Cells by Design: A Mini-Review of Targeting Cell Engineering Using DNA Microarrays

Recent studies have demonstrated the utility of DNA microarray technology in engineering cellular properties. For instance, cellular adhesion, the necessity of cells to attach to a surface in order to to proliferate, was examined by comparing two distinct HeLa cell lines. Two genes, one encoding a type II membrane glycosylating sialyltransferase (siat7e) and the other encoding a secreted glycoprotein (lama4), were found to influence adhesion. The expression of siat7e correlated with reduced adhesion, whereas expression of lama4 correlated with increased adhesion, as shown by various assays. In a separate example, a gene encoding a mitochondrial assembly protein (cox15) and a gene encoding a kinase (cdkl3), were found to influence cellular growth. Enhanced expression of either gene resulted in slightly higher specific growth rates and higher maximum cell densities for HeLa, HEK-293, and CHO cell lines. Another investigated property was the adaptation of HEK-293 cells to serum-free media. The genes egr1 and gas6, both with anti-apoptotic properties, were identified as potentially improving adaptability by impacting viability at low serum levels. In trying to control apoptosis, researchers found that by altering the expression levels of four genes faim, fadd, alg-2, and requiem, apoptotic response could be altered. In the present work, these and related studies in microorganisms (prokaryote and eukaryote) are examined in greater detail focusing on the approach of using DNA microarrays to direct cellular behavior by targeting select genes.     

A Systematic Comparison of Mathematical Models for Inherent Measurement of Ciliary Length: How a Cell Can Measure Length and Volume

Cells control organelle size with great precision and accuracy to maintain optimal physiology, but the mechanisms by which they do so are largely unknown. Cilia and flagella are simple organelles in which a single measurement, length, can represent size. Maintenance of flagellar length requires an active transport process known as intraflagellar transport, and previous measurements suggest that a length-dependent feedback regulates intraflagellar transport. But the question remains: how is a length-dependent signal produced to regulate intraflagellar transport appropriately? Several conceptual models have been suggested, but testing these models quantitatively requires that they be cast in mathematical form. Here, we derive a set of mathematical models that represent the main broad classes of hypothetical size-control mechanisms currently under consideration. We use these models to predict the relation between length and intraflagellar transport, and then compare the predicted relations for each model with experimental data. We find that three models—an initial bolus formation model, an ion current model, and a diffusion-based model—show particularly good agreement with available experimental data. The initial bolus and ion current models give mathematically equivalent predictions for length control, but fluorescence recovery after photobleaching experiments rule out the initial bolus model, suggesting that either the ion current model or a diffusion-based model is more likely correct. The general biophysical principles of the ion current and diffusion-based models presented here to measure cilia and flagellar length can be generalized to measure any membrane-bound organelle volume, such as the nucleus and endoplasmic reticulum.     

Controlled Modulation of Folate Polyglutamyl Tail Length by Metabolic Engineering of Lactococcus lactis

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for γ-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.     

Studying Biomolecule Localization by Engineering Bacterial Cell Wall Curvature

In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i) the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii) the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.     

树木叶片面积与叶柄长、节间长和叶倾角的关系初探

树木的叶片面积与叶柄长、节间长之间有着明显的内在联系．本文分析研究了这个联系,在适当的理想假设下通过数学建模得到了反映树木叶片面积与叶柄长、节间长和叶倾角之间的关系模型．接着,文章对模型的不合理之处进行了分析与改进,并得到叶片面积与节间长、心茎距之间的关系表达式．最后,本文利用分别采集到的13种互生和对生树木叶子上的相关数据对模型进行了检验．检验结果表明建立的模型是正确的,能够反映树木的叶片面积与叶柄长、节间长和叶倾角之间的相互数量关系．     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Phene Synergism between Root Hair Length and Basal Root Growth Angle for Phosphorus Acquisition

