An artificial cell-cycle inhibitor isolated from a combinatorial library

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21Cip1, which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

中药蜘蛛香的化学成分研究及以羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞研究

本论文由三章组成。第一章介绍了中药蜘蛛香的化学成分的研究成果，第二章为羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞的研究，第三章综述了人工细胞在生物医学领域的应用。 第一章报道了中药蜘蛛香（Valeriana wallichii）根部乙醇提取物的化学成分，采用正、反相硅胶层析等分离方法和MS、NMR等多种波谱手段，从中共分离鉴定出17个化合物，分别为缬草素(valtrate，1)，valechlorine（2），homobadrinal（3），baldrinal（4），乙酰缬草素(acevaltrate 5)，valeriotetrate C（6），valeriotetrate B（7），对羟基苯乙酮(4'-hydroxy-acetophenone 8)，7-hydroxy valtrate（9），8-methylvalepotriate（10），1,5-dihydroxy-3,8-epoxyvalechlorine A（11），二氢缬草素(didrovaltrate 12)，胡萝卜苷（13），橙皮苷 (hesperidin 14)，prinsepiol-4-O-β-D-glucopyranoside（15），longiflorone（16），乙基糖苷（17）。其中化合物6、7、10、和11为新化合物，化合物9、15、16为首次从该植物中得到。新化合物11为含有氯原子的刚性骨架环烯醚萜，并且确定了其绝对构型。 第二章报道了以羧甲基魔芋葡苷聚糖（CKGM）和壳聚糖（CS）为膜的固定化L-天冬酰胺酶人工细胞研究成果。利用羧甲基魔芋葡苷聚糖和壳聚糖两种生物相容性很好的天然多糖之间的静电吸引力，在非常温和的条件下制备成具有半透过性膜的人工细胞，将治疗儿童急性成淋巴细胞性白血病(ALL)的药物L-天冬酰胺酶包裹在内。通过考察温度和pH对人工细胞的影响,结果表明以CKGM- CS为膜的L-天冬酰胺酶人工细胞对温度和pH的稳定性和耐受性均高于自由酶，说明CKGM-CS对酶具有保护作用，而且小分子底物和产物可以自由进出膜内外，而包裹在膜内的生物大分子则不能泄露出来。 第三章综述了微囊化人工细胞的研究进展。 This dissertation consists of three parts. In the first part, the chemical constituents from the root of Valeriana wallichii were reported. In the second part, preparation and characteristics of L-Asparaginase Artificial cell were reported. The third part is a review on progress of microcapsule artificial cell. The first chapter is about the isolation and identification of the chemical constituents from the root of V. wallichii. Seventeen compounds were isolated from the ethanol extract of roots of V. wallichii through repeated column chromatography on normal and reversed phase silica gel. By the spectroscopic and chemical evidence, their structures were elucidated as valtrate (1), valechlorine (2), homobadrinal (3), baldrinal (4), acevaltrate (5), valeriotetrate C (6), valeriotetrate B (7), 4'-hydroxy-acetophenone (8), 7-hydroxy valtrate (9), 8-methylvalepotriate (10), 1,5-dihydroxy-3,8-epoxyvalechlorine A (11), didrovaltrate (12), daucosterol (13), hesperidin (14), prinsepiol-4-O-β-D-glucopyranoside (15), longiflorone (16), and ethyl glucoside (17). Among them, 6, 7, 10, and 11 are new compounds. 15, 16 and 9 were isolated from this plant for the first time. The absolute configuration of compound 11, an unusual iridoid bearing a C-10 chlor-group and an oxo-bridge connecting C-3 and C-8 resulting in a rigid skeleton, was confirmed. The second chapter is about the semi-permeable microcapsule of carboxymethyl konjac glucomannan-chitosan for L-asparaginase immobilization. Carboxymethyl konjac glucomannan-chitosan (CKGM-CS) microcapsules, which have good biocompatibility, prepared under very mild conditions via polyelectrostatic complexation, were used for immobilize L-asparaginase-a kind of drug for acute lymphoblastic leukemia (ALL). The activity and stability under different temperature and pH of the enzyme loaded-microcapsules were studied. The results indicated the immobilized enzyme has better stability and activity contrasting to the native enzyme. The study illustrates that the L-asparaginase could be protected in CKGM-CS microcapsules, the substrate and product could pass through the system freely.

