Long-term liver cell cultures in bioreactors and possible application for liver support

Hybrid artificial liver systems are being developed as a temporary extracorporeal liver support therapy. A short overview is given which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies and the clinical application of hybrid liver support systems.An own bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells. The reactor is based on capillaries for hepatocyte aggregate immobilization, coated with biomatrix. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. There is a decentralized co-culture compartment for nonparenchymal liver cells. The use of identical parallel units to supply a few hepatocytes facilitates scale-up.     

Protein engineering and design

Rapid advances in site-directed mutagenesis and total gene synthesis combined with new expression systems in prokaryotic and eukaryotic cells have provided the molecular biologist with tools for modification of existing proteins to improve catalytic activity, stability and selectivity, for construction of chimeric molecules and for synthesis of completely novel molecules that may be endowed with some useful activity. Such protein engineering can be seen as a cycle in which the structures of engineered molecules are studied by X-ray analysis and two-dimensional nuclear magnetic resonance. The results are used in the improvement of the design by using knowledge-based procedures that exploit facts, rules and observations about proteins of known three-dimensional structure.     

The computation of structure from fixed-axis motion: rigid structures

We show that three distinct orthographic views of three points in a rigid configuration are compatibel with at most 64 interpretations of the three-dimensional structure and motion of the points. If, in addition, one assumes that the three points spin about a fixed axis over the three views, then with probability one there is a unique three-dimensional interpretation (plus a reflection). Moreover the probability of false targets is zero. In the special case that the axis of rotation is parallel to the image plane three views of the three points are sufficient to obtain at most two interpretations (plus reflections)-unless one assumes the angular velocity about the axis is constant, in which case three views of two points are sufficient to determine a unique interpretation. Closed form solutions are obtained for each of these cases. The systems of equations studied here are in each case overconstraining (i.e. there are more independent equations than unknowns) and are amenable to solution by nonlinear programming. These two properties make possible the construction of noise insensitive algorithms for computer vision systems. Our uniqueness proofs employ the Principle of upper semicontinuity, a principle which underlies a general mathematical framework for the analysis of solutions to overconstraining systems of equations.     

从景感生态学视角分析城市立体绿化内涵与功能——以深圳市为例

城市用地日益紧张,城市建(构)筑物高度不断抬升,理应赋予城市空间载体更多的生态功能。景感生态学倡导在保持、改善和增加城市生态系统服务的同时提升居民满意度和可持续发展能力。城市立体绿化具有降温、降噪、滞尘、固碳等功能,是节能减排的有效举措。通过对深圳市立体绿化实地调查和景感生态学分析,剖析了立体绿化在促进居民的视觉、听觉、嗅觉、味觉及触觉等物理感知及心理认知功能方面的作用,以及增强城市生态系统服务的机理。研究表明,立体绿化是城市景感营造的有效手段,既能增强城市生态系统服务价值,也能提高城市空间利用率;在增加城市绿视率的同时,丰富了居民的物理感知及心理认知。最后,讨论了景感生态学理论与方法在立体绿化中的实际应用,以及景感营造技术在城市绿色建筑中的作用,以期让景感营造技术为提升立体绿化水平,进而为促进城市绿地系统规划提供支撑。     

Microfluidic cell culture models for tissue engineering

Microfluidic systems have emerged as revolutionary new platform technologies for a range of applications, from consumer products such as inkjet printer cartridges to lab-on-a-chip diagnostic systems. Recent developments have opened the door to a new set of opportunities for microfluidic systems, in the field of tissue and organ engineering. Advances in the design of physiologically relevant structures and networks, fabrication processes for biomaterials suitable for in vivo use, and techniques for scaling towards large, three-dimensional constructs, are converging towards therapeutic applications of microfluidic technologies in engineering complex tissues and organs. These advances herald a new generation of microfluidics-based approaches designed for specific tissue and organ applications, incorporating microvascular networks, structures for transport and filtration, and a three-dimensional microenvironment suitable for supporting phenotypic cell behavior, tissue function, and implantation and host integration.     

