Potential of enzymatic kinetic resolution using solid substrates suspension: improved yield, productivity, substrate concentration, and recovery

In the literature the enzymatic kinetic resolution of a suspension of a solid substrate has largely been treated as a conventional kinetic resolution of a fully dissolved substrate. In this paper it is shown that this type of kinetic resolution is different in several important aspects. Quantitative models are developed for two types of such suspension processes. These models are used to compare the merits of these processes with the conventional kinetic resolution process where fully dissolved substrate is used. In the suspension processes the liquid phase concentration of substrate enantiomer that should be converted can be kept close to the maximum value, i.e., the solubility, when process conditions are properly chosen, whereas in a conventional process this concentration gradually decreases. Calculations show that this leads to a productivity that is about 6-fold higher in the suspension processes. Also, for enzymes with a low enantioselectivity, a severalfold increase in yield of remaining enantiopure substrate is predicted compared to the conventional kinetic resolution of dissolved enantiomers. Other potential advantages of using suspension reactions are that the initial substrate concentration may be higher (up to 25% (w/w)) and that the desired remaining substrate may be recovered by simply filtering off the solid crystals. Experimental evidence that these merits can be exploited is only partly given, using the few available examples from the literature.     

Unstructured models for suspension cultures of Taxus media cells in a bioreactor under substrate-sufficient conditions

For a better understanding of the simulation, optimization, and process control in cell cultures, good kinetic models are necessary for large scale plant cell culture. In this paper, the systematic kinetics of taxol production by Taxus media cell suspension cultures in a stirred 15-L bioreactor under substrate-sufficient conditions and the absence of inducer intervention were studied. A kinetic model of cell growth was established by logistic equation, and kinetic unstructured models of substrate consumption, product synthesis and rheological behavior were constituted, which incorporated energy spilling. These models were verified by comparing the simulation results with those obtained experimentally. These results showed that energy spilling was a key factor that must be considered in constructing unstructured kinetic models of Taxus media cell suspension cultures in a stirred bioreactor under substrate-sufficient conditions. Besides, an optimized operation measure of decreasing energy spilling was proposed. An increase of 17.64% in cell biomass and 14.88% in taxol concentration were obtained when the strategy of limiting added carbon several times was experimentally implemented in a 15-L bioreactor. Results demonstrated that these established models should be helpful in the process prediction and operation optimization to guide the production and amplification of Taxus media cell suspension cultures in a bioreactor.     

Theory of electric signals of membrane proteins in three dimensions

Transmembrane ion pumps are often investigated experimentally by photoelectric measurements in model systems. In addition to the most widely used systems based on model membranes, a fundamentally different class is represented by the so-called suspension methods. In this technique the electric signal is measured on a bulk suspension of oriented ion pumps in the form of a displacement current. On this system, electric and spectroscopic experiments can be performed simultaneously. Using the information from both types of measurements, and utilizing the three-dimensional nature of the system, it is possible to follow the intramolecular charge motions in all three spatial directions. The derivable dipole moment changes associated with conformational transitions allow the verification of molecular dynamic models. In this work a theory is presented to describe the suspension method; samples with different symmetry properties and the possibilities of photoselection to obtain the desired three-dimensional information are analyzed.     

不同方法建立多重耐药铜绿假单胞菌肠道感染动物模型的比较和评价

目的探讨建立肠道多重耐药细菌感染动物模型的方法,为实验研究和治疗因耐药菌引起的感染提供良好的模型。方法 24只6周龄SPF级雄性BALB/c小鼠分成4组:正常对照组、MDR-PA组、MDR-PA+抗生素组和MDR-PA+禁食组,6只/组。分别给予生理盐水灌胃、PA悬液灌胃、自由饮用含头孢曲松钠水后PA悬液灌胃和禁食后PA悬液灌胃。实验结束后对结肠组织进行病理学观察和炎症评分;检测炎症因子TNF-α和INF-γ浓度。结果结肠组织病理表现出不同程度的炎症变化;炎症因子TNF-α和INF-γ浓度较对照组均明显增加,有显著统计学意义。结论三种方法均可成功建立肠道多重耐药铜绿假单孢菌感染的动物模型,可根据不同实验目的选择不同的建立模型方法。     