Shallow basal root growth angle (BRGA) increases phosphorus acquisition efficiency by enhancing topsoil foraging because in most soils, phosphorus is concentrated in the topsoil. Root hair length and density (RHL/D) increase phosphorus acquisition by expanding the soil volume subject to phosphorus depletion through diffusion. We hypothesized that shallow BRGA and large RHL/D are synergetic for phosphorus acquisition, meaning that their combined effect is greater than the sum of their individual effects. To evaluate this hypothesis, phosphorus acquisition in the field in Mozambique was compared among recombinant inbred lines of common bean (Phaseolus vulgaris) having four distinct root phenotypes: long root hairs and shallow basal roots, long root hairs and deep basal roots, short root hairs and shallow basal roots, and short root hairs and deep basal roots. The results revealed substantial synergism between BRGA and RHL/D. Compared with short-haired, deep-rooted phenotypes, long root hairs increased shoot biomass under phosphorus stress by 89%, while shallow roots increased shoot biomass by 58%. Genotypes with both long root hairs and shallow roots had 298% greater biomass accumulation than short-haired, deep-rooted phenotypes. Therefore, the utility of shallow basal roots and long root hairs for phosphorus acquisition in combination is twice as large as their additive effects. We conclude that the anatomical phene of long, dense root hairs and the architectural phene of shallower basal root growth are synergetic for phosphorus acquisition. Phene synergism may be common in plant biology and can have substantial importance for plant fitness, as shown here.Suboptimal phosphorus availability is a primary limitation to plant growth in terrestrial ecosystems (Vance et al., 2003). Large areas of tropical and subtropical soils in Africa, Latin America, and Asia have phosphorus availability limited by low total phosphorus content as well as high phosphorus fixation (Sanchez and Uehara, 1980). The use of phosphorus fertilizer to correct phosphorus deficiency is only a partial solution, since phosphorus fertilizers are costly, nonrenewable, potentially harmful to the environment, and often marginally effective in tropical soils because of immobilization by the soil (Cathcart, 1980). Therefore, the development of crop cultivars with enhanced ability to acquire phosphorus is an important strategy to increase agricultural productivity in low-input agroecosystems and to reduce input requirements in intensive agriculture (Vance et al., 2003; Gahoonia and Nielsen, 2004; Lambers et al., 2006; Lynch, 2007, 2011).Several root phenes (i.e. basic units of the phenotype; Serebrovsky, 1925; Lynch, 2011; for discussion, see York et al., 2013) enhance phosphorus acquisition, including root architectural phenes for topsoil foraging (Lynch and Brown, 2001), such as shallow root growth angles (Liao et al., 2004; Ho et al., 2005), increased basal root whorl number (Lynch and Brown, 2012; Miguel et al., 2013), and adventitious rooting (Miller et al., 2003); phenes to enhance soil exploitation, including root hair length and density (RHL/D; Bates and Lynch, 2000a, 2000b, 2001; Ma et al., 2001a; Gahoonia and Nielsen, 2004; Yan et al., 2004) and phosphorus-solubilizing root exudates (Ryan et al., 2001); mycorrhizal symbioses (Smith and Read, 2008); and phenes that reduce the metabolic cost of soil exploration (Lynch and Ho, 2005), such as root etiolation and root cortical aerenchyma (Fan et al., 2003; Postma and Lynch, 2010, 2011). It is probable that interactions among these phenes are important in determining the phosphorus acquisition of integrated phenotypes. Results from the structural-functional model SimRoot indicate that RHL/D, the distance from the root tip to the first appearance of root hairs, and the pattern of root hair-bearing epidermal cells (trichoblasts) among non-hair-bearing cells (atrichoblasts) are synergetic for phosphorus acquisition in Arabidopsis (Arabidopsis thaliana; Ma et al., 2001b). Another SimRoot study showed that on low-phosphorus soils, the utility of root cortical aerenchyma in maize (Zea mays) may be 2.9 times greater in plants with increased lateral branching density than in plants with normal branching (Postma and Lynch, 2011). Morphological, anatomical, symbiotic, and biochemical phenes expressed by root axes should have significant synergies with architectural phenes, since architectural phenes determine the position of root axes in time and space and, therefore, the soil domain in which spatially localized phenes are expressed (Lynch, 2011).Phosphorus availability is greater in the topsoil, with a steep decline with depth. Therefore, root architectural phenes that increase topsoil foraging can improve phosphorus acquisition (Lynch and Brown, 2001). Root shallowness regulated by basal root growth angle (BRGA) has been demonstrated to be of particular importance for topsoil foraging (Bonser et al., 1996; Liao et al., 2001; Rubio et al., 2001; Ho et al., 2005). These studies show that common bean (Phaseolus vulgaris) genotypes with smaller BRGA (i.e. shallower roots) have better performance in low-phosphorus soils. Shallow root distribution is also important for phosphorus acquisition in maize (Zhu et al., 2005).RHL/D are also important for phosphorus acquisition (Bates and Lynch, 2000a, 2000b, 2001; Gahoonia and Nielsen, 2004). Since phosphorus mobility in soil is governed by diffusion rather than mass flow, phosphorus uptake by roots is limited by localized phosphorus depletion in the rhizosphere (Barber, 1995). Long root hairs extend the phosphorus depletion zone surrounding the root, thereby increasing the total amount of phosphorus accessible by the roots and phosphorus acquisition. In many plant species, the length and density of root hairs increase in response to low phosphorus availability (Bates and Lynch, 1996; Ma et al., 2001a). Increased RHL/D increases phosphorus accumulation in Arabidopsis growing in low-phosphorus conditions (Bates and Lynch, 2000a, 2000b), and mutants lacking root hairs have reduced phosphorus acquisition (Bates and Lynch, 2000b; Gahoonia et al., 2001). Species that develop more and/or longer root hairs (e.g. Lolium perenne) are more efficient in accessing inorganic phosphorus from soils and thus show greater growth response to phosphorus fertilization than species that lack these traits (e.g. Podocarpus totara). Genotypic variation for root hairs is associated with increased phosphorus acquisition in several species, including barley (Hordeum vulgare; Gahoonia and Nielsen, 2004), common bean (Miguel, 2004; Yan et al., 2004), and maize (Zhu et al., 2010).We hypothesize that the utilities of BRGA and RHL/D for phosphorus acquisition are synergetic. Root hairs will be more valuable for phosphorus acquisition if located in surface soil horizons by arising from roots with a shallow growth angle; shallow roots will have greater benefit for phosphorus acquisition if they have long and dense hairs. Therefore, genotypes possessing long, dense root hairs on shallow roots should have greater phosphorus acquisition than genotypes with either long root hairs on deep roots or short root hairs on shallow roots. We expect the combined benefit of long root hairs and shallow root growth angles to exceed the sum of their individual effects, since they permit greater exploitation of soil strata with the greatest phosphorus availability.In this study, we evaluated the potential synergism between the architectural phene of BRGA and the morphological phene of RHL/D for phosphorus acquisition by comparison of contrasting phenotypes of common bean growing in a weathered tropical soil.     