血管内皮细胞和心脏组织块的立体培养

本文旨在对比研究二维平面与三维立体培养模式下,内皮细胞和心脏组织形态学的差异。采用胶内、胶上、三明治模式、玻片培养小室模型等多种I型胶原立体培养模型,通过免疫荧光技术及显微形态学观察组织和细胞的生长情况。在二维平面培养中,原代心脏血管内皮细胞呈铺路石样排列;而在三维胶原培养模式中,内皮细胞呈长梭状形态,并迁入胶原培养介质中,和体内血管新生及血管生成过程中的内皮细胞活化表型相似。加入血管内皮生长因子(vascular endo- thelial growth factor VEGF)能增强内皮细胞管状结构的形成。在三维胶原中,心脏组织块生长良好,迁出的细胞将相邻组织块连接起来,组织块有自发的搏动。本工作表明,改进的薄层胶原培养、玻片培养小室模型和动脉条模型是较好的研究血管生成和血管新生的工具。在三维培养的情况下,内皮细胞通过空间增殖、迁移和锚定,可形成管状结构,比二维平面培养更适合用于血管新生的研究。不同的立体培养模型可用于不同目的的研究。     

Tissue engineering: a new frontier in physiological genomics.

Considerable progress has been made in the last decade in the engineering and construction of a number of artificial tissue types. These constructs are typically viewed from the perspective of possible sources for implant and transplant materials in the clinical arena. However, incorporation of engineered tissues, often referred to as three-dimensional (3D) cell culture, also offers the possibility for significant advancements in research for physiological genomics. These 3D systems more readily mimic the in vivo setting than traditional 2D cell culture, and offer distinct advantages over the in vivo setting for some organ systems. As an example, cardiac cells in 3D culture 1) are more accessible for siRNA studies, 2) can be engineered with specific cell types, and 3) offer the potential for high-throughput screening of gene function. Here the state-of-the-art is reviewed and the applications for engineered tissue in genomics research are proposed. The ability to use engineered tissue in combination with genomics creates a bridge between traditional cellular and in vivo studies that is critical to enabling the transition of genetic information into mechanistic understanding of disease processes.     

Identification of novel mammalian squalene synthase inhibitors using a three-dimensional pharmacophore

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerisation of farnesyl diphosphate in a [1-4] head to head fashion to form squalene, and is the first committed step in cholesterol biosynthesis. Specific inhibitors of squalene synthase would inhibit cholesterol formation and allow production of other important compounds derived from the cholesterol biosynthetic pathway, namely the ubiquinones (co-enzyme Q(10)), dolichol, and would also allow the isoprenylation process of ras by farnesyl-protein transferase. The construction of a hypothetical squalene synthase three-dimensional pharmacophore is presented. It serves as a template for the identification of several new potential classes of inhibitors. The synthesis, anti-microbial and mammalian pig liver squalene synthase activities of analogues based on the bicyclo[3.2.0]heptane and bicyclo[3.3.0]octane ring systems are reported. Analogues of the latter system are pro-drug type inhibitors and exhibit promising biological activity.     