甘草悬浮细胞的玻璃化法超低温保存研究

以甘草悬浮细胞为材料,进行了超低温保存技术的研究。高产黄酮的甘草悬浮细胞是一种极具价值但又难于保存的材料,为解决甘草细胞超低温保存中存在的关键问题,即如何减少渗透压的突然改变对细胞造成损伤,通过优化预培养时间、细胞年龄、预处理时间、脱水时间、洗涤液蔗糖浓度等影响因素,建立了一套适合于甘草悬浮细胞的保存方法。采用改良的2,3,5-氯化三苯基四氮唑(TTC)法检测,此法存活率可达82.9%。将保存后的细胞进行恢复培养,其恢复生长率可达80.4%。     

Identification of Growth Phases and Influencing Factors in Cultivations with AGE1.HN Cells Using Set-Based Methods

Production of bio-pharmaceuticals in cell culture, such as mammalian cells, is challenging. Mathematical models can provide support to the analysis, optimization, and the operation of production processes. In particular, unstructured models are suited for these purposes, since they can be tailored to particular process conditions. To this end, growth phases and the most relevant factors influencing cell growth and product formation have to be identified. Due to noisy and erroneous experimental data, unknown kinetic parameters, and the large number of combinations of influencing factors, currently there are only limited structured approaches to tackle these issues. We outline a structured set-based approach to identify different growth phases and the factors influencing cell growth and metabolism. To this end, measurement uncertainties are taken explicitly into account to bound the time-dependent specific growth rate based on the observed increase of the cell concentration. Based on the bounds on the specific growth rate, we can identify qualitatively different growth phases and (in-)validate hypotheses on the factors influencing cell growth and metabolism. We apply the approach to a mammalian suspension cell line (AGE1.HN). We show that growth in batch culture can be divided into two main growth phases. The initial phase is characterized by exponential growth dynamics, which can be described consistently by a relatively simple unstructured and segregated model. The subsequent phase is characterized by a decrease in the specific growth rate, which, as shown, results from substrate limitation and the pH of the medium. An extended model is provided which describes the observed dynamics of cell growth and main metabolites, and the corresponding kinetic parameters as well as their confidence intervals are estimated. The study is complemented by an uncertainty and outlier analysis. Overall, we demonstrate utility of set-based methods for analyzing cell growth and metabolism under conditions of uncertainty.     

Non‐Newtonian rheology in suspension cell cultures significantly impacts bioreactor shear stress quantification

The fields of regenerative medicine and tissue engineering require large‐scale manufacturing of stem cells for both therapy and recombinant protein production, which is often achieved by culturing cells in stirred suspension bioreactors. The rheology of cell suspensions cultured in stirred suspension bioreactors is critical to cell growth and protein production, as elevated exposure to shear stress has been linked to changes in growth kinetics and genetic expression for many common cell types. Currently, little is understood on the rheology of cell suspensions cultured in stirred suspension bioreactors. In this study, we present the impact of three common cell culture parameters, serum content, cell presence, and culture age, on the rheology of a model cell line cultured in stirred suspension bioreactors. The results reveal that cultures containing cells, serum, or combinations thereof are highly shear thinning, whereas conditioned and unconditioned culture medium without serum are both Newtonian. Non‐Newtonian viscosity was modeled using a Sisko model, which provided insight on structural mechanisms driving the rheological behavior of these cell suspensions. A comparison of shear stress estimated by using Newtonian and Sisko relationships demonstrated that assuming Newtonian viscosity underpredicts both mean and maximum shear stress in stirred suspension bioreactors. Non‐Newtonian viscosity models reported maximum shear stresses exceeding those required to induce changes in genetic expression in common cell types, whereas Newtonian models did not. These findings indicate that traditional shear stress quantification of cell or serum suspensions is inadequate and that shear stress quantification methods based on non‐Newtonian viscosity must be developed to accurately quantify shear stress.     

Effects of the inability of suspension-feeding protozoa to collect all cell sizes of a bacterial population

The effects of the inability of suspension-feeding protozoa to collect bacteria over the whole range of sizes in the bacterial size distribution were examined by constructing mathematical models based on this assumption. Systems of suspension-feeding protozoa grown on both growing and nongrowing bacteria were examined in both batch and continuous culture. The models were able to predict three experimental observations common in such systems. Some additional features of the systems which should be useful in interpreting results of experiments with suspension feeding protozoa and in designing new experiments were predicted, also.     

