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Molecular, Cellular and Tissue Engineering, 2002. Proceedings of the IEEE-EMBS Special Topic Conference on

Date 9-9 June 2002

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  • Proceedings of the IEEE-EMBS Special Topic Conference on Molecular, Cellular and Tissue Engineering (Cat. No.02EX596)

    Publication Year: 2002
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  • Index

    Publication Year: 2002 , Page(s): 202 - 211
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    Freely Available from IEEE
  • Direct visualization of unwinding activity of Duplex RNA by Dhpa, a dead box helicase, at single molecule resolution

    Publication Year: 2002
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    The Eschericia coli protein, DbpA, is unique in its subclass of DEAD box RNA helicases because it possesses ATPase specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity is well defined toward various substrates, its RNA helicase activity remained to be characterized. We show by using biochemical assays and atomic force microscopy (AFM) that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. The work presents an attempt to investigate the action of DEAD-box proteins by a single-molecule visualization methodology. Our AFM images enabled us to directly observe the unwinding reaction of a DEAD box helicase on long stretches of double stranded RNA (dsRNA). Specifically, we could differentiate between defined steps in the unwinding reaction. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidences and the results reported here suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases. Although the biological function of DbpA and many RNA helicases from the DEAD box family remain to be elucidated, this work provides means to understand the structural bases of the motoric and enzymatic activity of DEAD box proteins. In addition, application of AFM to the study of these motor proteins open a new way to investigate the intimate relationship between these enzymes and their co-factors needed for the biological catalysis. View full abstract»

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  • Development of an interface model for the generation and engineering of tissue interfaces in vitro

    Publication Year: 2002
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    Summary form only given. The interface between any newly engineered tissue and pre-existing tissue is absolutely key to tissue engineering, yet this process has so far largely ignored with only a few published reports of the mechanical strength of newly integrated surfaces between connective tissues with other engineered tissues or simply cell-free substrates. Although some correlation between cell migration and matrix deposition have been found, the cellular mechanism of tissue integration is still poorly understood. Work in our laboratory on a tendon-collagen gel interface model has shown measurable adhesion strength increasing with cultivation time dependent on cell migration and surface injury (though not cell proliferation). This model has been evolved to generate a better-defined interface between two collagen lattices, one pre-contracted by resident fibroblasts and the other cell free. A new culture chamber has been designed and fabricated to allow a vertical casting of the cell free gel and then horizontal cultivation immediately after the interface is formed. This can be cultivated for prolonged period of time (> 2 weeks) and can be fitted onto a computer-driven mechanical testing system to perform indentation studies or apply predefined tensile loading. In this new geometry, stress and strain can be precisely measured, allowing for further modelling of the mechanics of the system by finite element modelling. Baseline (time zero) adhesion force measurements showed good uniformity and reproducibility. The current experimental design permits solid interface formation in a controlled manner with a well-defined geometry and the possibility to measure mechanical linkage and loading effects. The long-term findings of this research will be beneficial to the development of a new generation of tissue bioreactors. View full abstract»

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  • Electrochemical biosensors for on-site analysis

    Publication Year: 2002 , Page(s): 54 - 55
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    An electrochemical biosensor is an analytical device in which biological components, in close contact with electrodes, are used to detect a target analyte in solution. We present here the development and application of several electrochemical biosensors based on enzyme amplification and amperometric detection, with screen-print electrodes. The sensors are easy to operate, and the procedures are rapid, accurate, reproducible and inexpensive, requiring neither special skills and training nor complicated instrumentation. The basic configuration of the sensor can be adapted to and applied in various analytical determinations. Examples for the fast identification and quantitative determination of hazardous environmental chemical pollutants, food components, and certain bacteria and viruses that adversely affect human health will be discussed. View full abstract»

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  • A preliminary study on optical measurements of glucose concentration using a surface plasmon resonance system

    Publication Year: 2002 , Page(s): 68 - 70
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    Optical glucose measurement is an attractive research topic for years. One of the goals is to develop a portable device for continuous and non-invasive monitoring of blood glucose for diabetics. In this work, a surface plasmon resonance (SPR) system, which has a high detection sensitivity of 7.61×10-7 refractive index unit (RIU), is utilized for measuring the glucose concentration in aqueous solution. The experimental results show that this approach, which measures the optical behavior of glucose solution, may possibly reveal a new pathway for the non-invasive measurement of blood glucose. View full abstract»