Delivery of bone morphogenetic proteins for orthopedic tissue regeneration

Carriers for bone morphogenetic proteins (BMPs) are used to increase retention of these factors at orthopedic treatment sites for a sufficient period of time to allow regenerative tissue forming cells to migrate to the area of injury and to proliferate and differentiate. Carriers can also serve as a matrix for cell infiltration while maintaining the volume in which repair tissue can form. Carriers have to be biocompatible and are often required to be bioresorbable. Carriers also have to be easily, and cost-effectively, manufactured for large-scale production, conveniently sterilized and have appropriate storage requirements and stability. All of these processes have to be approvable by regulatory agencies. The four major categories of BMP carrier materials include natural polymers, inorganic materials, synthetic polymers, composites of these materials. Autograft or allograft carriers have also used. Carrier configurations range from simple depot delivery systems to more complex systems mimicking the extracellular matrix structure and function. Bone regenerative carriers include depot delivery systems for fracture repair, three-dimensional polymer or ceramic composites for segmental repairs and spine fusion and metal or metal/ceramic composites for augmenting implant integration. Tendon/ligament regenerative carriers range from depot delivery systems to three-dimensional carriers that are either randomly oriented or linearly oriented to improve regenerative tissue alignment. Cartilage regenerative systems generally require three-dimensional matrices and often incorporate cells in addition to factors to augment the repair. Alternative BMP delivery systems include viral vectors, genetically altered cells, conjugated factors and small molecules.     

Motor protein decoration of microtubules grown in high salt conditions reveals the presence of mixed lattices

We have used back-projection methods to obtain three-dimensional maps of motor-protein decorated nine and ten protofilament microtubules polymerized in the presence of high salt and preserved in vitreous ice. The resulting three-dimensional maps show that the vast majority of these microtubules have multiple seams, rather than being helical as would be expected according to the lattice accommodation model. These results indicate that microtubules should be analyzed by back-projection before using helical reconstruction approaches, and that nine and ten protofilament microtubules polymerized in high salt conditions are not suitable for helical analysis.     

Silk as an innovative biomaterial for cancer therapy

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.     

Cell interactions with three-dimensional matrices

Signaling and other cellular functions differ in three-dimensional compared with two-dimensional systems. Cell adhesion structures can evolve in vitro towards in-vivo-like adhesions with distinct biological activities. In this review, we examine recent advances in studies of interactions of fibroblasts with collagen gels and fibronectin-containing matrices that mimic in vivo three-dimensional microenvironments. These three-dimensional systems are illuminating mechanisms of cell-matrix interactions in living organisms.     

Three-dimensional tissue constructs built by bioprinting

Bioprinting is an evolving tissue engineering technology. It utilizes computer controlled three-dimensional printers for rapid and high-precision construction of three-dimensional biological structures. We employed discrete and continuous bioprinting to build three-dimensional tissue constructs. In the former case bioink particles - spherical cell aggregates composed of many thousands of cells - are delivered one by one into biocompatible scaffolds, the biopaper. Structure formation takes place by the subsequent fusion of the bioink particles due to their liquid-like and self-assembly properties. In the latter case a mixture of cells and scaffold material is extruded from the biocartridge akin to toothpaste to arrive at the desired construct. Specifically, we built rectangular tissue blocks of several hundred microns in thickness as well as tubular structures of several millimeters in height. The physical basis of structure formation was studied by computer simulations.     

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.     

Life sciences require the third dimension

Novel technologies are required for three-dimensional cell biology and biophysics. By three-dimensional we refer to experimental conditions that essentially try to avoid hard and flat surfaces and favour unconstrained sample dynamics. We believe that light-sheet-based microscopes are particularly well suited to studies of sensitive three-dimensional biological systems. The application of such instruments can be illustrated with examples from the biophysics of microtubule dynamics and three-dimensional cell cultures. Our experience leads us to suggest that three-dimensional approaches reveal new aspects of a system and enable experiments to be performed in a more physiological and hence clinically more relevant context.     

Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.

The vascular smooth muscle cell (VSMC) is surrounded by a complex extracellular matrix that provides and modulates a variety of biochemical and mechanical cues that guide cell function. Conventional two-dimensional monolayer culture systems recreate only a portion of the cellular environment, and therefore there is increasing interest in developing more physiologically relevant three-dimensional culture systems. This review brings together recent studies on how mechanical, biochemical, and extracellular matrix stimulation can be applied to study VSMC function and how the combination of these factors leads to changes in phenotype. Particular emphasis is placed on in vitro experimental studies in which multiple stimuli are combined, especially in three-dimensional culture systems and in vascular tissue engineering applications. These studies have provided new insight into how VSMC phenotype is controlled, and they have underscored the interdependence of biochemical and mechanical signaling. Future improvements in creating more complex in vitro culture environments will lead to a better understanding of VSMC biology, new treatments for vascular disease, as well as improved blood vessel substitutes.     