植物细胞工程技术生产紫杉醇研究进展

紫杉醇是一种二萜类生物碱,主要从红豆杉树皮中分离提取。目前采用植物细胞工程技术是提高紫杉醇产率、保护稀缺资源红豆杉、解决紫杉醇药源紧缺的一种最有效方法。该文对近十年来国内外有关采用植物细胞工程技术生产紫杉醇的研究进展,包括红豆杉细胞系的建立、悬浮培养条件的研究、生物反应器培养以及紫杉醇合成代谢调控方面的最新研究进展进行综述。并重点论述了前体、诱导子和代谢支路抑制剂对红豆杉细胞悬浮培养生产紫杉醇的影响,为植物细胞工程技术生产紫杉醇提供借鉴和参考。     

成长记录袋评价法提升细胞工程课堂教学质量

首次将成长记录袋评价法引入到高等院校生物类专业课程《细胞工程》的课堂教学改革中,构建了较为完备的课堂评价体系,将成长记录袋课堂评价体系的建立分为4个阶段,即准备阶段、演练阶段、实施阶段和作品展示阶段.并从实施成长记录袋评价法的可行性与必要性、评价体系的构建、执行过程中应注意的要点及事项分别展开论述.结果 表明:通过该评...     

以HaCaT细胞为种子细胞构建新型人组织工程皮肤

目的:评估以人永生化角朊细胞HaCaT为种子细胞构建新型人组织工程皮肤的可行性。方法:对HaCaT进行无血清培养(DK-SFM)及传代培养;取处于对数生长期的细胞进行接种;利用物理化学方法,采用SD大鼠网状层真皮制备(Acelluar dermal matrix,ADM);应用MTT比色法作生长曲线观察HaCaT细胞长满真皮支架所需时间。HE染色法观察脱细胞前后真皮支架和细胞单层及初步形成的细胞多层。结果:脱细胞真皮的细胞成分消失,组织疏松,为理想的组织皮肤支架;将HaCaT细胞按2×10~5/cm~2密度接种到ADM上,细胞长满支架的时间为5天。结论:在SD大鼠脱细胞真皮支架上以HaCaT细胞为种子细胞可以构建新型人组织工程皮肤     

Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus luminescens Cell Populations

Phenotypic heterogeneity in bacterial cell populations allows genetically identical organisms to different behavior under similar environmental conditions. The Gram‐negative bacterium Photorhabdus luminescens is an excellent organism to study phenotypic heterogeneity since their life cycle involves a symbiotic interaction with soil nematodes as well as a pathogenic association with insect larvae. Phenotypic heterogeneity is highly distinct in P. luminescens. The bacteria exist in two phenotypic forms that differ in various morphologic and phenotypic traits and are therefore distinguished as primary (1°) and secondary (2°) cells. The 1 cells are bioluminescent, pigmented, produce several secondary metabolites and exo-enzymes, and support nematode growth and development. The 2° cells lack all these 1°-specific phenotypes. The entomopathogenic nematodes carry 1° cells in their upper gut and release them into an insect's body after slipping inside. During insect infection, up to the half number of 1° cells undergo phenotypic switching and convert to 2° cells. Since the 2° cells are not able to live in nematode symbiosis any more, they cannot re-associate with their symbiosis partners after the infection and remain in the soil. Phenotypic switching in P. luminescens has to be tightly regulated since a high switching frequency would lead to a complete break-down of the nematode-bacteria life cycle. Here, we present the main regulatory mechanisms known to-date that are important for phenotypic switching in P. luminescens cell populations and discuss the biological reason as well as the fate of the 2° cells in the soil.     