No change in myonuclear number during muscle unloading and reloading

Muscle fibers are the cells in the body with the largest volume, and they have multiple nuclei serving different domains of cytoplasm. A large body of previous literature has suggested that atrophy induced by hindlimb suspension leads to a loss of "excessive" myonuclei by apoptosis. We demonstrate here that atrophy induced by hindlimb suspension does not lead to loss of myonuclei despite a strong increase in apoptotic activity of other types of nuclei within the muscle tissue. Thus hindlimb suspension turns out to be similar to other atrophy models such as denervation, nerve impulse block, and antagonist ablation. We discuss how the different outcome of various studies can be attributed to difficulties in separating myonuclei from other nuclei, and to systematic differences in passive properties between normal and unloaded muscles. During reload, after hindlimb suspension, a radial regrowth is observed, which has been believed to be accompanied by recruitment of new myonuclei from satellite cells. The lack of nuclear loss during unloading, however, puts these findings into question. We observed that reload led to an increase in cross sectional area of 59%, and fiber size was completely restored to the presuspension levels. Despite this notable growth there was no increase in the number of myonuclei. Thus radial regrowth seems to differ from de novo hypertrophy in that nuclei are only added during the latter. We speculate that the number of myonuclei might reflect the largest size the muscle fibers have had in its previous history.     

A comparison of enumeration techniques for Cryptosporidium parvum oocysts

A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspension density is not reported by authors. To confound the problem, each method of counting has large inherent variation. There is a relationship between suspension density, overall number of organisms counted, and counting mechanism accuracy that should be accounted for when selecting a counting mechanism. This study selected a maximum acceptable coefficient of variation (CV) to be 10%. A method was considered unreliable if this standard was not achieved. Flow cytometry achieved this standard at 486 oocysts/ml. Counting with a Coulter counter achieved this level of reliability at about 1,230 oocysts/ml. Neither chamber slides nor fluorescent antibody-stained well slides ever demonstrated less than 10% CV. However, estimates of the minimum required concentrations were 5,100 oocysts/ml and approximately 6,500 oocysts/ml, respectively. The hemacytometer provided counts accurate to a 10% CV at a concentration of at least 60,000 organisms/ml. Of the methods tested, flow cytometry provided the least amount of variability at low suspension densities.     

Cinefilms of particle capture by an induced local change of beat of lateral cilia of a bryozoan

A local disruption of the metachronal wave always accompanies capture of algal cells by tentacles of Flustrellidra hispida (Fabricius). Beat changes for ≈0.2 s over ≈100μm of the ciliated band during capture of a 10-μm particle. The halted parcel of water is therefore larger than the particle of food but much smaller than the flow that continues past the tentacles elsewhere. These events are consistent with the hypothesis that an induced local reversal of beat concentrates particles for those suspension feeders that retain particles upstream from a band of simple cilia (adults or larvae of bryozoans, brachiopods, phoronids, hemichordates, and echinoderms). These events are not explained by other hypotheses that have been advanced for concentration of particles by these suspension feeders. Aerosol filtration models of direct interception are not applicable to this type of ciliary suspension feeder because retention depends on the magnitude of a stimulus and response to it. The stimulus will not be the same function of diameter of the food particle, and response is unlikely below a threshold stimulus.     

Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte.

The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately 10(4) fold by the addition of 2 X 10(6) normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG8 myeloma tumor cells secreting IgG1. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth-supporting factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cell abolishes their growth-enhancing properties. Thymus cells can be syngeneic and allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-8 week old C3H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of XB3-AG8 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shown that these B cells divide every 18 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immonoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.     

CHO工程细胞 (11G-S) 悬浮培养的无血清培养基的设计

以悬浮适应的表达重组尿激酶原 (Pro-urokinase,pro-UK) CHO工程细胞系11G-S为对象,采用Plackett-Burman实验设计及响应面分析法,设计支持CHO工程细胞 (11G-S) 悬浮生长的无血清培养基。以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计对影响细胞生长的培养基添加成分进行考察,确定了3种对细胞生长明显促进作用的培养基添加成分：胰岛素、转铁蛋白及腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种适用于CHO工程细胞 (11G-S) 悬浮培养的无血清培养基SFM-CHO-S。11G-S细胞在SFM-CHO-S批次悬浮培养的细胞最大生长密度达到4.12×106 cells/mL,pro-UK的最大累积活性达到5 614 IU/mL,培养效果优于商品化的同类无血清培养基。     