Statistical analysis of double NOE transfer pathways in proteins as measured in 3D NOE-NOE spectroscopy

Summary The recent development of three-dimensional NMR spectroscopy has alleviated the problem of overlap of resonances. However, also for the 3D experiments resonance assignment strategies have usually relied upon knowledge about spin systems, combined with information about short (sequential) distances. For doubly (¹⁵N/¹³C)-labelled molecules, a novel assignment strategy has been developed. In this paper we address the possibilities of an assignment strategy for proteins, based solely upon the use of NOE data. For this, the 3D NOE-NOE experiment seems most suitable. Therefore, we have made a theoretical evaluation of double NOE transfer pathways in 28 protein crystal structures. We identify 95 connectivities which are most likely to be observed as cross peaks in a 3D NOE-NOE spectrum of a protein. Given the occurrence of one of these 95 connectivities, we evaluate the chances of occurrence for the others. Analysis of these conditional probabilities allowed the construction of five patterns of related, highly correlated cross peaks which resemble the conventional idea of spin systems to some extent and may provide a basis for assignment and secondary structure analysis from 3D NOE-NOE data alone.Dedicated to the memory of Professor V.F. Bystrov     

Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39 ± 2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.     

基于二维与三维信息的南京市主城区生态网络格局对比分析

生境破碎化导致生物多样性锐减,影响生境的生态系统服务,合理而有效的生态网络建设可以恢复城市内破碎生境斑块之间的连接。现有城市生态网络的构建与模拟多基于二维数据信息,未能很好地考虑植被三维结构信息所表征的多元生态位对生境质量的影响,致使生态网络的功能有效性有待验证。选取南京市主城区为研究区,基于高分辨率遥感影像与机载激光雷达数据,对研究区三维绿地植被结构进行了量化,在二维、三维两种情景下分别进行生态源地的提取与景观阻力面的设定,基于最小费用路径识别出两种情景下的生态廊道,并依据电路理论方法识别出廊道中需要重点保护的生态关键节点,进而分析使用二维、三维生态空间信息对生态网络构建结果产生的影响。研究结果表明:1)研究区不同情景下提取的生态源地共有11处不同,与传统二维情景下仅使用绿地面积指标作为斑块属性相比,使用三维植被参数可以更准确地识别具有丰富植被结构的生态源地;2)二维与三维情景下构建的生态廊道分别有137条与129条,平均每条廊道的单位距离阻力值分别为18.2与24.0,运用传统二维信息的模拟结果会在一定程度上高估研究区的景观连通性;3)不同情境下生态关键节点的空间分布基本一致,主要...     

Chemical substructures that enrich for biological activity

MOTIVATION: Certain chemical substructures are present in many drugs. This has led to the claim of 'privileged' substructures which are predisposed to bioactivity. Because bias in screening library construction could explain this phenomenon, the existence of privilege has been controversial. RESULTS: Using diverse phenotypic assays, we defined bioactivity for multiple compound libraries. Many substructures were associated with bioactivity even after accounting for substructure prevalence in the library, thus validating the privileged substructure concept. Determinations of privilege were confirmed in independent assays and libraries. Our analysis also revealed 'underprivileged' substructures and 'conditional privilege'-rules relating combinations of substructure to bioactivity. Most previously reported substructures have been flat aromatic ring systems. Although we validated such substructures, we also identified three-dimensional privileged substructures. Most privileged substructures display a wide variety of substituents suggesting an entropic mechanism of privilege. Compounds containing privileged substructures had a doubled rate of bioactivity, suggesting practical consequences for pharmaceutical discovery.