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  • Investigation of the mesoscopic contact mechanics of sexithienyl thin films

    Publication Year: 2002 , Page(s): 141 - 142
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    We demonstrate that the mechanical properties of self-affine fractal thin films can be investigated on mesoscopic scale with an atomic force microscope. Sexithienyl films have been studied by acquiring load-displacement curves with flat micrometric tips. It is shown that the mechanical response of these samples strongly depends on their surface morphology, the contact stiffness varying an order of magnitude upon small but significative changes of fractal parameters. This indicates a general route to tailor films properties at the stage of their deposition and growth. View full abstract»

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  • Prediction of protein coarse contact maps using recursive neural networks

    Publication Year: 2002 , Page(s): 130 - 131
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (282 KB) |  | HTML iconHTML  

    Tools for predicting topological features such as residue contacts can serve as a crucial step towards prediction of protein folding from sequence. In this paper we focus on the prediction of contacts between secondary structure segments. We introduce a new machine learning approach for scoring candidate contact maps and we show that the predicted score can be effectively used to guide a graph search algorithm. View full abstract»

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  • Optical measurement of 3D collagen gel constructs by elastic scattering spectroscopy

    Publication Year: 2002
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    Summary form only given. Analysis of the formation and organisation of new connective tissue formed in tissue-engineered constructs is a major requirement for tissue bioreactor technology. We have analysed early stage responses in collagen lattices using elastic scattering spectroscopy to assess its potential to monitor tissue structural changes in structures up to 3 mm thick, under normal culture conditions. optical system (medOptica TM) is based on the principle that elastically scattered light carries information about structure, density, composition and architecture of a tissue. For all three mechanical conditions imposed to the lattices (i.e. (1) relaxed untethered, (2) restrained tethered/released and (3) tethered under the Culture Force Monitor (CFM)), correlation could be found between changes in the spectral signatures and changes in the collagen gels. Measurements of the influence of isolated factors (e.g. cell concentration, gel thickness etc.) on the spectral signatures have been used to generate a hypothesis of how matrix alters during fibroblasts mediated lattice remodelling to influence scatter. Further work is needed before the key biological events may be correlated with specific areas of the composite spectral signatures observed but results indicate that elastic scattering spectroscopy will be an effective tool in monitoring tissue engineered constructs or early repair in collagenous tissues. Acknowledgements: This study has been supported by the Fifth Framework Programme of the European Commission, "Biomechanical Interactions in Tissue Engineering and Surgical Repair (BITES)". View full abstract»

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  • A nano-scale theoretical model of the myosin head mechanics

    Publication Year: 2002 , Page(s): 125 - 127
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    The conformational changes of the myosin head due to the ATP hydrolysis determine the tilting of the head and the consequent sliding of the actin filament. A mathematical model of the myosin head tilting mechanism is performed here. In particular the myosin head mechanics has been explored starting from recent findings about the myosin ultrastructure, morphology and energetics in order to calculate the working stroke and the force transmitted to the actin filament during muscle contraction. Two different working stroke mechanisms have been investigated, assuming that the tilting of the myosin head occurs in one step or in two steps. Our results show that force and working stroke values vary markedly between the two models. The maximum force generated is about 10 pN for the one step tilting model and 27 pN for the two step model, and the working stroke is about 13 nm and 5 nm respectively. The model results are in agreement with experimental measurements. A sensitivity analysis has also been performed. This research project is part of an overall model of sarcomere mechanics recently developed in our Laboratory (Redaelli et al., J. Biomech, 2001). View full abstract»

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  • Immobilization of RNA: application to single molecule spectroscopy

    Publication Year: 2002 , Page(s): 71 - 72
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (280 KB) |  | HTML iconHTML  

