倍体雄性不育三系材料，并实现水稻同源四倍体的三系配套，开展水稻同源四倍体杂种优势利用和四倍体杂交水稻选育研究，建立水稻同源四倍体杂种优势利用的新技术体系。这不仅有助于倍性水平杂种优势的开拓和利用，同时也将为我国新世纪“超级稻”育种研究开辟一条新的技术途径。 水稻幼穗诱导愈伤组织并分化成苗是一项成熟、简单的组织培养技术。本研究以普通二倍体水稻亲本为材料，用秋水仙素进行水稻的多倍体化诱导，创制同源四倍体水稻三系亲本材料并对其进行鉴定。多倍体化以秋水仙素诱导的愈伤组织培养为基础，研究不同秋水仙素浓度梯度和愈伤组织诱导培养基组合对诱导四倍体植株的影响。结果表明在MS+2,4 D 1.0mg/L+ KT0.2mg/L+ IAA0.2mg/L 和500mg/L的秋水仙素处理下，水稻愈伤组织染色体加倍（有最高的效率）效果较好，平均加倍频率可达25.26%，其中，材料CDR22和IR26诱导较易成功，加倍频率分别达到75%和26.5%；相对材料94109 1.3%加倍频率和冈46B 10.8%加倍频率，诱导率差异极显著。 对水稻四倍体材料进行了形态学鉴定结果表明，与二倍体水稻对照相比其株高、穗长、花粉育性等主要农艺性状，确定四倍体材料在穗长和千粒重两方面极显著提高，种子的长度和宽度也显著增长。对花粉育性鉴定，确认水稻四倍体不育系材料仍为不育，保持系材料自交和杂交可育，恢复系材料自交和杂交可育。对四倍体材料进行细胞形态、染色体数目等方面进行细胞学鉴定，经核型分析表明水稻四倍体材料具有48条染色体，是二倍体水稻的两倍。水稻四倍体材料根尖分生组织细胞与二倍体的根尖分生组织细胞相比，细胞体积、细胞核和核仁显著增大。四倍体三系材料在细胞有丝分裂中期均可规则排列在赤道板，并能均等地移向两极；后期观察中没有发现染色体分离滞后现象，分裂末期细胞能够形成大小相对均一的子细胞。水稻同源四倍体三系材料细胞分裂未见异常，植株生长发育正常。 从1996年至2006年，针对结实率、有效分蘖、着粒数和穗长等主要农艺性状，通过系谱选育的方法，对培育的同源四倍体水稻亲本材料进行了连续选择和改良，取得较好成效。表现为结实率的改良效果极佳，所有改良材料的平均结实率均呈上升趋势，如D237（29.70％→72.70％）、DTB（19.55％→53.21％）等。有效分蘖总体呈现上升趋势，但在不同的年份，如1998和2002存在较大的负向波动。部分材料改良效果明显，如D19B（5.87→13.50）、D什香 (7.00→12.00)等；同时一些材料如DTB和D明恢63虽然总体略有提高，但在不同的年份波动很大，因此存在较大改良阻力，原因还有待进一步研究。着粒数的改良上升趋势比较显著，除保持系的DTB之外，其余材料的平均着粒数有显著提高。穗长的改良阻力较大，虽然不同材料总体上有所提高，但效果并不显著，并且不同年份有较大负向波动（2001）。此外还对株高、剑叶长等性状也进行了选择，但效果不显著，原因有待进一步提高。同源四倍体材料产量相关性状遗传改良幅度不一致，保持系和恢复系间的遗传改良效果也存在差异。这为同源四倍体水稻的进一步利用打下了良好的基础。 籼稻和粳稻亚种间杂交及杂种优势利用的主要障碍就是其低的结实率。而同源四倍体杂交水稻的研究为提高杂交水稻的杂种优势利用创造了新的途径。本研究通过随机区组设计方案，挑选性状优良的二倍体水稻材料，包括雄性不育系，保持系和恢复系进行秋水仙素诱导加倍，从而获得同源四倍体水稻对应的三系材料。利用选育的优良水稻同源四倍体三系材料，配制7个杂交组合，杂交F1代与其恢复系亲本进行比较，用于计算超亲优势（HB）；而杂交F1代与生产上大面积推广的二倍体杂交品种汕优63进行比较，用于计算杂种优势。结果显示，同源四倍体杂交水稻的超亲优势表现为：每株有效穗变化幅度为1.4%至105.9%，总粒数为0.5%至74.3%，每穗实粒数为17.6%至255.7%，结实率为9.6%至130.4%。这些农艺性状的改良使得这7个杂种F1的理论产量的超亲优势高达64.8%至672.7%。小区试验中四倍体杂交水稻组合T461A/T4002和T461A/T4193分别比二倍体对照汕优63提高46.3%和38.3%以上，除一个品种以外所有品种产量均接近或高于汕优63的产量。同源四倍体水稻强大的杂种优势表明，亚种间杂交育性低的问题可通过四倍体化及强化选择来解决。此外，同源四倍体杂交水稻器官的巨大性也是其产量提高的有利因素，水稻同源四倍体三系杂种优势利用研究具有一定的理论价值和商业生产潜力。 Rice is one of the major food crops, the improvement of the production and quality of it is an important thing related to the people's livelihood. On China's current national conditions, steadily increase of the rice yield based on the current level is still one of the most important goals in the next decade or even decades of agricultural production. According to the "super hybrid rice breeding" the strategic and rice breeding practice, improvement of the use of hybrid rice heterosis through continuous improvements in breeding technology is expected to get a higher level of rice yield, there are also a great yield potential can be exploited. However, there are also some difficulties to increase rice yield obviously and implement the goal of "super hybrid rice" based on the existing germplasm: Rice varieties in promotion are diploid, although there are many varieties, but their genome are from a comparatively single source; Meanwhile, the rice genome DNA are the least among the crops, it is difficult to exploit the development potential within the genome; Rice as C3 plants, photosynthetic efficiency is not high, it is one of the factors constraint rice yield. Therefore, seeking technological breakthroughs or innovative methods different from conventional means is the urgent needs to reach the target of "super hybrid rice". Using colchicine inhibit spindle contraction during cell division, double the cell chromosome, we induced callus cells from rice panicle to be doubled, and differentiated regeneration; we created a new autotetraploid rice germplasm material, and on that basis we bred male sterility three line autotetraploid rice materials, and the achieved the three line rice autotetraploid matchmaking, researched in autotetraploid rice heterosis usage and tetraploid hybrid rice breeding, constituted a new technology system of autotetraploid hybrid rice heterosis utilization. This not only helps the tetraploid rice heterosis exploration and use, but also inaugurates a new technical means for China in the new century "super rice" breeding research. We chose ordinary diploid rice as materials, using colchicine to induce the polyploidization, created the autotetraploid rice three-line materials and identified them. The polyploidization was based on the colchicine-induced callus tissue culture, and we experimented different colchicine concentrations and culture mediums to induce tetraploid plants, confirmed that the optimal concentration for inducement was 500 mg/L, the average induce rate was 25.26 %. Among all the materials, CDR22 