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.     

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.     

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     

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.     

Cohesin's DNA Exit Gate Is Distinct from Its Entrance Gate and Is Regulated by Acetylation

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal?a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.     

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。     

A Differential Cargo-Loading Model of Ciliary Length Regulation by IFT

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Bacillus subtilis Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that of E. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.     

A Combined Approach for the Assessment of Cell Viability and Cell Functionality of Human Fibrochondrocytes for Use in Tissue Engineering

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5–P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5–P6 for cell therapy protocols.     

Quantifying the Length and Variance of the Eukaryotic Cell Cycle Phases by a Stochastic Model and Dual Nucleoside Pulse Labelling

A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases and and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase-specific biosensor-based fluorescent imaging.     

细胞梯度力学微环境体外构建的研究

细胞微环境与细胞的相互作用日益成为细胞生物学领域研究热点。微环境中物理信号(如基底的力学性能、形貌和牵张力)在控制细胞命运中的作用更不容忽视。其中力学刺激常以不均一的梯度形式参与调节发育、炎症、伤口愈合以及癌症过程中不同细胞的增殖、迁移和分化等行为。水凝胶是模拟细胞外基质(extracellular matrix, ECM)二维/三维组织支架的理想材料。先进的微纳制造技术已被广泛应用于支撑或包裹细胞的仿生水凝胶的合成和微环境的个性化定制研究中。本文阐述了体内细胞力学微环境中刚度和拉压应力刺激的构建方法与表征手段的研究现状,并着重综述了近年来水凝胶在细胞梯度力学微环境体外构建中的应用研究,同时也对未来研究中所面临的挑战提出了新的展望。这些工作对于组织工程及再生医学具有重要意义。     

Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

Age-Related Declines and Disease-Associated Variation in Immune Cell Telomere Length in a Wild Mammal

Immunosenescence, the deterioration of immune system capability with age, may play a key role in mediating age-related declines in whole-organism performance, but the mechanisms that underpin immunosenescence are poorly understood. Biomedical research on humans and laboratory models has documented age and disease related declines in the telomere lengths of leukocytes (‘immune cells’), stimulating interest their having a potentially general role in the emergence of immunosenescent phenotypes. However, it is unknown whether such observations generalise to the immune cell populations of wild vertebrates living under ecologically realistic conditions. Here we examine longitudinal changes in the mean telomere lengths of immune cells in wild European badgers (Meles meles). Our findings provide the first evidence of within-individual age-related declines in immune cell telomere lengths in a wild vertebrate. That the rate of age-related decline in telomere length appears to be steeper within individuals than at the overall population level raises the possibility that individuals with short immune cell telomeres and/or higher rates of immune cell telomere attrition may be selectively lost from this population. We also report evidence suggestive of associations between immune cell telomere length and bovine tuberculosis infection status, with individuals detected at the most advanced stage of infection tending to have shorter immune cell telomeres than disease positive individuals. While male European badgers are larger and show higher rates of annual mortality than females, we found no evidence of a sex difference in either mean telomere length or the average rate of within-individual telomere attrition with age. Our findings lend support to the view that age-related declines in the telomere lengths of immune cells may provide one potentially general mechanism underpinning age-related declines in immunocompetence in natural populations.