    RNA molecules play important roles in many biological processes including the storage and transfer of information in the cell. Importantly, RNA folds associated with biological functions. The development of new single-molecule methodologies allows to study native RNA molecules, independent in their sizes, in realtime. This requires the immobilization of RNA molecules on a surface. At the present time, however, there is insufficient knowledge on how to optimize the attachment of these molecules to a plane. We report a direct approach to immobilize long RNA on a glass surface. Importantly, these procedures can be applied to both native and synthetic RNA molecules to be probed by various single molecule methodologies. View full abstract»

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  • Comparative evaluation of autologous chondrocyte implantation and mosaic plasty: a controlled randomized clinical study

    Publication Year: 2002
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    Summary form only given. Mosaic plasty and autologous chondrocyte implantation (ACI) may hold particular promise as strategies to repair focal chondral defects of the knee. Both procedures can give 60-90% success as has emerged from separate short/medium-term clinical studies. To assess their real respective efficacy, we have performed a 4-year controlled randomized clinical trial. Only lesions localized on the femoral condyles were considered. Previous treatment(s) of those lesions by alternative traditional means was retained as an exclusion factor. Out of 25 patients (16 to 40 year old), 13 were randomized for mosaic plasty and 12 for ACI. In conclusion, 8 patients have greater than 36 months follow-up, 9 have greater than 24 months follow-up and 6 have 12-18 months follow-up. Patient assessment instruments included arthroscopy, nuclear magnetic resonance, the Lyshohlm Knee Scoring Scale and the Standard IKDC Evaluation Form. The outcome of the study is now being statistically evaluated and results of the analysis will be presented. View full abstract»

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  • AgeWa: An integrated approach for antisense experiment design

    Publication Year: 2002 , Page(s): 120 - 121
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (232 KB) |  | HTML iconHTML  

    The new experimental methods for gene expression analysis, such as those based on the microarray technology, require a subsequent validation of the gene function, that can be carried out by means of either in vitro or in vivo models. One of the most promising methodologies that have been proposed in the last decade for the investigation of gene function is based on antisense oligonucleotides [ASO]. In antisense experiment design, the crucial step is the characterization of nucleotide domains that can efficiently be targeted by this synthetic molecule. At present, no standard procedures are available for target selection. In this paper, we propose an integrative approach for ASO target selection. The proposed tool, AgeWa (Automatic Gene Walk), combines a neural filter with database mining in order to predict the optimal target for antisense action. View full abstract»

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  • Cytomechanics and cell-level cues in tissue engineering

    Publication Year: 2002
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    Summary form only given. Many of the most common targets for tissue engineering involve replacement of substantive connective tissue function. To achieve rational strategies for engineering of such mechanically active tissues it is essential to consider the importance and application of mechanical loads as cellular control cues. Inasmuch as our aim is to develop biomimetic systems for tissue growth and repair, it will not be possible to ignore the key mechanical signals, which govern function and form in tissues such as skin, cartilage, tendon, bone, muscle. Indeed, for most adult mammalian, connective tissues, the default repair pathway results in scar tissue and this may be, in large part, a result of inappropriate cell-level mechanical signalling. The key architectural cues in CTs are thought to be mechanical but as long as these regulatory mechanisms remain unclear they cannot be used predictably in tissue engineering. Importantly signals must act at the cell (cytomechanical), rather than the tissue level. We have developed model systems based on 3D cell-seeded collagen gel cultures and uniaxial tensile loading and measuring devices (the culture force monitors - CFMs) to help to identify and quantify key cytomechanic control mechanisms. This approach has emphasised that external loads can only be used to regulate cell function through the mediation of the material properties of their extracellular matrix. At the same time cells change shape and attachment during tissue remodelling and alter the structure of the very matrix, which propagates those mechanical cues. Consequently, cytomechanical control cues must be delivered in a dynamic and vectored manner, taking account of the progressive responses of resident fibroblastic cells. It is concluded that cell responses to external loads can be regulated through: loading patterns, relative to planes of maximum and minimum matrix compliance; cell-matrix interactions (substrate structure, ligand density, integrin modification); cell shape and cytoskeletal structure (regulating cell motility & contraction). View full abstract»

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  • A wavelet based method to predict the alpha helix content in the secondary structure of globular proteins