Monosaccharide-Responsive Phenylboronate-Polyol Cell Scaffolds for Cell Sheet and Tissue Engineering Applications

Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.     

5-Aza-C 对鼻咽癌细胞端粒长度及细胞增殖的影响

目的：研究去甲基化药物5-氮杂胞嘧啶核苷（5-Azacytidine,5-Aza-C）对鼻咽癌细胞端粒长度及细胞生长增殖的影响。方法： 常规培养鼻咽癌CNE,CNE1,CNE2 及5-8F细胞系,5-Aza-C 处理鼻咽癌细胞后,甲基化测序聚合酶链反应(MSP)法检测亚端粒 区D4Z4 甲基化,端粒限制性片断检测端粒长度,CCK-8 检测细胞增殖。结果：2.5 uM浓度的5-Aza-C处理后,亚端粒区D4Z4 序 列的甲基化水平明显下降,约在15-20%之间,与处理前的甲基化水平（35-48%）有明显差异,差异均有统计学意义（P < 0.05)。在1 uM和2.5 uM浓度的5-Aza-C 处理后,四种细胞的端粒长度明显缩短,长度在2-4.5kb 之间,差异具有统计学意义(P < 0.05)。5uM 的5-Aza-C处理72 h 后,CNE,CNE1,CNE2 和5-8F 的生存率分别为51.27%,50.46%,48.85%,48.83%,10 uM 的5-azaC 处理72 h 后,CNE,CNE1,CNE2 和5-8F的生存率分别为31.64%,32.34%,30.01%,32.10%,与对照组比较差异有统计学意义（P<0.01）。结 论：5-Aza-C 能缩短端粒长度,抑制鼻咽癌细胞生长增殖活性。     

Cell-Cell Adhesion and Cortical Actin Bending Govern Cell Elongation on Negatively Curved Substrates

Physiologically, cells experience and respond to a variety of mechanical stimuli such as rigidity and topography of the extracellular matrix. However, little is known about the effects of substrate curvature on cell behavior. We developed a novel, to our knowledge, method to fabricate cell culture substrates with semicylindrical grooves of negative curvatures (radius of curvature, R_c = 20–100 μm). We found that negative substrate curvatures induced elongation of mesenchymal and epithelial cells along the cylinder axis. As R_c decreases, mesenchymal National Institutes of Health 3T3 fibroblasts increasingly elongate along the long axis of the grooves, whereas elongation of epithelial Madin-Darby Canine Kidney (MDCK) cells is biphasic with maximal cell elongation when R_c = 40 μm. Addition of blebbistatin to MDCK cells to reduce cortical actin rigidity resulted in a decrease in cell elongation across all curvatures while preserving the biphasic trend. However, addition of calyculin A or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, to increase cortical rigidity or reduce intercellular adhesion, respectively, resulted in a monotonic increase in MDCK cell elongation with decreasing R_c. Using an energy minimization model, we showed that cell elongation in epithelial cell sheet is governed by the competition between two energies as R_c decreases: curvature-dependent intercellular adhesion that prevents elongation; and intracellular cortical actin bending that enhances elongation. Therefore, our results of cellular elongation induced by negatively curved substrates offer insights into how tubule elongation or growth of tubular structures such as kidney tubules can be controlled by the substrate curvature in vivo.     

石竹科植物组织培养与细胞工程

近年来,植物组织培养与细胞工程研究在石竹科植物上取得了一定进展。现从组织培养、原生质体培养和体细胞杂交、单倍体育种、试管开花、转基因等5个方面对其进行综述,并展望了石竹科植物在组织培养和细胞工程研究方面的发展前景。     

魔芋组织培养与细胞工程

近几年来,魔芋组织培养研究进展较快,其细胞工程领域也取得了一定的研究成果。现对魔芋组织培养过程中外植体取材、愈伤组织诱导与分化的激素应用进行了概述,重点介绍了魔芋离体形态建成的几种模式及其调控机制的研究新进展。有关魔芋种质资源离体保存和突变体的筛选的研究工作已经展开,其遗传转化体系也逐渐完善,外源基因如抗病基因、抗除草剂基因等现已转化成功。最后还对魔芋今后的研究方向进行了讨论,指出了目前存在的主要问题并提出了相应的对策。