Interactions of gold nanoparticles with polymeric membranes

This thesis focuses on the interactions of nanoparticles with artificial membranes. The synthesis of the block copolymer poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PDMS-b-PMOXA) is described, as well as the formation of polymersomes in water. These polymersomes act as minimal cell models, consisting of an artificial bilayer membrane only, allowing the study of the interactions between nanoparticles and polymeric membranes. Both spherical and rod-shaped gold nanoparticles (AuNPs) were used in this study and they were characterized using light scattering (PCS), transmission electron microscopy (TEM), UV/Vis spectroscopy, and polarization anisotropy measurements. The polymer grafting on the spherical cores is asymmetric (shell asphericity) but is parallel to the inherent, due to polycrystallinity, core anisotropy, resulting in a characteristic scattering of the AuNPs in PCS.rnInteractions of polymersomes and AuNPs were investigated by PCS, cryo-TEM and UV/Vis. Three possible scenarios upon mixing of polymersomes and AuNPs can be distinguished by using only PCS: (i) no interactions between particles and vesicles, (ii) attachment of the particles to the outer side of the vesicles (decoration), and (iii) uptake of particles into the vesicles. It is shown that all three scenarios are possible, solely depending on the particle’s surface functionalization. In addition, it was revealed that the AuNPs need to be attached to the inner side of the membrane instead of diffusing freely within the vesicle. The present experimental findings essentially help with the understanding of the interactions of nanoparticles with membranes and show that the process of endocytosis can be attributed to physical processes only. rn

An Immune Inspired Approach to Anomaly Detection

The immune system provides a rich metaphor for computer security: anomaly detection that works in nature should work for machines. However, early artificial immune system approaches for computer security had only limited success. Arguably, this was due to these artificial systems being based on too simplistic a view of the immune system. We present here a second generation artificial immune system for process anomaly detection. It improves on earlier systems by having different artificial cell types that process information. Following detailed information about how to build such second generation systems, we find that communication between cells types is key to performance. Through realistic testing and validation we show that second generation artificial immune systems are capable of anomaly detection beyond generic system policies. The paper concludes with a discussion and outline of the next steps in this exciting area of computer security.

Synthetic biology in droplet-based microfluidics

Droplet microfluidics is an active multidisciplinary area of research that evolved out of the larger field of microfluidics. It enables the user to handle, process and manipulate micrometer-sized emulsion droplets on a micro- fabricated platform. The capability to carry out a large number of individual experiments per unit time makes the droplet microfluidic technology an ideal high-throughput platform for analysis of biological and biochemical samples. The objective of this thesis was to use such a technology for designing systems with novel implications in the newly emerging field of synthetic biology. Chapter 4, the first results chapter, introduces a novel method of droplet coalescence using a flow-focusing capillary device. In Chapter 5, the development of a microfluidic platform for the fabrication of a cell-free micro-environment for site-specific gene manipulation and protein expression is described. Furthermore, a novel fluorescent reporter system which functions both in vivo and in vitro is introduced in this chapter. Chapter 6 covers the microfluidic fabrication of polymeric vesicles from poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) tri-block copolymer. The polymersome made from this polymer was used in the next Chapter for the study of a chimeric membrane protein called mRFP1-EstA∗. In Chapter 7, the application of microfluidics for the fabrication of synthetic biological membranes to recreate artificial cell- like chassis structures for reconstitution of a membrane-anchored protein is described.

Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

Protein-free cell culture on an artificial substrate with covalently immobilized insulin.

Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation. In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin. Immobilized insulin activated the insulin receptor and downstream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin. Finally the immobilized-insulin film was usable repeatedly without marked loss of activity.

The role of insect cell lines in an artificial diet for the parasitold wasp, Trichogramma pretiosum