    Publication Year: 2002 , Page(s): 132 - 133
    Cited by:  Papers (2)
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (272 KB) |  | HTML iconHTML  

    A single sequence based method is proposed to predict the α-helix content of a protein from its primary sequence. The analysis of the secondary structure of the protein is performed using the information derived from its hydrophobicity profile and the amino acid composition. The method is applied to a test set of 347 proteins obtaining a residue by residue accuracy of 72%. View full abstract»

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  • The development of an artificial, biodegradable nerve guide

    Publication Year: 2002
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    Summary form only given. Over a period of approximately ten years, a biodegradable nerve guide was developed at the University of Groningen (The Netherlands). It comprised the combined research of the departments of Polymer Chemistry, Medical Physiology, Medical Biology, Pathology and Plastic Surgery. At first a polymer was developed and tested. A prototype tube was then used in an animal model (rat sciatic nerve) to establish axonal regeneration short-term and long-term. At the same time cytotoxicity and biocompatibility tests of the polymer were conducted. Once the axonal regeneration and safety of this material were proven, a functional outcome study was performed using several parameters (walking track analysis, withdrawal reflex) to monitor sensory and motor function recovery. We are now at a stage were a clinical pilot study will be initiated to find clinical regeneration of short nerve defects. Future research will be directed towards faster regeneration as well as the bridging of longer gaps using tissue engineered devices. The whole process of integrating this research will be discussed. View full abstract»

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  • Investigation of protein similarity using the Kolmogrov-Smirnov test

    Publication Year: 2002 , Page(s): 38 - 39
    Cited by:  Papers (1)
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (229 KB) |  | HTML iconHTML  

    The near completion of the sequencing of the human and other genomes has provided a wealth of information that is yet to be interpreted. Similarity studies of proteins can often provide insights into protein function, as functionally similar proteins are often similar at some level of their primary, secondary, tertiary or quaternary organization. We propose the Kolmogorov-Smirnov statistic as a metric of protein similarity and demonstrate its use by comparing functionally related and unrelated proteins. View full abstract»

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  • Modelling hydrophobicity to identify secondary structure signals in globular proteins

    Publication Year: 2002 , Page(s): 134 - 135
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    In globular proteins the hydrophobic effect is a well-known phenomenon that is more and more investigated in relation with protein folding. The Discrete Fourier Transform of the hydrophobic profile of the primary structure, resulting in the so-called amphipatic plots, allow to identify some regular structures. We present a modelling method, which aims to improve both the user-friendliness and the quality of the representation of the amphipatic plots. View full abstract»

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  • Exploring the mechanics of endothelial cells one cell at a time

    Publication Year: 2002
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    Summary form only given. Research during the past 20 years on the biology of the vascular wall, and the endothelial layer lining the major arteries in particular, has been dominated by measurements and theories that treat the layer as being homogeneous. This is not the case, and more recent measurements have shown that the behavior of individual cells can differ markedly from the mean. In this address, I will review some of the new results that provide information at the level of individual cells and suggest how these differences between cells can provide clues regarding the biophysical processes that are at work. View full abstract»

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  • Cooperative versus key residues in globular protein folding: an artificial neural network approach

    Publication Year: 2002 , Page(s): 128 - 129
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (258 KB) |  | HTML iconHTML  

    Neural networks have been applied with success for protein sequence analysis. Besides, the protein folding process involves many scientific aspects that are not completely understood yet. We present two new artificial neural network approaches (forward and reverse) focusing on the capability of each amino acid to cause a specific folding-either by itself or with the cooperativity of other residues that are close in the primary structure. The forward approach looks for an association between the protein primary structure fragments and its folded structure; this approach should retain the cooperative features of residues. The reverse approach looks for an association between a fragment of protein tertiary structure and the amino acid placed in the center of the fragment; this approach should retain the singular influence that a key residue by itself has on that folding. The results obtained emphasize the cooperative nature of regular structures and the specific role that some residues plays in the folded structures. View full abstract»

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  • Biomolecular optical storage

    Publication Year: 2002 , Page(s): 73 - 76
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (467 KB) |  | HTML iconHTML  