Automated,in vitro harvest of somatic embryos

This work has demonstrated the aseptic, automated harvest of somatic embryos from a bioreactor suspension culture. Machine vision, emulating the selection criteria of an experienced biologist, classified embryos as harvestable or non-harvestable as they flowed through a 3 mm glass conduit. Embryos classified as harvestable were separated in a sealed harvest chamber. The system harvested 60% of the embryos at a rate of 2.4 embryos/h and incorrectly harvested less than 1% of the non-harvest objects. The low harvest rate precludes the applicability of this technique to research and commercial tissue culture laboratories. The suspension feed-rate, culture population density and culture homogeneity were identified as the most important factors influencing embryo harvest rate. The performance of this technique on more densely populated cultures was projected using anticipated improvements in suspension feedrate. It was concluded that, under the conditions of this analysis, the harvester would be of limited value in a commercial propagation environment but could be beneficial to many research labs working with plant somatic embryos.     

Assessing metabolic constraints on the maximum body size of actinopterygians: locomotion energetics of Leedsichthys problematicus (Actinopterygii,Pachycormiformes)

Maximum sizes attained by living actinopterygians are much smaller than those reached by chondrichthyans. Several factors, including the high metabolic requirements of bony fishes, have been proposed as possible body‐size constraints but no empirical approaches exist. Remarkably, fossil evidence has rarely been considered despite some extinct actinopterygians reaching sizes comparable to those of the largest living sharks. Here, we have assessed the locomotion energetics of Leedsichthys problematicus, an extinct gigantic suspension‐feeder and the largest actinopterygian ever known, shedding light on the metabolic limits of body size in actinopterygians and the possible underlying factors that drove the gigantism in pachycormiforms. Phylogenetic generalized least squares analyses and power performance curves established in living fishes were used to infer the metabolic budget and locomotion cost of L. problematicus in a wide range of scenarios. Our approach predicts that specimens weighing up to 44.9 tonnes would have been energetically viable and suggests that similar body sizes could also be possible among living taxa, discarding metabolic factors as likely body size constraints in actinopterygians. Other aspects, such as the high degree of endoskeletal ossification, oviparity, indirect development or the establishment of other large suspension‐feeders, could have hindered the evolution of gigantism among post‐Mesozoic ray‐finned fish groups. From this perspective, the evolution of anatomical innovations that allowed the transition towards a suspension‐feeding lifestyle in medium‐sized pachycormiforms and the emergence of ecological opportunity during the Mesozoic are proposed as the most likely factors for promoting the acquisition of gigantism in this successful lineage of actinopterygians.     

A Highlights from MBoC Selection: Cell type–dependent mechanisms for formin-mediated assembly of filopodia

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.     

Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors

The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells.     

The correspondence between proximal phalanx morphology and locomotion: implications for inferring the locomotor behavior of fossil catarrhines

Phalanges are considered to be highly informative in the reconstruction of extinct primate locomotor behavior since these skeletal elements directly interact with the substrate during locomotion. Variation in shaft curvature and relative phalangeal length has been linked to differences in the degree of suspension and overall arboreal locomotor activities. Building on previous work, this study investigated these two skeletal characters in a comparative context to analyze function, while taking evolutionary relationships into account. This study examined the correspondence between proportions of suspension and overall substrate usage observed in 17 extant taxa and included angle of curvature and relative phalangeal length. Predictive models based on these traits are reported. Published proportions of different locomotor behaviors were regressed against each phalangeal measurement and a size proxy. The relationship between each behavior and skeletal trait was investigated using ordinary least-squares, phylogenetic generalized least-squares (pGLS), and two pGLS transformation methods to determine the model of best-fit. Phalangeal curvature and relative length had significant positive relationships with both suspension and overall arboreal locomotion. Cross-validation analyses demonstrated that relative length and curvature provide accurate predictions of relative suspensory behavior and substrate usage in a range of extant species when used together in predictive models. These regression equations provide a refined method to assess the amount of suspensory and overall arboreal locomotion characterizing species in the catarrhine fossil record.     

大蒜体细胞胚胎发生研究进展

大蒜生产主要靠无性繁殖 ,因此 ,进行大蒜体细胞胚发育研究具有重要意义。本文对大蒜体细胞胚发育的影响因子进行了综述 ,其中较高浓度的维生素B1 及还原态氮源可能有利于胚胎发生 ,而大蒜体细胞内含物则不利于胚胎发生。此外 ,对大蒜体细胞胚培养中存在的主要问题进行了讨论 ,并认为系统开展体细胞胚发生的细胞分子生物学机理研究、建立悬浮培养体系以及进行大蒜体细胞胚无性系变异的研究等 ,具有广阔的前景。