Trichogramma species are mass-produced for biological control using host eggs. Artificial diets have been developed to reduce production costs, however, most include insect haemolymph as a major component, which still results in a significant expense. Medium conditioned with insect cell lines has produced some success as a haemolymph replacement in artificial diets for several parasitoid wasp species. Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) was the first species to develop successfully to the adult stage on diets containing concentrated HeliothiS zea (Boddie) (Lepidoptera: Noctuidae) cells. Tricho-gramma pretiosum Riley (Hymenoptera: Trichogrammatidae) was subsequently grown to the adult stage on a similar cell line diet. This success encouraged a systematic investigation into the use of insect cell lines in Trichogramma artificial diets. We compared the effect of diets containing insect cells with diets containing conditioned cell line media. Diets containing insect cells produced significantly more pupae than diets containing conditioned medium and, although not significant, produced a higher number of adults. Second, we compared the effect of diets containing cell lines established from ovary-associated tissue of H. zea and embryo tissue of Aedes albopictus (Skuse) (Diptera: Culicidae) on T pretiosum development. Trichogramma pretiosum development was not significantly different on diets containing cells from the two origins and tissue types. Third, the effect of cell storage on T pretiosum development was observed. HeliothiS zea cells in medium were stored at 4 degrees C and room temperature (22 degrees C for one, two, four and seven days before addition to artificial diets. Cell viability was calculated for these storage treatments. HeliothiS zea cells could be stored at 4 degrees C for up to seven days with no detrimental effect on T pretiosum development. Tricho-gramma pretiosum development did not depend on cell viability. The use of insect cell lines as a haemolymph replacement has the potential to significantly reduce production costs and simplify Trichogramma artificial diets with the eventual aim of replacing host production in mass rearing facilities. (c) 2005 Elsevier Inc. All rights reserved.

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Tricho-grammatidae) was successfully used to rear Trichogramm pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace's insect medium, a common component of artificial diets, was found to markedly improve the development of T pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts. (c) 2005 Elsevier Inc. All rights reserved.

Artificial Skin - Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres

Background: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. Methodology/Principal Findings: Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross-sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses. Conclusion/Significance: Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration.

Optimal control of non-linear systems through hybrid cell-mapping/artificial-neural-networks techniques

In this paper, a real-time optimal control technique for non-linear plants is proposed. The control system makes use of the cell-mapping (CM) techniques, widely used for the global analysis of highly non-linear systems. The CM framework is employed for designing approximate optimal controllers via a control variable discretization. Furthermore, CM-based designs can be improved by the use of supervised feedforward artificial neural networks (ANNs), which have proved to be universal and efficient tools for function approximation, providing also very fast responses. The quantitative nature of the approximate CM solutions fits very well with ANNs characteristics. Here, we propose several control architectures which combine, in a different manner, supervised neural networks and CM control algorithms. On the one hand, different CM control laws computed for various target objectives can be employed for training a neural network, explicitly including the target information in the input vectors. This way, tracking problems, in addition to regulation ones, can be addressed in a fast and unified manner, obtaining smooth, averaged and global feedback control laws. On the other hand, adjoining CM and ANNs are also combined into a hybrid architecture to address problems where accuracy and real-time response are critical. Finally, some optimal control problems are solved with the proposed CM, neural and hybrid techniques, illustrating their good performance.

Porous polyurethane coatings bonded onto surface-modified titanium substrates for the growth of an endothelial cell layer