    A new application of bacteriorhodopsin (BR) in optical storage is presented. BR is used as the active layer of a Write-Once-Read-Many (WORM) storage which may be easily applied to a wide variety of substrates. The recording process uses polarization-sensitive two-photon absorption. As an example for this new BR application an ID-card equipped with an optical recording strip is presented. The capacity is about 1 MB of data per card. The recording density used currently is 125 kB/cm2 which is far from the optical and materials limits but allows operation with cheap terminals. Data are stored in pages of 10 kB each. A special optical encryption procedure, which is first described here, allows to protect the stored data from unauthorized reading. The molecular basis of this process is described. A special property of BR in this application is that by biomolecular design storage, photochromism and traceability of the material are combined in a single molecule. BR introduces a new quality of storage capability for applications with increased security and anti-counterfeiting requirements. View full abstract»

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  • In situ synthesis of oligonucleotide arrays by using the molecular stamp method

    Publication Year: 2002 , Page(s): 34 - 35
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    This work developed a new technique for in situ synthesis of oligonucleotide arrays on glass surfaces. In this method, based on the standard phosphoramidites chemistry protocol, the coupling was conducted through contacting glass surfaces with a set of molecular stamps on which surfaces spread nucleoside monomer and tetrazole mixed acetonitrile solution. It was shown that 20-mer oligonucleotide on the glass slide was successfully synthesized using the modified polydimethylsiloxane (PDMS) stamps and showed the high synthetic efficiency. An effective method has been used to eliminate residual reactive nucleosides on chip with small molecules containing hydroxyl group. A specific oligonucleotide arrays of four probes including matched and mismatched with the target sequence was fabricated to identify the perfect match and mismatch sequences. It indicated that the perfect match and mismatch probes hybridization fluorescent signals had clearly difference, and may be used to identify rapidly screen single-nucleotide polymorphisms. View full abstract»

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  • The development of Web-based clinical information systems

    Publication Year: 2002
    Cited by:  Papers (1)
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    The requirements for clinical information systems are converging onto a universally accepted model. The key question is how can such systems be realised in practice. The presentation addresses this key question in a number of ways. The key components of a generic model for clinical information systems are described. How each of the various standards which have been developed, or are in the process of being developed, fit the generic model is discussed. The key difference today is that open architecture hardware is sufficiently powerful that, when coupled to broadband telecommunication systems and Web-based technology, the generic model for clinical information systems is fully realisable. View full abstract»

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  • Spongy hydrogel capsules as ideal immunoprotective system for cell encapsulation

    Publication Year: 2002 , Page(s): 62 - 63
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    It was shown that the living cell inclusion inside bulk alginate hydrogel matrix is a good method to protect them from external environment. But a lot of unsolved problems derived from this technical procedure when the cells, suspended inside the hydrogel matrix, are subjected to mechanical stress during the crosslinking process of polymer solution. To avoid this unfavourable compression on the cell surface, a new spongy alginate hydrogel matrix was made. To test this new spongy capsule performance, fibroblasts were included in this new spongy matrix. With these experiments it was showed that the cell growth inside these new hydrogel sponges, compared with a cell growing inside a conventional hydrogel bulk matrix, was very different. View full abstract»

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  • Growth factors contained in platelets enhance proliferation of human mesenchymal stem cells

    Publication Year: 2002 , Page(s): 155 - 156
    Save to Project icon | Request Permissions | Click to expandQuick Abstract | PDF file iconPDF (241 KB) |  | HTML iconHTML  

    Mesenchymal stem cells (MSC) contained in the bone marrow provide an invaluable source for for bone reconstruction. Major limitations in their use are: the relative low number of MSC in the marrow cell population and the small volume of bone marrow available. There are emerging data demonstrating clinical applications of ex-vivo expanded cells, whose expansion is supported by several growth factors. We investigated whether growth factors (GFs) released by platelets stimulated MSC proliferation. MTT assays showed that the effect of GFs on MSC proliferation was dose dependent and was maximal at 10%. Also frozen GFs were able to stimulate proliferation. In conclusion we identified a method to promote ex-vivo expansion of MSC to be used in bone reconstruction. View full abstract»

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