and IR26 had higher induced rate; in contrary, 94109 and GANG46B had lower induced rate, the difference was significant. Autotetraploid materials was identified of both morphological and cytological, compared plant height, length of pollen sterility, and other major agronomic traits with a diploid rice as the control plant, identified that the autotetraploid materials had very significant advantages in ear length and thousand-grain weight, as well as the size of the seeds. Cytology identification included observation of the cell morphology, the number of chromosomes, and karyotype analysis on the autotetraploid materials confirmed that their chromosome number was 48, twice of the diploid rice. Mitoses in the three lines were common: chromosomes arrayed normally in metaphase and separated balanced into the two poles, chromosome moved without lagging in anaphase and daughter cells normally formed in telophase except one. It has been proved that tetraploid rice has normal meiosis as their diploid relatives, which usually including series of sub-phases as interphase, prophase I (five sub-phases), prophase II, metaphase I, II, anaphase I, II and telophase I, II. However, abnormal phenomena, such as formation of tetravalent, trivalent and univalent, chromosome lagging and so on, which would finally block meiosis. Configurations of chromosome in metaphaseⅠwere versatile in structure and form accept the bivalent. That condition varied in different strain, suggesting more complex paring configurations and more versatile genetic characters in tetraploid rice. All these abnormalities in meiosis contributed to low fertility of gamete and might consequently resulted in low seed setting. Successive selection and improvement on seed set, productive tiller per plant, total grains per panicle, panicle length and so on had been carried out from 1996 to 2006. The raise of seed sets was significant in both restorers and maintainers. Seed sets of some strains were improved more significantly than others, for example D237（29.70％→72.70％）、DTB（19.55％→53.21％）and et al.. Productive tiller per plant was improved to some extant. The tendency of improvement was rising on the whole but changed in some years such as 1998 and 2002. Part of the stains increased greatly, such as D19B（5.87→13.50）、Dshixiang (7.00→12.00) and so on, but some strains including DTB and Dminghui63 only increased little and decreased in some years by unknown reason. Total grains per panicle increased significantly and all strains except DTB increased. Improvement of panicle length termed to be hard. Different strains showed different capacities for improvement and floating existed in different years for example 2001. It has been proved that other agronomical traits including plant length, flag leaf length and so on could be improved but not significantly by selection also. In a word, agronomical traits could be raised by successive selection that is prerequisite for further utility of autotetraploid rice. Poor fertility is the main barrier for utilizing heterosis between the two rice (Oryza stiva L.) sub-species, indica and japonica. Recently, the development of autotetraploid hybrids (2n=4x=48) has been suggested as a new method for increasing heterosis in hybrid rice. Using standard experimental protocols, the elite diploid rice male sterile, maintainer, and restorer lines were colchine-doubled and autotetraploid counterparts were obtained. Seven resulting hybrids were analyzed for heterobeltiosis (HB), where the F1 was compared to the male parent, and the degree of heterosis, where the F1 was compared to the diploid commercial hybrid, Shanyou 63. The HB among the autotetraploid hybrids ranged from 1.4 to 105.9% for the productive panicles per plant, 0.5 to 74.3% for total kernels per panicle, 17.6 to 255.7% for filled kernels per panicle, and 9.6 to 130.4% for seed set. Improvements in these yield components resulted in the HB for kernel yield ranging from 64.8 to 672.7% among the seven hybrids. Hybrids T461A/T4002 and T461A/T4193 yielded 46.3 and 38.3% more, respectively than Shanyou 63, and all other hybrids but one yielded the same or more than Shanyou 63. The high heterosis for yield suggests that hybrid sterility between two rice sub-species may be overcome by using tetraploid lines followed by intensive selection. Also, the gigantic features of the autotetraploid hybrids may establish a plant structure able to support the higher yield.