Cardiovascular diseases refer to the class of diseases that involve the heart or blood vessels (arteries and veins). Examples of medical devices for treating the cardiovascular diseases include ventricular assist devices (VADs), artificial heart valves and stents. Metallic biomaterials such as titanium and its alloy are commonly used for ventricular assist devices. However, titanium and its alloy show unacceptable thrombosis, which represents a major obstacle to be overcome. Polyurethane (PU) polymer has better blood compatibility and has been used widely in cardiovascular devices. Thus one aim of the project was to coat a PU polymer onto a titanium substrate by increasing the surface roughness, and surface functionality. Since the endothelium of a blood vessel has the most ideal non-thrombogenic properties, it was the target of this research project to grow an endothelial cell layer as a biological coating based on the tissue engineering strategy. However, seeding endothelial cells on the smooth PU coating surfaces is problematic due to the quick loss of seeded cells which do not adhere to the PU surface. Thus it was another aim of the project to create a porous PU top layer on the dense PU pre-layer-coated titanium substrate. The method of preparing the porous PU layer was based on the solvent casting/particulate leaching (SCPL) modified with centrifugation. Without the step of centrifugation, the distribution of the salt particles was not uniform within the polymer solution, and the degree of interconnection between the salt particles was not well controlled. Using the centrifugal treatment, the pore distribution became uniform and the pore interconnectivity was improved even at a high polymer solution concentration (20%) as the maximal salt weight was added in the polymer solution. The titanium surfaces were modified by alkli and heat treatment, followed by functionlisation using hydrogen peroxide. A silane coupling agent was coated before the application of the dense PU pre-layer and the porous PU top layer. The ability of the porous top layer to grow and retain the endothelial cells was also assessed through cell culture techniques. The bonding strengths of the PU coatings to the modified titanium substrates were measured and related to the surface morphologies. The outcome of the project is that it has laid a foundation to achieve the strategy of endothelialisation for the blood compatibility of medical devices. This thesis is divided into seven chapters. Chapter 2 describes the current state of the art in the field of surface modification in cardiovascular devices such as ventricular assist devices (VADs). It also analyses the pros and cons of the existing coatings, particularly in the context of this research. The surface coatings for VADs have evolved from early organic/ inorganic (passive) coatings, to bioactive coatings (e.g. biomolecules), and to cell-based coatings. Based on the commercial applications and the potential of the coatings, the relevant review is focused on the following six types of coatings: (1) titanium nitride (TiN) coatings, (2) diamond-like carbon (DLC) coatings, (3) 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer coatings, (4) heparin coatings, (5) textured surfaces, and (6) endothelial cell lining. Chapter 3 reviews the polymer scaffolds and one relevant fabrication method. In tissue engineering, the function of a polymeric material is to provide a 3-dimensional architecture (scaffold) which is typically used to accommodate transplanted cells and to guide their growth and the regeneration of tissue. The success of these systems is dependent on the design of the tissue engineering scaffolds. Chapter 4 describes chemical surface treatments for titanium and titanium alloys to increase the bond strength to polymer by altering the substrate surface, for example, by increasing surface roughness or changing surface chemistry. The nature of the surface treatment prior to bonding is found to be a major factor controlling the bonding strength. By increasing surface roughness, an increase in surface area occurs, which allows the adhesive to flow in and around the irregularities on the surface to form a mechanical bond. Changing surface chemistry also results in the formation of a chemical bond. Chapter 5 shows that bond strengths between titanium and polyurethane could be significantly improved by surface treating the titanium prior to bonding. Alkaline heat treatment and H2O2 treatment were applied to change the surface roughness and the surface chemistry of titanium. Surface treatment increases the bond strength by altering the substrate surface in a number of ways, including increasing the surface roughness and changing the surface chemistry. Chapter 6 deals with the characterization of the polyurethane scaffolds, which were fabricated using an enhanced solvent casting/particulate (salt) leaching (SCPL) method developed for preparing three-dimensional porous scaffolds for cardiac tissue engineering. The enhanced method involves the combination of a conventional SCPL method and a step of centrifugation, with the centrifugation being employed to improve the pore uniformity and interconnectivity of the scaffolds. It is shown that the enhanced SCPL method and a collagen coating resulted in a spatially uniform distribution of cells throughout the collagen-coated PU scaffolds.In Chapter 7, the enhanced SCPL method is used to form porous features on the polyurethane-coated titanium substrate. The cavities anchored the endothelial cells to remain on the blood contacting surfaces. It is shown that the surface porosities created by the enhanced SCPL may be useful in forming a stable endothelial layer upon the blood contacting surface. Chapter 8 finally summarises the entire work performed on the fabrication and analysis of the polymer-Ti bonding, the enhanced SCPL method and the PU microporous surface on the metallic substrate. It then outlines the possibilities for future work and research in this area.

The use of artificial MicroRNA technology to control gene expression in Arabidopsis thaliana

In plants, double-stranded RNA (dsRNA) is an effective trigger of RNA silencing, and several classes of endogenous small RNA (sRNA), processed from dsRNA substrates by DICER-like (DCL) endonucleases, are essential in controlling gene expression. One such sRNA class, the microRNAs (miRNAs) control the expression of closely related genes to regulate all aspects of plant development, including the determination of leaf shape, leaf polarity, flowering time, and floral identity. A single miRNA sRNA silencing signal is processed from a long precursor transcript of nonprotein-coding RNA, termed the primary miRNA (pri-miRNA). A region of the pri-miRNA is partially self-complementary allowing the transcript to fold back onto itself to form a stem-loop structure of imperfectly dsRNA. Artificial miRNA (amiRNA) technology uses endogenous pri-miRNAs, in which the miRNA and miRNA*(passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/ amiRNA*sequences that direct highly efficient RNA silencing of the targeted gene. Here, we describe the rules for amiRNA design, as well as outline the PCR and bacterial cloning procedures involved in the construction of an amiRNA plant expression vector to control target gene expression in Arabidopsis thaliana. © 2014 Springer Science+Business Media New York.