MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Biotechnology' source. Thu, 09 Feb 2012 18:15:16 +0100 http://www.medworm.com/index.php?rid=5375751&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_9 Electrospinning is one of the most versatile and effective tools to produce nanostructured fibers in the biomedical science fields. The nanofibrous structure with diameters from tens to hundreds of nanometers largely mimics the native extracellular matrix (ECM) of many tissues. Thus far, a range of compositions including polymers and ceramics and their composites/hybrids have been successfully applied for generating electrospun nanofibers. Different processing tools in electrospinning set-ups and assemblies are currently developed to tune the morphology and properties of nanofibers. Herein, we demonstrate the electrospinning process and the electrospun biomaterials for specific use in tissue regeneration with some examples, involving different material combinations and fiber morphologies. ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375751 Fri, 04 Nov 2011 22:14:48 +0100 5375751 http://www.medworm.com/index.php?rid=5375750&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_8 The large number of cytokines and growth factors implicated in the regulation of liver regeneration has led to the possibility of using these molecules in therapy, namely, in the case of recombinant human hepatocyte growth factor (rhHGF). The importance and potential clinical usefulness of rhHGF has been extensively studied and documented, with results suggesting that this molecule could be a powerful tool toward increased success in hepatic regenerative therapy. However, the peptidic nature of this drug presents several challenges toward its effective administration and targeting. The possibility of encapsulating rhHGF in dextran sulfate/chitosan nanoparticles to allow its oral administration and direct liver-targeting is discussed in this manuscript. Details of a rapid and simple method ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375750 Fri, 04 Nov 2011 22:14:48 +0100 5375750 http://www.medworm.com/index.php?rid=5375749&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_7 Microwell arrays are cell culture and imaging platforms to assess cells at a single cell level and in high-throughput. They allow the spatial confinement of single cells in microfabricated cavities on a substrate and thus the continuous long-term observation of single cells and their progeny. The recent development of microwell arrays from soft, biomimetic hydrogels further increases the physiological relevance of these platforms, as it substantially enhances stem cell survival and the efficiency of self-renewal or differentiation. This protocol describes the microfabrication of such hydrogel microwell arrays, as well as the cell handling and imaging. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375749 Fri, 04 Nov 2011 22:14:48 +0100 5375749 http://www.medworm.com/index.php?rid=5375748&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_6 There is a large interest in the use of nucleic acids covalently bound to surfaces for a variety of biomedical uses: biosensors, microarrays, drug delivery, lab-on-chip devices, and gene therapy, etc. Most of these applications require the covalent attachment of oligonucleotides via specific reactive groups on both modified oligonucleotide and/or surface. The purpose of this chapter is to provide experimental protocols for the synthesis of oligonucleotides and for the immobilization of these synthetic oligonucleotides onto surfaces such as gold and silicon oxide. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375748 Fri, 04 Nov 2011 22:14:48 +0100 5375748 http://www.medworm.com/index.php?rid=5375747&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_5 Micro and nanoscale protein patterning based on microcontact printing technique on large substrates have often resolution problems due to roof collapse of the poly(dimethylsiloxane) (PDMS) stamps used. Here, we describe a technique that overcomes these issues by using instead a stamp made of poly(methyl methacrylate) (PMMA), a much more rigid polymer that do not collapse even using stamps with very high aspect ratios (up to 300:1). Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density submicrometer scale patterns, or,... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375747 Fri, 04 Nov 2011 22:14:48 +0100 5375747 http://www.medworm.com/index.php?rid=5375746&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_4 There is an increasing demand on the development of “smart” switchable interfaces since controlling surface topography and chemical functionality on a nanometer scale is crucial for numerous biomedical applications. Those surfaces, which are based on stimuli responsive polymers (SRPs), are able to modify their interactions with cells, biomolecules responding to different physical (e.g., temperature) or chemical (e.g., pH) stimuli. Such behavior may partially mimic complex dynamic properties of natural systems that are regulated by many biological stimuli. This paper reviews major studies and applications of SRPs as biointerfaces in a form of thin polymeric films (gels) and surface tethered polymers (brushes). (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375746 Fri, 04 Nov 2011 22:14:48 +0100 5375746 http://www.medworm.com/index.php?rid=5375745&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_3 An important goal in the development of highly functional organic materials is to design self-assembling molecules that can reproducibly display chemical signals across length scales. Within the biomedical field, biomolecules are highly attractive candidates to serve as bioactive building blocks for the next generation of biomaterials. The peptide amphiphiles (PAs) developed by the Stupp Laboratory at Northwestern University generated a highly versatile self-assembly code to create well-defined bioactive nanofibers that have been proven to be very effective at signaling cells in vitro and in vivo. Here, we describe the basic steps necessary for synthesis and assembly of PA molecules into functional nanostructures. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375745 Fri, 04 Nov 2011 22:14:48 +0100 5375745 http://www.medworm.com/index.php?rid=5375744&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_2 In this chapter, we describe two methods for bio-producing recombinant repetitive polypeptide polymers for use in biomedical devices. These polymers, known as elastin-like recombinamers (ELRs), are derived from the repetition of selected amino acid domains of extracellular matrix proteins with the aim of recreating their mechanical and physiological features. The proteinaceous nature of ELRs allows us to make use of the natural biosynthetic machinery of heterologous hosts to express advanced and large polymers or “recombinamers.” Despite the essentially unlimited possibilities for designing recombinamers, the production of synthetic genes to encode them should allow us to overcome the difficulties surrounding bioproduction of these non-natural monotonous DNA and protein sequenc... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375744 Fri, 04 Nov 2011 22:14:48 +0100 5375744 http://www.medworm.com/index.php?rid=5375743&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_1 Regenerative medicine is an emerging field aiming to the development of new reparative strategies to treat degenerative diseases, injury, and trauma through developmental pathways in order to rebuild the architecture of the original injured organ and take over its functionality. Most of the processes and interactions involved in the regenerative process take place at subcellular scale. Nanotechnology provides the tools and technology not only to detect, to measure, or to image the interactions between the different biomolecules and biological entities, but also to control and guide the regenerative process. The relevance of nanotechnology for the development of regenerative medicine as well as an overview of the different tools that contribute to unravel and engineer biological systems are... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375743 Fri, 04 Nov 2011 22:14:48 +0100 5375743 http://www.medworm.com/index.php?rid=5375742&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_19 Advances in regenerative nanomedicine raise a host of ethical, legal, and social questions that healthcare providers and scientists will need to consider. These questions and concerns include definitions, appropriate applications, dual use, potential risks, regulations, and access. In this chapter, we provide an overview of the questions and concerns and recommend proactive consideration and solutions. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375742 Fri, 04 Nov 2011 22:14:48 +0100 5375742 http://www.medworm.com/index.php?rid=5375741&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_18 The molecular dynamics method is a powerful computer simulation technique which provides access to the detailed time evolution (trajectory) of a system in specified conditions, such as a particular temperature or pressure. The full trajectory of the system can be analyzed using statistical mechanics tools to obtain thermodynamical quantities and dynamical properties; the mechanism of chemical reactions and other time-dependent processes, such as diffusion, can also be revealed in high detail. When applied to model extended and complex system such as biomaterials, MD simulations represent an invaluable tool to discover structure–activity relationships and rationalize biomedical applications. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375741 Fri, 04 Nov 2011 22:14:48 +0100 5375741 http://www.medworm.com/index.php?rid=5375740&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_17 Cells respond to biochemical and mechanical stimuli through a series of steps that begin at the molecular, nanometre level, and translate finally in global cell response. Defects in biochemical- and/or mechanical-sensing, transduction or cellular response are the cause of multiple diseases, including cancer and immune disorders among others. Within the booming field of regenerative medicine, there is an increasing need for developing and applying nanotechnology tools to bring understanding on the cellular machinery and molecular interactions at the nanoscale. Nanotechnology, nanophotonics and in particular, high-resolution-based fluorescence approaches are already delivering crucial information on the way that cells respond to their environment and how they organize their receptors to perf... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375740 Fri, 04 Nov 2011 22:14:47 +0100 5375740 http://www.medworm.com/index.php?rid=5375739&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_16 Quantum Dots (QDs) are a new class of semiconductor nanoparticulate luminophores, which are actively researched for novel applications in biology and nanomedicine. In this review, the recent progress in the design and applications of QD labels for in vitro and in vivo imaging of cells is presented. Surface chemical engineering of hydrophobic QDs is required to render them water soluble and biocompatible. Further surface modification and attachment of bioactive molecules to the surface of QDs, such as peptides, aptamers, or antibodies are intensively explored for targeted imaging of living cells, and disease states in animals. Specially designed surface coatings can drastically decrease nonspecific interactions between QDs and cells, minimize degradation of QDs under in vivo physiological c... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375739 Fri, 04 Nov 2011 22:14:47 +0100 5375739 http://www.medworm.com/index.php?rid=5375738&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_15 Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. In this chapter, we examine the promise and shortcomings of “top-down” and “bottom-up” approaches for creating engineered biological tissues. In top-down approaches, the cells are expected to populate the scaffold and create the appropriate extracellular matrix and microarchitecture often with the aid of a bioreactor that furnish the set of stimuli required for an optimal cellular viability. Specifically, we survey the role of cell material interaction on oxygen metabolism in three-dimensional (3D) in vitro cultures as well as the time and space evolution of the transport and biophysical properties during the development of de novo synthesized tissue-engine... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375738 Fri, 04 Nov 2011 22:14:47 +0100 5375738 http://www.medworm.com/index.php?rid=5375737&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_14 This chapter presents as a proof of concept the development of a nanosensor based on the localized surface plasmon resonance for the analysis of biomolecules. The method presented take advantage of the plasmon generated in the surrounding of gold nanoparticles (i.e., 100 nm) for the specific interaction between antigen and antibody. The procedure for the optimization of an assay for the determination of biomolecules consisted mainly of four steps. First, the immobilization of gold nanoparticles over the glass surface using the appropriate ratio, concentration and time-contact of amino-sylilating agent, and nonreactive sylilating agent. Next, the suitable concentration of coating antigen in order to obtain the maximum signal LSPR. Following this step, the interaction between antigen and ant... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375737 Fri, 04 Nov 2011 22:14:47 +0100 5375737 http://www.medworm.com/index.php?rid=5375736&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_13 While in the last years applications of carbon nanotubes in the field of biotechnology have been largely proposed, so far biomedical applications of boron nitride nanotubes (BNNTs) are still totally unexplored. BNNTs show very interesting physical properties that might be exploited in the nanomedicine field. To fill up the lack of biocompatibility studies on BNNTs, our group has recently begun a rational investigation on interactions between BNNTs and different cell lines. This chapter reports on preliminary cytocompatibility studies carried out on human neuroblastoma (SH-SY5Y) and on mouse myoblast (C2C12) cell lines as model of neural and skeletal muscle cells, respectively, highlighting the methods that allowed us to evaluate the main parameters of interests for cytocompatibility, such ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375736 Fri, 04 Nov 2011 22:14:47 +0100 5375736 http://www.medworm.com/index.php?rid=5375735&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_12 This chapter describes a versatile approach to immobilize proteins and other biomolecules on reactive self-assembled monolayers on gold as a means to study interactions (forces) between these biomolecules and nonbiological entities. Biomolecules are either immobilized on the surface of flat substrates or on the surface of gold-coated atomic force microscopy (AFM) probe tips. In addition to the immobilization protocols, the actual AFM experiments in liquid and the quantitative analysis of adhesive forces receive attention. With these procedures and tools at hand, a wide variety of problems in the area of bio/nonbio interactions can be addressed experimentally. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375735 Fri, 04 Nov 2011 22:14:47 +0100 5375735 http://www.medworm.com/index.php?rid=5375734&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_11 Determining the contact angle of a liquid on a solid surface is a simple method to assess the surface wettability. The most common method to measure the contact angle of a liquid consists of capturing the profile of a sessile drop of a few microliters on the surface using an optical system. Currently, this is a widely used technique to analyze wettability both in researched materials and in products of multiple technological fields. However, the drop dispensed by a traditional macroscopic contact angle meter is too big to assess the wettability properties of individual topographical features and/or chemical patterns at the micro/nanoscale. Recently, contact angle meters that can discharge drops that are microscopic, with volumes in the range of 1 × 10−3 to 10−5 μL have... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375734 Fri, 04 Nov 2011 22:14:47 +0100 5375734 http://www.medworm.com/index.php?rid=5375733&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-388-2_10 Protein adsorption from (aqueous) solutions onto a (solid) surface is a common process that takes place at biological interfaces. This phenomenon, that spontaneously occurs, changes the properties of the surface and can induce structural modifications on proteins. Proteins in solution can be easily identified/quantified using classical biochemical methods. However, adsorbed proteins are more difficult to assess since they are always associated with a substrate. The selection of the analytical method depends on the type of substrate used, the amount of adsorbed protein, the type of solution (single protein solution vs. complex biological media), and the type of information that is demanded (quantification of the adsorbed protein, adsorption kinetics, conformation, and orientation of the ads... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5375733 Fri, 04 Nov 2011 22:14:47 +0100 5375733 http://www.medworm.com/index.php?rid=5035512&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_32 Laser-capture microdissection (LCM) allows for retrieval of distinct populations of cells from their closely surrounding neighbors in situ. As such, LCM is highly advantageous for investigating gene expression along the central nervous system (CNS) microvascular endothelium, a tissue that shows both ­considerable segmental and regional heterogeneity. Combining immunohistochemical staining of CNS microvascular endothelial cells with immunofluorescent staining of perivascular astrocytes or smooth muscle cells, immune-guided LCM, immuno-LCM, may be coupled to downstream qRT-PCR to probe varied expression of the endothelium along the CNS microvascular tree during health and disease. Immuno-LCM/qRT-PCR has been used to highlight contributions of the respective segments of the CNS microvascu... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035512 Thu, 30 Jun 2011 23:00:00 +0100 5035512 http://www.medworm.com/index.php?rid=5035511&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_31 Neurulation is a critical process in the formation of the central nervous system during embryonic ­development. Closure of the neural tube is driven by forces that originate from both the neuroepithelium and the surrounding tissues. In this chapter, we describe the use of laser capture microdissection to ­isolate and separately collect cells from the neuroepithelium and the underlying mesenchyme. We provide protocols for processing of samples for downstream comparison of the transcriptomes of two cell populations using high-density oligonucleotide microarrays, with an emphasis on important technical issues that are to be borne in mind when carrying out these experiments. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035511 Thu, 30 Jun 2011 23:00:00 +0100 5035511 http://www.medworm.com/index.php?rid=5035510&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_30 Cell specificity of gene expression analysis is essential to avoid tissue sample related artifacts, in particular when the relative number of target cells present in the compared tissues varies dramatically, e.g., when comparing dopamine neurons in midbrain tissues from control subjects with those from Parkinson’s disease (PD) cases. Here, we describe a detailed protocol that combines contact-free UV-laser microdissection and quantitative PCR of reverse-transcribed RNA of individual neurons from postmortem human midbrain tissue from PD patients and unaffected controls. Among expression changes in a variety of dopamine neuron marker, maintenance, and cell-metabolism genes, we found that α-synuclein mRNA levels were significantly elevated in individual neuromelanin-positive dopam... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035510 Thu, 30 Jun 2011 23:00:00 +0100 5035510 http://www.medworm.com/index.php?rid=5035509&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_2 Microarray-based gene expression profiling is revolutionizing biomedical research by allowing expression profiles of thousands of genes to be interrogated in a single experiment. In cancer research, the use of laser microdissection (LM) to isolate RNA from tissues provides the ability to accurately identify molecular profiles from different cell types that comprise the tumor and its surrounding microenvironment. Because RNA is an unstable molecule, the quality of RNA extracted from tissues can be affected by sample preparation and processing. Thus, special protocols have been developed to isolate research-quality RNA after LM. This chapter provides detailed descriptions of protocols used to generate micro­array data from high-quality frozen breast tissue specimens, as well as challenge... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035509 Thu, 30 Jun 2011 23:00:00 +0100 5035509 http://www.medworm.com/index.php?rid=5035508&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_29 This chapter describes the procedure of in situ hybridization-guided laser-capture microdissection performed on postmortem human brain tissue. This procedure permits the precise collection of brain tissue within anatomically defined brain nuclei that is enriched with mRNA. The chapter emphasizes the specific handling of postmortem tissue and preservation of RNA integrity to ensure high-quality gene profiling. Downstream procedures including mRNA amplification, gene profiling using high-density microarray chips, and confirmation with quantitative real-time polymerase chain reaction (qPCR) are described. PCR primer design and cDNA quantification required for qPCR are delineated. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035508 Thu, 30 Jun 2011 23:00:00 +0100 5035508 http://www.medworm.com/index.php?rid=5035507&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_28 The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a c... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035507 Thu, 30 Jun 2011 23:00:00 +0100 5035507 http://www.medworm.com/index.php?rid=5035506&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_27 Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this chapter, we describe a technique for isolating individual spiral ganglion cells from archival formalin-fixed, celloidin-embedded (FFCE) human temporal bone sections. The DNA isolated from these single cells is suitable for analysis with a duplex real-time polymerase chain reaction (PCR) methodology to quantify the mitochondrial DNA (mtDNA) deletion level present. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035506 Thu, 30 Jun 2011 23:00:00 +0100 5035506 http://www.medworm.com/index.php?rid=5035505&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_26 Laser microdissection permits isolation of specific cell types from tissue sections or cell cultures. This may be beneficial when investigating the role of specific cells in a complex tissue or organ. In tissues with easily distinguishable morphology, a simple hematoxylin staining is sufficient, but in most cases a more specific staining is required to identify which cells to microdissect. We have established two staining protocols for frozen sections (1) Oil red O, which stains lipid droplet in fat cells and steroid-producing cells and (2) NBT BCIP, which stains cells expressing an alkaline phosphatase enzyme, such as fetal germ cells, testicular carcinoma in situ cells, and putatively also other early stem cell populations. We have applied these protocols for microdissection of rat Leydi... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035505 Thu, 30 Jun 2011 23:00:00 +0100 5035505 http://www.medworm.com/index.php?rid=5035504&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_25 In this study, we investigated the expression of these enzymes in normal prostate gland (NP), hormone-sensitive prostate cancer (HSPC), and hormone-refractory prostate cancer (HRPC). The prostatic tissue specimens were obtained from patients who had undergone prostate needle biopsies without any treatments or with PSA failure after initial androgen deprivation. The tissue samples derived from formalin-fixed, paraffin-embedded (FFPE) sections were prepared by laser-capture microdissection, and from them RNA was extracted. The levels of OPRT and DPD mRNA expression were examined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The level of OPRT mRNA expression in the HSPC or the HRPC specimens was significantly higher than that in the NP specimens. There was a signif... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035504 Thu, 30 Jun 2011 23:00:00 +0100 5035504 http://www.medworm.com/index.php?rid=5035503&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_24 Prognostic markers can improve prediction of the behaviour of a cancer at the point of diagnosis. A key value of any prognostic marker is at the point of tumour diagnosis. In the context of prostate cancer, this implies profiling in the diagnostic formalin-fixed, paraffin-embedded (FFPE) transrectal ultrasound-guided (TRUS) needle biopsy. TRUS needle biopsies commonly contain both stromal and epithelial cells, and malignant glands are found as isolated foci within this tissue. Using the entire biopsy for genetic analysis inevitably results in a significant contamination of malignant cells with benign tissue. This combination of minimal tumour yields and tissue heterogeneity have so far prohibited prognostic transcript and microarray molecular studies in needle biopsies. Laser-capture micro... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035503 Thu, 30 Jun 2011 23:00:00 +0100 5035503 http://www.medworm.com/index.php?rid=5035502&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_23 Kidney cancer is characterized by significant morphological and molecular heterogeneity. Evaluation of mechanisms involved in the development and progression of kidney cancer require comprehensive analyses of genomes, transcriptomes, proteomes, and methylation profiles in normal and tumor tissue. To date, indiscriminate homogenates of tumor tissue or biopsy samples have been used as a source for DNA, RNA, or protein isolation. A major technical improvement has been the development of laser-assisted microdissection that allows the isolation of morphologically similar cells. The applications of this techno­logy to kidney cancer research are outlined. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035502 Thu, 30 Jun 2011 23:00:00 +0100 5035502 http://www.medworm.com/index.php?rid=5035501&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_22 Nucleoside reverse transcriptase inhibitors (NRTIs) are drugs used in the treatment of HIV/AIDS. Despite the distinct benefits of NRTI-based therapies, tissue specific toxicity is a limiting factor. Although the mechanisms of these specific antiretroviral drug-related toxicities remain unclear, it has been hypothesized that as analogs to native nucleosides, NRTIs may potentially inhibit mammalian DNA polymerases, including mitochondrial DNA (mtDNA) polymerase γ. Tenofovir disoproxil fumarate (TDF) is a nucleotide analog of adenosine monophosphate and the only NRTI that is associated with renal disease. The inherent heterogeneity of kidney tissues could affect the outcome and interpretation of molecular studies to define the mechanism(s) of tenofovir tubular toxicity. Laser-capture mi... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035501 Thu, 30 Jun 2011 23:00:00 +0100 5035501 http://www.medworm.com/index.php?rid=5035500&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_21 Laser-capture microdissection and transcriptional profiling have enabled compartment- and cell-specific analysis of gene expression in chronic kidney disease, thus facilitating the investigation of pathophysiological associations between glomerular, tubular, and interstitial structures. Due to the pico- and nanogram amounts of RNA isolated from LCM-captured material linear RNA amplification protocols are necessary prior to real-time PCR and microarray analysis. In this chapter, we describe the isolation of renal tubule cells from cryocut sections from routine kidney biopsies, and the isolation and linear amplification of RNA for downstream purposes. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035500 Thu, 30 Jun 2011 23:00:00 +0100 5035500 http://www.medworm.com/index.php?rid=5035499&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_20 Pure populations of tumor cells are essential for the identification of tumor-associated proteins for the development of targeted therapy. In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. The polycomb group (PcG) protein, enhancer of zeste homolog 2 (EzH2), a methyl-transferase that plays a key role in ­transcriptional gene repression, is frequently overexpressed in several malignant tumors. High levels of EzH2 are often associated with advanced disease stage in many solid tumors; however, its role in the pathogenesis of pancreatic ductal adeno-carcinoma (PDAC) is poorly understood. Because of the limited sample availability and the absence of in vitro amplification steps for prote... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035499 Thu, 30 Jun 2011 23:00:00 +0100 5035499 http://www.medworm.com/index.php?rid=5035498&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_1 Laser microdissection is a nonmolecular, minimally disruptive method to obtain cytologically and/or phenotypically defined cells or groups of cells from heterogeneous tissues. It is a versatile technology and allows the preparation of homogenous isolates of specific subpopulations of cells from which RNA/DNA or protein can be extracted for RT-polymerase chain reaction (PCR), quantitative PCR, Western blot analyses, and mass spectrophotometry. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035498 Thu, 30 Jun 2011 23:00:00 +0100 5035498 http://www.medworm.com/index.php?rid=5035497&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_19 Laser microdissection (LMD) is a robust well-established technology for the isolation of chosen cell populations from surrounding tissues and cells. This technique is particularly useful to minimize bias inherent in the molecular analysis of highly heterogeneous whole tissue sections. The aim of this study was to identify the pattern of mRNA expression in hepatocellular carcinoma (HCC) arising in cirrhotic liver and compare it to the pattern of expression in HCC arising from non-cirrhotic liver. The expression profiles of the tumors were also compared to that of the surrounding liver (either cirrhotic or non-cirrhotic) from the same patient. In addition, the expression pattern of each of the four tissues were compared to normal hepatic tissue. Samples of HCC tissue and surrounding cirrhoti... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035497 Thu, 30 Jun 2011 23:00:00 +0100 5035497 http://www.medworm.com/index.php?rid=5035496&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_18 Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease characterized by severe gastro-intestinal (GI) dysmotility caused by mutations in the thymidine phosphorylase gene. Thymidine phosphorylase (TP) is involved in the control of the pyrimidine nucleoside pool of the cell. Reduced TP activity induces nucleotide pool imbalances that in turn affect both the rate and fidelity of mtDNA replication, leading to multiple deletions and depletion of mtDNA. By using laser capture microdissection and quantitative real-time-polymerase chain reaction technique, we showed that depletion of mitochondrial DNA (mtDNA) is the most prominent molecular defect in the gut wall of MNGIE patients. Depletion affects severely the smooth muscle cells of muscularis propria and the skeletal mu... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035496 Thu, 30 Jun 2011 23:00:00 +0100 5035496 http://www.medworm.com/index.php?rid=5035495&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_17 This chapter refers to the application of laser-capture microdissection with oligonucleotide microarray analysis. The protocol described has been successfully used to identify differential transcript expression between contrasting colorectal cancer invasive phenotypes. Tissue processing, RNA extraction, quality control, amplification, fluorescent labelling, purification, hybridisation, and elements of data analysis are covered. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035495 Thu, 30 Jun 2011 23:00:00 +0100 5035495 http://www.medworm.com/index.php?rid=5035494&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_16 Tissues are complicated three-dimensional structures, composed of different types of interacting cells. Since the cell population of interest might constitute only a minor fraction of the total tissue volume, the problem of tissue heterogeneity has been a major barrier to the molecular analysis of normal versus diseased tissue. Thus, tissue microdissection represents one of the most promising techniques in molecular pathology offering the link between morphology and genetic analysis since it was established in the early 1970s. These first applications and further developments in the techniques enable preparation of morphologically well described and circumscribed cell populations of either tumor cells or surrounding tissue or even cytology specimens without contamination of unwanted cells.... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035494 Thu, 30 Jun 2011 23:00:00 +0100 5035494 http://www.medworm.com/index.php?rid=5035493&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_15 The intestinal epithelium is at the front line when it comes to preserving mucosal immune homeostasis. There is growing evidence that the epithelium plays a crucial role in the pathogenesis of inflammatory bowel disease. Laser captured microdissection techniques offer a promising approach to investigating the underlying molecular mechanisms. This would require reliable protocols for the extraction of high quality RNA from intestinal mucosa samples acquired by laser microdissection. However, such protocols are not around. Therefore our objective was to establish a feasible protocol which supports the study of the involvement of intestinal epithelium in the pathogenesis of IBD. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035493 Thu, 30 Jun 2011 23:00:00 +0100 5035493 http://www.medworm.com/index.php?rid=5035492&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_14 Barrett’s esophageal adenocarcinoma (BEAC) arises from Barrett’s esophagus (BE), a premalignant lesion caused by acid reflux (heartburn). Although the cancer is uncommon, its incidence is rapidly rising in western countries. Like most other cancers, BEAC cells also have elevated telomerase activity which maintains telomere length and supports continued proliferation of these cells. It is not clear if telomerase is activated early at premalignant (BE) stage, because reports of telomerase activity in Barrett’s and normal esophagi have been controversial. We have shown that detection of telomerase and telomeres becomes easier and much more reliable if purified BE cells are used instead of tissue specimens. This chapter, therefore, emphasizes the importance of laser capture m... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035492 Thu, 30 Jun 2011 23:00:00 +0100 5035492 http://www.medworm.com/index.php?rid=5035491&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_13 Expression array analysis of epithelial mRNA to identify biomarkers of premalignant and malignant conditions in the gastrointestinal (GI) tract is an area of intense study. Archived formalin-fixed paraffin-embedded (FFPE) tissues documenting these changes are readily available and should be a valuable resource for retrospective analysis. Laser capture microdissection of defined areas of epithelial cells at different stages of neoplastic progression is described together with methods for prequalification of RNA in FFPE tissue blocks selected for analysis. Paradise reagents specifically designed for isolation and amplification of RNA from FFPE archival tissue specimens are used to prepare probes for the human X3P microarray from Affymetrix. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035491 Thu, 30 Jun 2011 23:00:00 +0100 5035491 http://www.medworm.com/index.php?rid=5035490&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_12 The evolution of ovarian cancer will highly depend on platinum sensitivity or resistance. In an attempt to better understand this mechanism of resistance, we combined laser microdissection (LMD) of ovarian tumor cells with mass spectrometric techniques. To obtain disease-specific markers we isolated ovarian tumor cells with LMD without contamination of surrounding cells. Proteins were extracted by chemical lysis and subsequent peptide and protein information was gathered using surface-enhanced laser desorption/ionization-time of flight mass spectrometry. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035490 Thu, 30 Jun 2011 23:00:00 +0100 5035490 http://www.medworm.com/index.php?rid=5035489&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_11 Breast cancer tissues are characterized by cellular heterogeneity, representing a mixture of, e.g., healthy epithelial ducts, invasive or in situ tumor cells, surrounding stroma, infiltrating immune cells, blood vessels, and capillaries. As a consequence, protein extracts from whole tissue lysates also represent a variety of cell types present in the tissues under examination. This, however, seriously hampers the analysis of tumor cell-specific signals, which is of interest when performing biomarker discovery-type of studies. Therefore, laser capture microdissection is a perfect tool to isolate a relatively pure population of cells of interest, such as tumor cells. In this chapter, we describe the use of the PALM MicroBeam system for laser microdissection and pressure catapulting. Protocol... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035489 Thu, 30 Jun 2011 23:00:00 +0100 5035489 http://www.medworm.com/index.php?rid=5035488&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-163-5_10 MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs that regulate gene expression post-transcriptionally through targeting protein-coding mRNAs for cleavage or translational repression, and thus play key roles in cellular fate-determinant pathways. Both profiling and functional studies demonstrated derangement of miRNA repertoire in many human cancers, including breast tumours. Discovery of miRNAs provided new insights into cancer pathogenesis and led the scientific community to approach novel diagnostic and therapeutic strategies in cancer management. Research in this field is increasing, and the potential for miRNAs being used in clinical settings emphasises the need for high-throughput and sensitive detection techniques. In this chapter, techniques for the analysis of miR... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=5035488 Thu, 30 Jun 2011 23:00:00 +0100 5035488 http://www.medworm.com/index.php?rid=4991874&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_16 DNA is one of the most promising molecules for nanoscale bottom-up fabrication. For both scientific studies and fabrication of devices, it is desirable to be able to manipulate DNA molecules, or self-­assembled DNA constructions, at the single unit level. Efficient methods are needed for precisely attaching the single unit to the external measurement setup or the device structure. So far, this has often been too cumbersome to achieve, and consequently most of the scientific studies are based on a statistical analysis or measurements done for a sample containing numerous molecules in liquid or in a dry state. Here, we explain a method for trapping and attaching nanoscale double-stranded DNA (dsDNA) molecules between nanoelectrodes. The method is based on dielectrophoresis and gives a hi... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991874 Sun, 29 May 2011 23:00:00 +0100 4991874 http://www.medworm.com/index.php?rid=4991873&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_15 Current in vitro techniques cannot accurately identify small differences in concentration in samples containing few molecules in single or few cells. Nanotechnology overcomes these limitations with the possibility of measuring protein amounts down to a hundred molecules and subnanomolar concentrations and in nanoliter to picoliter volumes. The nanoscale approach, therefore, permits measurements in samples consisting of single or few cells. Atomic force microscopy (AFM) nanografting can be utilized to prepare DNA nanopatches of different sizes (from few hundreds of nanometers to few microns in size) onto which DNA–antibody conjugates can be anchored through DNA-directed immobilization. AFM height measurements are used to assess the binding of the proteins as well as their subsequent i... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991873 Sun, 29 May 2011 23:00:00 +0100 4991873 http://www.medworm.com/index.php?rid=4991872&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_14 The functionalization of silicon as elemental crystalline wafer, nanoporous layers, or nanocrystalline particles with DNA oligonucleotides using automated solid phase synthesis is described. The procedures provide semiconductor surfaces covalently modified with oligomers suitable for capturing complementary oligonucleotide strands. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991872 Sun, 29 May 2011 23:00:00 +0100 4991872 http://www.medworm.com/index.php?rid=4991871&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_13 Artificial DNA nanostructures such as DNA origami have garnered significant interest as templates for sub-20 nm lithography because their rational design allows for the incorporation of binding sites to assemble nanocomponents with 6 nm resolution. In addition, their overall size of 100 nm is easily accessible by top-down lithographic methods. Combining the strengths of top-down lithography and bottom-up self-assembly using DNA nanostructures may provide a commercially viable route to fabricating electronic and photonic devices with nanometer-scale features. We have demonstrated just such a comprehensive process in which 5 nm gold nanoparticles are first assembled in high yield on DNA origami. The constructs are then organized, rinsed, and dried on patterned silicon substrates, yielding la... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991871 Sun, 29 May 2011 23:00:00 +0100 4991871 http://www.medworm.com/index.php?rid=4991870&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_12 Nanopatterned arrays of biomolecules are a powerful tool to address fundamental issues in many areas of biology. DNA nanoarrays, in particular, are of interest in the study of DNA–protein interactions and for biodiagnostic investigations. In this context, achieving a highly specific nanoscale assembly of oligonucleotides at surfaces is critical. In this chapter, we describe a method to control the immobilization of DNA on nanopatterned surfaces; the nanofabrication and the bio-functionalization involved in the process will be discussed. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991870 Sun, 29 May 2011 23:00:00 +0100 4991870 http://www.medworm.com/index.php?rid=4991869&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_11 A long one-dimensional single-stranded DNA (ssDNA) molecule with a periodic sequence motif is an attractive building block for DNA nanotechnology because it allows the positioning of oligonucleotide-labeled particles or molecules with high spatial resolution via molecular self-assembly simply by hybridization reactions. In vitro enzymatic isothermal rolling circle amplification (RCA) produces such long concatemeric ssDNA molecules. These are complementary in sequence to their circular template. In this chapter, the preparation of stretched and surface-attached RCA products at the single molecule level is described. The methods presented comprise the enzymatic circularization of a ssDNA oligonucleotide, the covalent coupling of amino-modified primers to carboxylated fluorescence beads, the ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991869 Sun, 29 May 2011 23:00:00 +0100 4991869 http://www.medworm.com/index.php?rid=4991868&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-142-0_10 DNA molecules are well known for containing the genetic information of an individual. Furthermore, DNA is a biopolymer with the potential of building up nanoscale structures. These structures can be addressed sequence specifically and, therefore, they allow connecting and arranging with subnanometer accuracy. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4991868 Sun, 29 May 2011 23:00:00 +0100 4991868 http://www.medworm.com/index.php?rid=4801683&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_9 A hybrid system comprising a hydrogenase and a photosensitizer co-attached to a nanoparticle serves as a rational model for fast dihydrogen (H2) production using visible light. This chapter describes a stepwise procedure for preparing TiO2 nanoparticles functionalized with a hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) and a tris(bipyridyl)ruthenium photosensitizer (RuP). Upon irradiation with visible light, these particles produce H2 from neutral water at room temperature in the presence of a sacrificial electron donor – a test-system for the cathodic half reaction of water splitting. In particular, we describe how a hydrogenase and a photosensitizer with desired properties, including strong adsorption on TiO2, can be selected by electrochemical methods. The catalyst ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801683 Mon, 09 May 2011 23:14:31 +0100 4801683 http://www.medworm.com/index.php?rid=4801682&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_8 Common enzyme immobilization methods on nanomaterials (adsorption, covalent binding, crosslinking, encapsulation) often generate problems in enzyme leaching, 3D structure change and diffusion resistance. We show here a detailed site-specific enzyme immobilization method that overcomes the foresaid limitations. It is based on the specific interaction between His-tagged enzyme and single-walled carbon nanotubes modified with N α ,N α-bis(carboxymethyl)-l-lysine hydrate. This method does not require enzyme purification and the resulting nanoscale biocatalyst can maintain high enzyme activity and stability. The enzyme-loading capacity is also comparable with the reported immobilization capacity on carbon nanotubes by either covalent binding or adsorption. Furth... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801682 Mon, 09 May 2011 23:14:30 +0100 4801682 http://www.medworm.com/index.php?rid=4801681&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_7 This chapter describes a method for the formation of novel protein–nanotube hybrid conjugates. Specifically, we took advantage of the self-assembly and self-recognition properties of tubulin cytoskeletal protein immobilized onto carbon nanotubes to form nanotube-based biohybrids. Further biohybrid hierarchical integration in assemblies enabled molecular-level manipulation on engineered surfaces, as demonstrated with biocatalyst kinesin 1 ATPase molecular motor. The method presented herein can be extended for the preparation of biocatalyst-based or protein-based assemblies to be used as sensors or biological templates for nanofabrication. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801681 Mon, 09 May 2011 23:14:30 +0100 4801681 http://www.medworm.com/index.php?rid=4801680&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_6 Glutamate is the principal excitatory neurotransmitter in the brain, and its excessive release plays a key role in neuronal death associated with a wide range of neural disorders. Real-time monitoring of extracellular glutamate levels would be very helpful in understanding the excitotoxic process of neurotransmitters on brain injury. Toward the detection of l-glutamate, we describe in this chapter the preparation of carbon nanotube (CNT) network probes with immobilized l-glutamate oxidase (GLOD) by using a non-covalent functionalized method. Such GOLD-CNT network probes are evaluated with real-time electronic responses corresponding to standard glutamate solutions in vitro and a 11-vessel occlusion (11 VO) rat model in vivo. The ultrahigh sensitivity, selectivity, and fast response time of... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801680 Mon, 09 May 2011 23:14:30 +0100 4801680 http://www.medworm.com/index.php?rid=4801679&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_5 The solubilization of enzymes in organic solvents for non-aqueous biocatalysis has attracted considerable attention since the homogeneous distribution accounts for a drastically improved reaction efficiency compared to enzymes dispersed as aggregates in an organic phase. This chapter highlights ion-pairing as a valuable and facile method to make enzymes soluble in organic solvents. Ion-pairing denotes the formation of a nano-complex, in which a single enzyme molecule in the core is surrounded by counter-charged surfactant molecules. The special architecture of this nano-complex exposes the surfactant hydrophobic group toward the bulk solvent and renders the complex sufficiently soluble in organic media. This chapter also describes the underlying principle of ion-pairing as well as simple p... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801679 Mon, 09 May 2011 23:14:29 +0100 4801679 http://www.medworm.com/index.php?rid=4801678&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_4 Room temperature ionic liquids (RTILs), having no measurable vapor pressure, represent an interesting class of tunable designer solvents. Due to their many unique properties, ILs have been used as attractive alternatives to environmentally harmful ordinary organic solvents in a wide range of applications including enzymatic biotransformation. Compared to conventional organic solvents, ILs offer many advantages for biocatalysis such as enhanced conversion rates, high enantioselectivity, better enzyme stability, and improved catalyst recoverability and recyclability. However, biocatalysis in ILs has not yet fully achieved its potential because many biocatalysts are insoluble in most ILs. This limitation could be overcome by the formation of nano/micrometer-sized aqueous microemulsion droplet... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801678 Mon, 09 May 2011 23:14:28 +0100 4801678 http://www.medworm.com/index.php?rid=4801677&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_3 Many biocatalysts have been shown powerful in enabling reactions among a broad range of substrates possessing very different hydrophilicity/hydrophobicity. Biphasic reaction systems, especially oil–water biphasic systems, have been commonly adopted to mediate such reactions. The greatest challenge in conducting an efficient reaction between two substrates that have to be hosted in two immiscible liquid phases is the mass transfer resistance across interfaces. Imaginably, the substrates afford the most extensive interactions at the interfacial region. The interfacial assembled enzymes, developed by conjugating water-soluble enzymes with hydrophobic polymers, are therefore expected to be efficient in catalyzing biotransformation at the organic–aqueous interfaces. This chapter des... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801677 Mon, 09 May 2011 23:14:27 +0100 4801677 http://www.medworm.com/index.php?rid=4801676&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_2 Continued exploration into the field of chemically induced dimerization (CID) has revealed a number of applications for its use in a broader context as a method of structural assembly (1–4). In particular, the use of CID technology to generate self-assembled (and selectively disassembled) protein toroids serves as a key advancement toward developing stable and controllable protein-based platforms. Such structures have broad application to the development of novel therapeutics, lab-on-a-chip technologies, and multi-enzyme assemblies (5, 6). This chapter describes a method of developing an enzymatically active protein nanostructure incorporating both a CID-based assembly region containing dihydrofolate reductase (DHFR) and an enzymatic region consisting of histidine triad nucleotide bi... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801676 Mon, 09 May 2011 23:14:27 +0100 4801676 http://www.medworm.com/index.php?rid=4801675&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_1 Most of the bacterial cytochrome P450 s require two kinds of electron transfer proteins, ferredoxin and ferredoxin reductase, and thus P450 s do not show catalytic activity by themselves. A microbial transglutaminase-mediated site-specific cross-linking enables the formation of fusion P450 protein with a branched structure, which is generated from a genetic fusion protein of P450–ferredoxin reductase and ferredoxin, an interactive nanoscale protein structure. This fusion P450 system is self-sufficient due to intramolecular electron transfer, which means the system does not require additional electron-transferring proteins. Because some components of bacterial cytochrome P450 system are interchangeable, this self-sufficient system can be applied to non-natural combination of... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801675 Mon, 09 May 2011 23:14:27 +0100 4801675 http://www.medworm.com/index.php?rid=4801674&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_18 The prerequisite for many successful enzyme-based biotechnologies is the preparation of highly stable and active biocatalysts, which can be achieved effectively by immobilization. This chapter introduces the immobilization of enzymes by entrapment in nanoporous silica particles made in a sol–gel process. These easily tailorable materials have been proven very beneficial for a broad variety of applications of biocatalysts. Besides the spatial confinement in silica sol–gels, another advantage is given by the easy possibility of fine-tuning the physicochemical properties of the matrix itself to provide the ideal environment for the reaction and the biocatalyst. Preparation details are demonstrated using the process of immobilizing a lipase in a sol–gel matrix, which is chemi... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801674 Mon, 09 May 2011 23:14:26 +0100 4801674 http://www.medworm.com/index.php?rid=4801673&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_17 This chapter describes a method for generating uniform lab-scale biocatalytic nanoporous latex coatings. Nearly everything we come into contact with on a daily basis has been coated with some polymer material. High-speed waterborne polymer coating and ink-jet printing techniques are mature technologies. Methods for immobilizing microorganisms in lab-scale waterborne latex biocatalytic coatings draw on existing coating technologies for generating precision industrial paint and paper coatings and would therefore be amenable to scale up in future applications. An inherent problem for many lab-scale techniques is coating uniformity. The method described here has been developed to dramatically increase the uniformity of multiple individual small surface area coatings derived from a single coati... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801673 Mon, 09 May 2011 23:14:26 +0100 4801673 http://www.medworm.com/index.php?rid=4801672&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_16 Compared to other nanomaterials as supports for enzyme immobilization, nanofibers provide a promising configuration in balancing the key factors governing the catalytic performance of the immobilized enzymes including surface area-to-volume ratio, mass transfer resistance, effective loading, and the easiness to recycle. Synthetic and natural polymers can be fabricated into nanofibers via a physical process called electrospinning. The process requires only simple apparatus to operate, yet has proved to be very flexible in the selection of feedstock materials and also effective to control and manipulate the properties of the resulting nanofibers such as size and surface morphology, which are typically important parameters for enzyme immobilization supports. This chapter describes a protocol ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801672 Mon, 09 May 2011 23:14:25 +0100 4801672 http://www.medworm.com/index.php?rid=4801671&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_15 This chapter provides an overview of key tools and methodologies available to practitioners of biocatalysis interested in using microorganisms to carry out biotransformations and describes specific examples of applying genetic modification strategies for strain design. We focus on the use of the polymerase chain reaction (PCR) for gene amplification, plasmid DNA for recombinant gene cloning and expression, and homologous recombination and phage transduction for modifying chromosomal DNA. Specifically we use Escherichia coli as the host organism, and the overproduction of xylitol by reduction of xylose represents the biotransformation of interest. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801671 Mon, 09 May 2011 23:14:24 +0100 4801671 http://www.medworm.com/index.php?rid=4801670&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_14 Synthetic biology promises to enhance our ability to control biological systems by creating a systematic approach for the construction of genetic circuits that reliably program cellular function. As part of this approach, efficient methods are needed for the tuning of genetic circuits so as to allow for optimization of a design despite varying cellular contexts and incomplete understanding of in vivo biological interactions. Here we outline an optimization method that we have used to improve the logical responses of a genetic AND logic gate derived from components of the LuxI–LuxR bacterial quorum-sensing system. Basing our approach on the idea of evolutionary design, we improved the properties of our genetic AND logic gate by using directed evolution and a two-step screening process... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801670 Mon, 09 May 2011 23:14:24 +0100 4801670 http://www.medworm.com/index.php?rid=4801669&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_13 Supported or modified enzymes in the form of mobile nanoparticles are designed for enhanced activities and stabilities; however, their practical operations are dwarfed due to their tiny size which always makes recycling an arduous task and a potential risk to the environment. To overcome such drawbacks, this chapter describes a method for the preparation of a new form of microcapsules, possessing single-cavity compartments and nano-pores in the shell, to encage nanoparticle-based biocatalysts and form cell-like microreactors (CLMRs). The encaged nanoscale catalysts are maintained their high activities as in a bulk-phase solution, while they could be handled as materials of sizes hundreds-fold larger. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801669 Mon, 09 May 2011 23:14:23 +0100 4801669 http://www.medworm.com/index.php?rid=4801668&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_12 Enzymatic catalysis in nonaqueous media is considered as an attractive tool for the preparation of a variety of organic compounds of commercial interest. This approach is advantageous for numerous reasons including the enhanced stability of some substrates and products in solvents, sometimes improved selectivity of the enzyme, and reduction of unwanted water-dependent side reactions since little water is present. Due to the poor solubility of enzymes in these media, mass transfer limitations are sometimes present, leading to low apparent catalytic activity. Immobilization on solid supports has been successfully applied to overcome enzyme solubility issues by increasing the accessibility of substrates to the enzymes’ active sites. We have developed a simple immobilization protocol tha... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801668 Mon, 09 May 2011 23:14:22 +0100 4801668 http://www.medworm.com/index.php?rid=4801667&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_11 Here we describe a new method for the production of thiol-silicate particles and the entrapment of enzymes within the thiol particles as they are formed. When bio-inspired polymers (polyethyleneimine) are combined with a silicic acid source and phosphate buffer under pH neutral conditions, formation of silicate particles occurs. In the method presented here the silica source contains a thiol group and so therefore the silicate particles are pre-functionalized with thiol groups. We have termed the silicate particles produced “thiol particles” and the characterization of these thiol particles is also presented in this chapter. As enzymes can be entrapped during fabrication, it means that the thiol particles can not only attach to metal surfaces but also catalyse certain reactions... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801667 Mon, 09 May 2011 23:14:22 +0100 4801667 http://www.medworm.com/index.php?rid=4801666&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-132-1_10 Enzymes have been incorporated in nanostructures in order to provide robust catalysts for valuable reactions, particularly those performed under harsh and denaturing conditions. This chapter describes the encapsulation of enzymes in polyacrylamide nanogels by a two-step in situ polymerization process for preparing robust biocatalysts. The first step in this process is the generation of vinyl groups on the enzyme surface, while the second step involves in situ polymerization using acrylamide as the monomer. Encapsulation of the enzyme in the hydrophilic, porous, and flexible polyacrylamide gel of several nanometers thick would help to both give a significantly enhanced thermostability and prevent the removal of essential water by polar solvents. The hydrophilic flexible polymer shell also a... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4801666 Mon, 09 May 2011 23:14:21 +0100 4801666 http://www.medworm.com/index.php?rid=4741813&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_26 The potential clinical applications and the economic benefits of theranostics represent a tremendous incentive to push research and development forward. However, we should also carefully examine the possible downsides. In this chapter, we address the issue of how theranostics might challenge our current concept of informed consent, especially the disclosure of information concerning diagnosis and treatment options to human subjects. We argue that our lack of data concerning long-term effects and risks of nanoparticles on human health and the environment could undermine the process when it comes to weighing the risks against the benefits. Our lack of an agreed upon framework for risk management in nanomedicine may require us to adopt an “upstream” approach that emphasizes commun... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4741813 Sat, 23 Apr 2011 05:47:06 +0100 4741813 http://www.medworm.com/index.php?rid=4663326&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_25 This chapter, based on concepts developed for my book, NanoInnovation (Tomczyk, Nanoinnovation: What Every Manager Needs to Know, 2011), is one of the first attempts to evaluate nanotechnology in the context of the “marketing mix” – a conceptual challenge given that nanotechnology is not one product or even a set of products, but rather a technology that is incorporated in an expanding list exceeding a 1,000 products – encompassing materials, structures, processes, and devices. My purpose is to use this context to identify some of the critical issues and factors that will influence development of “nanotechnology markets” at this very early stage in the evolution of nanotechnology, and more specifically, bionanotechnology. As technological innovations con... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663326 Fri, 01 Apr 2011 09:13:19 +0100 4663326 http://www.medworm.com/index.php?rid=4663325&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_24 Scientists constantly generate great ideas in the laboratory and, as most of us were meant to believe, we should publish or perish. After all, what use is a great scientific idea if it is not shared with the rest of the scientific community? What some scientists forget is that a good idea can be worth something – sometimes it can be worth a lot (of money)! What do you do if you believe that your idea has some commercial potential? How do you turn this idea into a business? This chapter gives the aspiring scientific entrepreneur some (hopefully) valuable advice on topics like choosing the right people for your management team, determining inventorship of the technology and ownership shares in the new company, protecting your intellectual property, and others; finally, it describes som... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663325 Fri, 01 Apr 2011 09:13:19 +0100 4663325 http://www.medworm.com/index.php?rid=4663324&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_23 The patent landscape, like a garden, can tell you much about its designers and users: their motivations, biases, and general interests. While both patent landscapes and gardens may appear to the casual observer as refined and ordered, an in-depth exploration of the terrain is likely to reveal unforeseen challenges including, for example, alien species, thickets, and trolls. As this chapter illustrates, patent landscapes are dynamic and have been forced to continually evolve in response to technological innovation. While emerging technologies such as biotechnology and information communication technology have challenged the traditional patent landscape, the overarching framework and design have largely remained intact. But will this always be the case? The aim of this chapter is to highligh... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663324 Fri, 01 Apr 2011 09:13:18 +0100 4663324 http://www.medworm.com/index.php?rid=4663323&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_22 Legislation is a form of governance that directs attention and prescribes action. Within the domain of nanoscience, the US 21st Century Nanotechnology Research and Development Act contains mandates not only for rapid development for economic competitiveness but also for responsible implementation, which is required to take place by integrating societal considerations into research and development. This chapter investigates whether these two mandates tend more to coexist or compete with one another, both in the purview of nanoscience policy and in the venue of nanoscience practice. This chapter first reviews macrolevel analysis of the directives contained in the legislation. It then examines, drawing on an empirical case study, how these directives manifest at the microlevel of a nanoscienc... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663323 Fri, 01 Apr 2011 09:13:18 +0100 4663323 http://www.medworm.com/index.php?rid=4663322&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_21 The approval of drugs for human use by the US Food and Drug Administration (FDA) through the Center for Drug Evaluation and Research (CDER) is a time-consuming and expensive process, and approval rates are low (DiMasi et al., J Health Econ 22:151–185, 2003; Marchetti and Schellens, Br J Cancer 97:577–581, 2007). In general, the FDA drug approval process can be separated into preclinical, clinical, and postmarketing phases. At each step from the point of discovery through demonstration of safety and efficacy in humans, drug candidates are closely scrutinized. Advances in nanotechnology are being applied in the development of novel therapeutics that may address a number of shortcomings of conventional small molecule drugs and may facilitate the realization of personalized medicin... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663322 Fri, 01 Apr 2011 09:13:18 +0100 4663322 http://www.medworm.com/index.php?rid=4663321&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_20 Health risks following exposures to nanoparticle types are dependent upon two primary factors, namely, hazard and exposure potential. This chapter describes a pulmonary bioassay methodology for assessing the hazardous effects of nanoparticulates in rats following intratracheal instillation exposures; these pulmonary exposures are utilized as surrogates for the more physiologically relevant inhalation route of exposure. The fundamental features of this pulmonary bioassay are dose–response evaluations and time-course assessments to determine the sustainability of any observed effect. Thus, the major endpoints of this assay are the following: (1) time course and dose–response intensity of pulmonary inflammation and cytotoxicity, (2) airway and lung parenchymal cell proliferation, ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663321 Fri, 01 Apr 2011 09:13:18 +0100 4663321 http://www.medworm.com/index.php?rid=4663320&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_19 The field of nanotoxicology recently has emerged out of the need to systematically study the biocompatibility and potential adverse effects of novel nanomaterials. Carbon nanotubes (CNT) are one of the most interesting types of nanomaterials, and recently, their use in applications has dramatically increased. Their potential adverse impact on human health and the environment, however, have caused them to be viewed with apprehension in certain cases so further studies into their toxicology are justified. Current methodologies using cell culture (in vitro) models are unreliable and are not yet able to offer conclusive results about the toxicity profile of CNT. The need for reliable and rapid toxicity assays that will allow high throughput screening of nanotube materials is a prerequisite for... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663320 Fri, 01 Apr 2011 09:13:18 +0100 4663320 http://www.medworm.com/index.php?rid=4663319&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_9 Nanoelectromechanical systems (NEMS) correlate analyte-binding events with the mechanical motions of devices in nanometer scales, which in turn are converted into detectable electrical or optical signals. Biosensors based on NEMS have the potential to achieve ultimate sensitivity down to the single-molecule level, provide rapid and real-time detection signals, be operated with extremely low power consumption, and be mass produced with low cost and high reproducibility. This chapter reviews fundamental concepts in NEMS fabrication, actuation and detection, and device characterization, with examples of using NEMS for sensing DNA, proteins, viruses, and bacteria. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663319 Fri, 01 Apr 2011 09:13:18 +0100 4663319 http://www.medworm.com/index.php?rid=4663318&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_8 SnO2 nanowires are synthesized via the chemical-vapor-deposition process using gold as the catalyst and characterized by X-ray powder diffraction, field-emission scanning electron microscopy, high-resolution transmission electron microscopy, and cathodoluminescence. Finally, a new type of microelectrode based on a single SnO2 nanowire is fabricated. This microelectrode is expected to have promising applications in various chemical and biomedical nanosensors. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663318 Fri, 01 Apr 2011 09:13:18 +0100 4663318 http://www.medworm.com/index.php?rid=4663317&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_7 Semiconductor nanocrystals or quantum dots (QDs) have become well-established as a unique nanoparticle scaffold for bioapplications due to their robust luminescent properties. In order to continue their development and expand this technology, improved methodologies are required for the controllable functionalization and display of biomolecules on QDs. In particular, efficient routes that allow control over ligand loading and spatial orientation, while minimizing or eliminating cross-linking and aggregation are needed. Two conjugation approaches are presented that address these needs: (1) polyhistidine-based metal-affinity self-assembly to QD surfaces and (2) carbodiimide-based amide bond formation to carboxy-functionalized polyethylene glycol or PEGylated QDs. These approaches can be succe... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663317 Fri, 01 Apr 2011 09:13:17 +0100 4663317 http://www.medworm.com/index.php?rid=4663316&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_6 Microspheres and nanospheres are being used in many of today’s biosensing applications for automated sample processing, flow cytometry, signal amplification in microarrays, and labeling in multiplexed analyses. The surfaces of the spheres/particles need to be modified with proteins and other biomolecules to be used in these sensing applications. This chapter contains protocols to modify carboxyl- and amine-coated polymer spheres with proteins and peptides. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663316 Fri, 01 Apr 2011 09:13:17 +0100 4663316 http://www.medworm.com/index.php?rid=4663315&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_5 Semiconductor photocatalysis using nanoparticulate TiO2 has proven to be a promising technology for use in catalytic reactions, in the cleanup of water contaminated with hazardous industrial by-products, and in nanocrystalline solar cells as a photoactive material. Metal oxide semiconductor colloids are of considerable interest because of their photocatalytic properties. The coordination sphere of the surface metal atoms is incomplete and thus traps light-induced charges, but also exhibits high affinity for oxygen-containing ligands and gives the opportunity for chemical modification. We use enediol linkers, such as dopamine and its analogs, to bridge the semiconductors to biomolecules such as DNA or proteins. Nanobio hybrids that combine the physical robustness and chemical reactivity of ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663315 Fri, 01 Apr 2011 09:13:17 +0100 4663315 http://www.medworm.com/index.php?rid=4663314&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_4 We describe a single quantum dot tracking method that can be used to monitor individual proteins in the membrane of living cells. Unlike conventional fluorescent dyes, quantum dots (fluorescent semiconductor nanocrystals) have high quantum yields, narrow emission wavelengths, and excellent photostability, making them ideal probes in single-molecule detection. This technique has been applied to study the dynamics of various membrane proteins including glycine receptors, nerve growth factors, kinesin motors, and γ-aminobutyric acid receptors. In this chapter, a basic introduction and experimental setup for single quantum dot labeling of a target protein is given. In addition, data acquisition and analysis of time-lapse single quantum dot imaging with sample protocols are provided. (Sou... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663314 Fri, 01 Apr 2011 09:13:17 +0100 4663314 http://www.medworm.com/index.php?rid=4663313&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_3 The rapid and sensitive detection of molecular targets such as proteins, cells, and pathogens in biological specimens is a major focus of ongoing medical research, as it could promote early disease diagnoses and the development of tailored therapeutic strategies. Magnetic nanoparticles (MNP) are attractive candidates for molecular biosensing applications because most biological samples exhibit negligible magnetic susceptibility, and thus the background against which measurements are made is extremely low. Numerous magnetic detection methods exist, but sensing based on magnetic resonance effects has successfully been developed into a general detection platform termed diagnostic magnetic resonance (DMR). DMR technology encompasses numerous assay configurations and sensing principles, and to ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663313 Fri, 01 Apr 2011 09:13:17 +0100 4663313 http://www.medworm.com/index.php?rid=4663312&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_2 Applications in a variety of fields rely on the high-throughput ultrasensitive and multiplexed detection of oligonucleotides. However, the conventional microarray-based techniques that employ fluorescent dyes are hampered by several limitations; they require target amplification, fluorophore labeling, and complicated instrumentation, while the fluorophore-labeled species themselves exhibit slow binding kinetics, photo-bleaching effects, and overlapping spectral profiles. Among the emerging nanomaterials that are being used to solve these problems, oligonucleotide–gold nanoparticle conjugates (Oligo-AuNPs) have recently been highlighted due to their unique chemical and physical properties. In this chapter, a detection scheme for oligonucleotides that utilize Oligo-AuNPs is evaluated w... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663312 Fri, 01 Apr 2011 09:13:16 +0100 4663312 http://www.medworm.com/index.php?rid=4663311&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_18 We develop a shape-based coarse-grained (SBCG) model for DNA-functionalized gold nanoparticles (DNA-Au NPs) and use this to study the interaction of this potential antisense therapeutic with a lipid bilayer model of a cell membrane that is also represented using a coarse-grained model. Molecular dynamics simulations of the SBCG model of the DNA-Au NP show structural properties which coincide with our previous atomistic models of this system. The lipid membrane is composed of 30% negatively charged lipid (1,2-dioleoyl-sn-glycero-3-phosphoserine, DOPS) and 70% neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) in 0.15 M sodium chloride solution. Molecular dynamics (MD) simulations of the DNA-Au NP near to the lipid bilayer show that there is a higher density of DOPS than DOPC nea... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663311 Fri, 01 Apr 2011 09:13:16 +0100 4663311 http://www.medworm.com/index.php?rid=4663310&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_17 Many promising strategies have been developed for controlling the release of drugs from scaffolds, yet there are still challenges that need to be addressed in order for these scaffolds to serve as successful treatments. The RADA4 self-assembling peptide spontaneously forms nanofibers, creating a scaffold-like tissue-bridging structure that provides a three-dimensional environment for the migration of living cells. We have found that RADA4: (1) facilitates the regeneration of axons in the brain of young and adult hamsters, leading to functional return of behavior and (2) demonstrates robust migration of host cells and growth of blood vessels and axons, leading to the repair of injured spinal cords in rats. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663310 Fri, 01 Apr 2011 09:13:16 +0100 4663310 http://www.medworm.com/index.php?rid=4663309&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_16 Tissue-engineered medical implants, such as polymeric nanofiber scaffolds, are potential alternatives to autografts and allografts, which are short in supply and carry risks of disease transmission. These scaffolds have been used to engineer various soft connective tissues such as skin, ligament, muscle, and tendon, as well as vascular and neural tissue. Bioactive versions of these materials have been produced by encapsulating molecules such as drugs and growth factors during fabrication. The fibers comprising these scaffolds can be designed to match the structure of the native extracellular matrix (ECM) closely by mimicking the dimensions of the collagen fiber bundles evident in soft connective tissues. These nanostructured implants show improved biological performance over the bulk mater... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663309 Fri, 01 Apr 2011 09:13:16 +0100 4663309 http://www.medworm.com/index.php?rid=4663308&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_15 Carbon nanotubes (CNTs) are novel, one-dimensional nanomaterials with many unique physical and chemical properties that have been increasingly explored for biological and biomedical applications. In this chapter, we briefly summarize the intrinsic properties of single-walled carbon nanotubes (SWNTs), a special class of CNTs, and their corresponding applications in these fields. SWNTs have been utilized for the ultrasensitive detection of biological species, providing a label-free approach. SWNT-Raman tags have achieved detection sensitivity down to 1 fmol/L. SWNT-based drug delivery systems have shown promising potential based on preliminary in vitro and in vivo studies. Also, the remarkable optical properties of SWNTs have made them promising candidates as contrast agents for imaging in c... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663308 Fri, 01 Apr 2011 09:13:16 +0100 4663308 http://www.medworm.com/index.php?rid=4663307&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_14 Virus delivery vectors are one among the many nanomaterials that are being developed as drug delivery materials. This chapter focuses on methods utilizing plant virus nanoparticles (PVNs) synthesized from the Red clover necrotic mosaic virus (RCNMV). A successful vector must be able to effectively carry and subsequently deliver a drug cargo to a specific target. In the case of the PVNs, we describe two types of ways cargo can be loaded within these structures: encapsidation and infusion. Several targeting approaches have been used for PVNs based on bioconjugate chemistry. Herein, examples of such approaches will be given that have been used for RCNMV as well as for other PVNs in the literature. Further, we describe characterization of PVNs, in vitro cell studies that can be used to test th... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663307 Fri, 01 Apr 2011 09:13:15 +0100 4663307 http://www.medworm.com/index.php?rid=4663306&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_13 We describe a new application in the area of antibacterial nanomedicines where filamentous phages can be formulated as targeted drug-delivery vehicles of nanometric dimensions (phage nanomedicines) and used for therapeutic purposes. This protocol involves both genetic and chemical engineering of these phages. The genetic engineering of the phage coat, which results in the display of a target-specificity-conferring peptide or protein on the phage coat, can be used to design the drug-release mechanism and is not described herein. However, the methods used to chemically conjugate cytotoxic drugs at high density on the phage coat are described. Further, assays to measure the drug load on the surface of the phage and the potency of the system in the inhibition of growth of target cells as well ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663306 Fri, 01 Apr 2011 09:13:15 +0100 4663306 http://www.medworm.com/index.php?rid=4663305&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_12 We present a general method using water dissolvable hydrogel templates for the fabrication of homogeneous, shape-specific polymer/drug constructs in the size range of 200 nm to 50 μm. This hydrogel template strategy is mild, inexpensive, and readily scalable for the fabrication of multifunctional drug delivery vehicles. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663305 Fri, 01 Apr 2011 09:13:15 +0100 4663305 http://www.medworm.com/index.php?rid=4663304&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_11 Photodynamic therapy is a relatively new clinical therapeutic modality that is based on three key components: photosensitizer, light, and molecular oxygen. Nanoparticles, especially targeted ones, have recently emerged as an efficient carrier of drugs or contrast agents, or multiple kinds of them, with many advantages over molecular drugs or contrast agents, especially for cancer detection and treatment. This paper describes the current status of PDT, including basic mechanisms, applications, and challenging issues in the optimization and adoption of PDT; as well as recent developments of nanoparticle-based PDT agents, their advantages, designs and examples of in vitro and in vivo applications, and demonstrations of their capability of enhancing PDT efficacy over existing molecular drug-ba... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663304 Fri, 01 Apr 2011 09:13:15 +0100 4663304 http://www.medworm.com/index.php?rid=4663303&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_10 Some efficient diagnosis and therapy systems require the isolation and quantification of circulating tumor cells (CTCs), since these species are important “biomarkers” for monitoring cancer metastasis and prognosis. Existing techniques for isolating/counting CTCs include immunomagnetic-bead-based separation and microfluidic capture. However, some of these techniques have low capture efficiency and low specificity. Through the use of a three-dimensional (3D) nanostructured substrate – specifically, a silicon-nanowire (SiNW) array coated with epithelial-cell-adhesion-molecule antibodies (anti-EpCAM) – we show that CTCs can be captured efficiently and specifically. Unlike conventional methods for isolating CTCs that depend on collision frequency and contact duration, n... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663303 Fri, 01 Apr 2011 09:13:15 +0100 4663303 http://www.medworm.com/index.php?rid=4663302&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-052-2_1 This chapter summarizes the roles of nanomaterials in biomedical applications, focusing on those highlighted in this volume. A brief history of nanoscience and technology and a general introduction to the field are presented. Then, the chemical and physical properties of nanostructures that make them ideal for use in biomedical applications are highlighted. Examples of common applications, including sensing, imaging, and therapeutics, are given. Finally, the challenges associated with translating this field from the research laboratory to the clinic setting, in terms of the larger societal implications, are discussed. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4663302 Fri, 01 Apr 2011 09:13:15 +0100 4663302 http://www.medworm.com/index.php?rid=4104891&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_18 Bioinformatics has become an increasingly important tool for molecular biologists, especially for the analysis of microarray data. Microarrays can produce vast amounts of information requiring a series of consecutive analyses to render the data interpretable. The direct output of microarrays cannot be directly interpreted to show differences in settings, conditions of samples, or time points. To make microarray experiments interpretable, it is necessary that a series of algorithms and approaches be applied. After normalization of generated data, which is necessary to make a comparison feasible, significance analysis, clustering of samples and biological compounds of interest and visualization are generally performed. This chapter will focus on providing a basic understanding of the general... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104891 Wed, 31 Mar 2010 23:00:00 +0100 4104891 http://www.medworm.com/index.php?rid=4104890&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_17 This chapter describes a protocol for forming open endothelial tubes in vitro and quantifying their permeability to macromolecules. These tubes consist of confluent monolayers of human microvascular endothelial cells in perfused microfluidic collagen gels. The cylindrical geometry of the tubes mimics the shape of microvessels in vivo; it allows simultaneous and/or repeated measurements of permeability coefficients and detection of focal leaks. We have used these in vitro models to test the effects of agonists on microvascular permeability and are developing arrays of microvascular tubes to enable large-scale testing. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104890 Wed, 31 Mar 2010 23:00:00 +0100 4104890 http://www.medworm.com/index.php?rid=4104889&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_16 This book chapter discusses recent advances in the fabrication of microscale surface patterns using chemical vapor deposition polymerization. Reactive poly(p-xylylene) (PPX) coatings are useful for their ability to immobilize specific biomolecules, as determined by the PPX functional group. PPXs can either be modified postdeposition, or they can be patterned onto a substrate in situ. Specific methods discussed in this progress report include microcontact printing, vapor-assisted micropatterning in replica structures, projection lithography-based patterning, and selective polymer deposition. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104889 Wed, 31 Mar 2010 23:00:00 +0100 4104889 http://www.medworm.com/index.php?rid=4104888&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_15 The PRINT® (pattern replication in non-wetting templates) process has been developed as a simple, gentle way to pattern films or generate discrete particles in arrays out of either pure biological materials or biomolecules encapsulated within polymeric materials. Patterned films and particle arrays can be fabricated in a wide array of sizes and shapes using Fluorocur® (a UV-curable perfluoropolyether polymer) from the nanometer to micron scale. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104888 Wed, 31 Mar 2010 23:00:00 +0100 4104888 http://www.medworm.com/index.php?rid=4104887&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_14 Microcontact printing (μCP) is a useful technique for transferring certain molecules onto surfaces with high spatial resolution using elastomeric stamps. The stamp for μCP is fabricated by replica molding from a master made by microlithography. After wetting with a type of material as an “ink,” the stamp comes into contact with the substrate. The ink is selectively transferred onto parts of the substrate wherever the stamp makes direct contact, to generate patterns and structures with designated features. Self-assembled monolayers (SAMs) and μCP are useful in many different fields, e.g., in the studies of protein adsorption, cell attachment, and in the construction of sensors. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104887 Wed, 31 Mar 2010 23:00:00 +0100 4104887 http://www.medworm.com/index.php?rid=4104886&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_13 In their normal in vivo matrix milieu, tissues assume complex well-organized 3D architectures. Therefore, a primary aim in the tissue engineering design process is to fabricate an optimal analog of the in vivo scenario, in which the precise configuration and composition of cells and bioactive matrix components can establish the well-defined biomimetic microenvironments that promote cell–cell and cell–matrix interactions. With the advent and refinements in microfabricated systems which can present physical and chemical cues to cells in a controllable and reproducible fashion unrealizable with conventional tissue culture, high-fidelity, high-throughput in vitro models are achieved. The convergence of solid freeform fabrication (SFF) technologies, namely microprinting, along with ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104886 Wed, 31 Mar 2010 23:00:00 +0100 4104886 http://www.medworm.com/index.php?rid=4104885&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_12 The technique of selective immobilization of biomolecules in defined positions or areas using a simple procedure is essential for various applications such as biosensors, biochips, biomedical microdevices, and tissue engineering. For the generation of biomolecule microarrays, it is necessary to develop a functional surface retaining protein functionality and cell viability, and an efficient patterning tool having flexibility of size and shape. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104885 Wed, 31 Mar 2010 23:00:00 +0100 4104885 http://www.medworm.com/index.php?rid=4104884&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_11 Photolithographic patterning of proteins on surfaces has been used extensively in the past to define cell adhesion domains with micrometer-scale resolution. However, photolithographic patterning is not amenable to depositing several different proteins on the same surface. We propose to merge photolithography with robotic printing of proteins in order to create arrays of protein spots (∼300–500 μm diameters) with encoded micrometer-scale cell adhesive domains. This method for biointerface design can employ standard positive tone resist lithography to create temporary stencils for printing of protein arrays. Alternatively, nonfouling poly(ethylene glycol) hydrogels can be micropatterned on top of protein spots. In both cases, cells become adherent on the underlying protein domai... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104884 Wed, 31 Mar 2010 23:00:00 +0100 4104884 http://www.medworm.com/index.php?rid=4104883&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_10 In this chapter, we describe the fabrication and use of microfluidic capillary systems (CSs) made in soft, transparent polydimethylsiloxane (PDMS). Sixteen microfluidic CSs, each containing a loading pad, a microchannel, and a capillary pump are engraved in a single PDMS chip. The CSs are used for two applications, firstly to pattern fibronectin on glass surfaces to locally control the adhesion of cultured cells to the substrate, and secondly to carry out multiplexed miniaturized immunoassays. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104883 Wed, 31 Mar 2010 23:00:00 +0100 4104883 http://www.medworm.com/index.php?rid=4104882&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_9 We have developed a combinatorial platform for fabricating tissue scaffold arrays that can be used for screening cell–material interactions. Traditional research involves preparing samples one at a time for characterization and testing. Combinatorial and high-throughput (CHT) methods lower the cost of research by reducing the amount of time and material required for experiments by combining many samples into miniaturized specimens. In order to help accelerate biomaterials research, many new CHT methods have been developed for screening cell–material interactions where materials are presented to cells as a 2D film or surface. However, biomaterials are frequently used to fabricate 3D scaffolds, cells exist in vivo in a 3D environment and cells cultured in a 3D environment in vitr... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104882 Wed, 31 Mar 2010 23:00:00 +0100 4104882 http://www.medworm.com/index.php?rid=4104881&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_8 There is a growing interest on the fabrication of bacteria and virus microarray owing to their great potential in many biological applications ranging from diagnostic devices to advanced platforms for fundamental studies on molecular biology. Over the past decade, a number of studies with regard to the biomolecular patterning have been presented. Capillary force lithography (CFL) for polymeric thin films can provide well-ordered microarray structures over a large area in a facile and cost-efficient way while maintaining its biocompatibility during a process. Patterned polymeric structures can be utilized either to physical barriers for the confinement of bacteria or to physicochemical template for the subsequent binding of viruses. In this chapter, we have shown that the patterned structur... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104881 Wed, 31 Mar 2010 23:00:00 +0100 4104881 http://www.medworm.com/index.php?rid=4104880&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_7 Cell-based biosensors constitute a promising field that has numerous applications ranging from pharmaceutical screening to detection of pathogen and toxicant. The trends toward miniaturization of cell-based biosensor continue to spur development of cell microarray integrated into microfluidic devices. For cell-based biosensors to be useful for larger applications, several technical goals must be realized. First, the cell-patterning method used to generate multi-phenotypic array can accommodate multiple cell lines without major losses of cell viability, maintain total isolation of each cell phenotype, provide for the adequate mass transfer of dissolved gases and nutrients, and easy enough to allow for mass production. Second, cells on microarray must be cultured in three-dimensional environ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104880 Wed, 31 Mar 2010 23:00:00 +0100 4104880 http://www.medworm.com/index.php?rid=4104879&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_6 The mammalian cell surface is rich with carbohydrate polymers involved in a diversity of biological recognition events. Dynamic alterations of surface glycans mediate cell–cell communication in the immune system and host specificity of bacterial and viral pathogens. In addition, altered surface glycosylation has been implicated in disease progression of many cancers and may serve as important new targets for therapeutics. Despite the importance of glycosylation, the systematic analysis of sugars, i.e., glycomics, has lagged behind the well-studied disciplines of genomics and proteomics. This deficiency is due in part to the unique analytical challenges presented by glycans and the overwhelming diversity of sugars in nature. New microarray technologies have provided a high-throughput ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104879 Wed, 31 Mar 2010 23:00:00 +0100 4104879 http://www.medworm.com/index.php?rid=4104878&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_5 Traditional methods to study normal and pathological development of tissues have been limited by ­difficulties in controlling experimental conditions and quantifying biological processes of interest. Here we describe methods to create microarrays of engineered tissues that enable controlled and quantitative investigations. Using soft lithography-based techniques, extracellular matrix proteins can be microcontact printed or micromolded to make two- and three-dimensional micropatterned scaffolds. The ultimate form and resulting properties of the tissue construct are dictated by the geometry of the patterned extracellular matrix components. This chapter describes elastomeric stamp fabrication, microcontact printing and micromolding of extracellular matrix proteins, cell culture in micropa... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104878 Wed, 31 Mar 2010 23:00:00 +0100 4104878 http://www.medworm.com/index.php?rid=4104877&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_4 Protein arrays are miniaturised and highly parallelised formats of interaction-based functional protein assays. Major bottlenecks in protein microarraying are the limited availability and high cost of purified, functional proteins for immobilisation and the limited stability of immobilised proteins in their functional state. In contrast, protein-coding DNA is readily available by PCR, and DNA arrays can be stored over prolonged times without deterioration. This chapter presents a method for the rapid and economical “printing” of replicate protein microarrays directly from a single DNA array template using cell-free protein synthesis, termed “DNA array to protein array,” DAPA. The procedure is a truly enabling technology, making customised protein microarrays afforda... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104877 Wed, 31 Mar 2010 23:00:00 +0100 4104877 http://www.medworm.com/index.php?rid=4104876&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_3 Microarrays are spatially ordered arrays with ligands chemically immobilized in discrete spots on a solid matrix, usually a microscope slide. Microarrays are a high-throughput large-scale screening system enabling simultaneous identification of a large number of labeled target molecules (up to several hundred thousand) that bind specifically to the immobilized ligands of the array. DNA microarrays represent a promising tool for clinical, environmental, and industrial microbiology since the technology allows relatively rapid identification of large number of genetic determinants simultaneously, providing detailed genomic level information regarding the pathogen species, including identification of their virulence-associated factors and the presence of antibiotic resistance genes. In this ch... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104876 Wed, 31 Mar 2010 23:00:00 +0100 4104876 http://www.medworm.com/index.php?rid=4104875&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_2 In less than 40 years, aptamers have consolidated their role in biosensor development. Chemically related to nucleic acid probes, production of aptamers against targets of various sizes and compositions places them as ideal capture elements, alternative to more consolidated molecules such as antibodies. Thanks to their chemical simplicity and production, as well as their unique characteristics, aptamers have been successfully integrated in several innovative approaches. The incorporation of aptamers into the existing microarray technologies has lead to the reporting of various detection strategies, including direct fluorescence detection of fluorescent reporters, fluorescence anisotropy, FRET, SPR imaging, and electrochemical detection. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104875 Wed, 31 Mar 2010 23:00:00 +0100 4104875 http://www.medworm.com/index.php?rid=4104874&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-551-0_1 The development of microarray technology has revolutionized RNA and deoxyribonucleic acid (DNA) research. In contrast with traditional biological assays, microarrays allow the simultaneous measurement of tens of thousands of messenger RNA (mRNA) transcripts for gene expression or of genomic DNA fragments for copy number variation analysis. Over the past decade, genome-wide RNA or DNA microarray analysis has become an essential component of biology and biomedical research. The successful use of microarrays requires attention to unique issues of experimental design and execution. This chapter provides an overview of the methodology and applications of RNA and DNA microarrays in various areas of biological research. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=4104874 Wed, 31 Mar 2010 23:00:00 +0100 4104874 http://www.medworm.com/index.php?rid=3011891&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_9 DNA-stable isotope probing, a method to identify active microorganisms without the prerequisite of cultivation, has been widely applied in the study of microorganisms involved in the degradation of environmental pollutants. Recent advances and technique considerations in applying DNA-SIP in bioremediation are highlighted. A detailed protocol of a DNA-SIP experiment is provided. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011891 Fri, 23 Oct 2009 00:00:00 +0100 3011891 http://www.medworm.com/index.php?rid=3011890&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_8 Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011890 Fri, 23 Oct 2009 00:00:00 +0100 3011890 http://www.medworm.com/index.php?rid=3011889&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_7 Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011889 Fri, 23 Oct 2009 00:00:00 +0100 3011889 http://www.medworm.com/index.php?rid=3011888&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_6 Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in composting systems. This analysis is based on the restriction endonuclease digestion of fluorescently end-labeled PCR products. The digested product is mixed with a DNA size standard, itself labeled with a distinct fluorescent dye, and the fragments are then separated by capillary or gel electrophoresis using an automated sequencer. Upon analysis, only the terminal end-labeled restriction fragments are detected. An electropherogram is produced, which shows a profile of compost microbial community as a series of peaks of varying height. This technique has also been effectively used in the exploration of complex microbial environments and in the study of bac... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011888 Fri, 23 Oct 2009 00:00:00 +0100 3011888 http://www.medworm.com/index.php?rid=3011887&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_5 Bioremediation has been identified as a beneficial and effective strategy for the removal of recalcitrant environmental contaminants. Bioaugmentation of polluted environments with exogenous degrading microorganisms constitutes a major strategy of bioremediation. However, the ecological role of these strains and their impact on the endogenous microbial community of the micro-ecosystems where they are released should be known. Fingerprinting PCR-based methods, like denaturating gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP), could be used in studies exploring the ecology of pollutant-degrading microorganisms and their effects on the structure of the soil microbial community. This chapter provides a brief outline of the technical details invo... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011887 Fri, 23 Oct 2009 00:00:00 +0100 3011887 http://www.medworm.com/index.php?rid=3011886&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_4 Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic–biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) c... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011886 Fri, 23 Oct 2009 00:00:00 +0100 3011886 http://www.medworm.com/index.php?rid=3011885&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_3 Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail. (Source: Springer protocols feed by Biotechnology... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011885 Fri, 23 Oct 2009 00:00:00 +0100 3011885 http://www.medworm.com/index.php?rid=3011884&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_2 This chapter considers the use of a variety of approaches to assess either the bioavailability or the bioaccessibility of metals in soil. The bioavailability of metals from soils is considered with respect to a series of single-extraction methods, including the use of ethylenediaminetetraacetic acid (EDTA), acetic acid, diethylenetriaminepentaacetic acid (DTPA), ammonium nitrate, calcium chloride and sodium nitrate. Then, a procedure for the recovery of metals using a three-stage sequential extraction protocol is described. Two alternate approaches for assessing the environmental health risk to humans by undertaking in vitro gastrointestinal extraction (also known as the physiologically based extraction test, PBET) are considered. Finally, two acid digestion protocols that allow the pseudo... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011884 Fri, 23 Oct 2009 00:00:00 +0100 3011884 http://www.medworm.com/index.php?rid=3011883&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_1 We present a rapid and easily reproducible HPCD shake extraction technique that has been experimentally demonstrated to directly predict microbial availability and degradation in soil. This method can provide practitioners with both an indication of bioremediation end-points and may be valuable in the risk assessment of contaminated land. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011883 Fri, 23 Oct 2009 00:00:00 +0100 3011883 http://www.medworm.com/index.php?rid=3011882&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_16 Community-level physiological profiling (CLPP) is a technique which offers an easily applied protocol yielding information regarding mixed microbial community function and functional adaptations over space and time. Different communities can be compared and classified based on sole carbon source utilization patterns (CSUPs) gathered using BIOLOG™ microplates. One of the most challenging aspects associated with the CLPP method is in the data analysis. This chapter describes the relatively simple CLPP laboratory protocol and provides a detailed description of different data analysis techniques. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011882 Fri, 23 Oct 2009 00:00:00 +0100 3011882 http://www.medworm.com/index.php?rid=3011881&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_15 The use of bioassays for soil characterization is receiving significant attention as a complementary tool to chemical analysis. Bioassays consist of direct toxicity assays of environmental samples that are transferred to the laboratory and analyzed for toxicity against selected organisms. Such soil samples contain the combination of the different pollutants present in situ and enable factors such as the bioavailability of contaminants or the interactions (synergic and antagonic) between them to be simultaneously studied. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011881 Fri, 23 Oct 2009 00:00:00 +0100 3011881 http://www.medworm.com/index.php?rid=3011880&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_14 Monitored natural attenuation (MNA) is an in situ remediation technology that relies on naturally occurring and demonstrable processes in soil and groundwater which reduce the mass and concentration of the contaminants. Natural attenuation (NA) involves both aerobic and anaerobic degradation of the contaminants due to the fact that oxygen is used up near the core of the contaminant plume. The aerobic and anaerobic microbial processes can be assessed by microbial activity measurements and molecular biology methods in combination with chemical analyses. The sampling and knowledge on the site conditions are of major importance for the linkage of the results obtained to the conditions in situ. Rates obtained from activity measurements can, with certain limitations, be used in modeling of the f... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011880 Fri, 23 Oct 2009 00:00:00 +0100 3011880 http://www.medworm.com/index.php?rid=3011879&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_13 Despite the widespread availability of state-of-the-art biological techniques, remediation practitioners have been slow to adopt these technologies to assist in designing or indeed monitoring remediation strategies. In part, this is because practitioners are driven by cost and fail to see the benefit of emerging technologies, and in part because most companies have only a small portfolio of procedures available to them. Here, we review the component parts required to design a decision support tool, appraise one that the authors have developed and critically evaluate its application to case studies. If bioremediation is to become adopted, then it is likely to have to operate in parallel with other remediation methods. Furthermore, remediation strategies must couple effective technology with... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011879 Fri, 23 Oct 2009 00:00:00 +0100 3011879 http://www.medworm.com/index.php?rid=3011878&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_12 Xenobiotic degradation during biological wastewater treatment can be established or enhanced by bioaugmentation − the addition of biological agents carrying biodegradation genes required to perform the task. Whereas the addition of microbial cells carrying chromosomally encoded catabolic genes can be impaired by limited survival of the added microorganisms, the addition of donor organisms carrying a transmissible catabolic plasmid is a promising alternative. This plasmid can spread within the indigenous microbial community of the system, circumventing the need for extended survival of the introduced bacterial strain. Here we discuss how the catabolic plasmid pNB2 can be evaluated towards its potential to facilitate the degradation of a xenobiotic compound, 3-chloroaniline, and demons... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011878 Fri, 23 Oct 2009 00:00:00 +0100 3011878 http://www.medworm.com/index.php?rid=3011877&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_11 Wastewater treatment systems tend to be engineered to select for a few functional microbial groups that may be organized in various spatial structures such as activated sludge flocs, biofilm or granules and represented by single coherent phylogenic groups such as ammonia-oxidizing bacteria (AOB) and polyphosphate-accumulating organisms (PAO). In order to monitor and control engineered microbial structure in wastewater treatment systems, it is necessary to understand the relationships between the microbial community structure and the process performance. This review focuses on bacterial communities in wastewater treatment processes, the quantity of microorganisms and structure of microbial consortia in wastewater treatment bioreactors. The review shows that the application of molecular tech... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011877 Fri, 23 Oct 2009 00:00:00 +0100 3011877 http://www.medworm.com/index.php?rid=3011876&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-439-5_10 Environmental contamination has been viewed as an ecological malaise for which bioremediation can be prescribed as a “perfect medicine.” The solution to the problems with bioremediation lies in analyzing to what extent the microbes’ physiological machinery contributes to the degradation process and which biomolecules and their mechanisms are responsible for regulatory factors within the degradation system, such as protein, metabolite, and enzymatic chemical transformation. In the post-genomic era, recent advances in proteomics have allowed us to elucidate many complex biological mechanisms. Two-dimensional gel electrophoresis (2DE) in conjunction with mass spectrometry (MS) can be utilized to identify the biomolecules and their molecular mechanisms in bioremediation. A se... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=3011876 Fri, 23 Oct 2009 00:00:00 +0100 3011876 http://www.medworm.com/index.php?rid=2823601&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_9 Lime pretreatment has proven to be a useful method for selectively reducing the lignin content of lignocellulosic biomass without significant loss in carbohydrates, thus realizing an important increase in biodigestibility. In lime pretreatment, the biomass is pretreated with calcium hydroxide and water under different conditions of temperature and pressure. It can be accomplished in one of three fashions: (1) short-term pretreatment that lasts up to 6 h, requires temperatures of 100–160°C, and can be applied with or without oxygen (pressure ~200 psig); (2) long-term pretreatment taking up to 8 weeks, requiring only 55–65°C, and capable of running with or without air (atmospheric pressure); and (3) simple pretreatment requiring 1 h in boiling water, without air or oxygen... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823601 Wed, 23 Sep 2009 18:01:11 +0100 2823601 http://www.medworm.com/index.php?rid=2823600&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_8 Exposure of cellulosic biomass to temperatures of about 120–210°C can remove most of the hemicellulose and produce cellulose-rich solids from which high glucose yields are possible with cellulase enzymes. Furthermore, the use of dilute sulfuric acid in this pretreatment operation can increase recovery of hemicellulose sugars substantially to about 85–95% of the maximum possible versus only about 65% if no acid is employed. The use of small-diameter tubes makes it possible to employ high solids concentrations similar to those preferred for commercial operations, with rapid heat-up, good temperature control, and accurate closure of material balances. Mixed reactors can be employed to pretreat larger amounts of biomass than possible in such small-diameter tubes, but solids con... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823600 Wed, 23 Sep 2009 18:01:11 +0100 2823600 http://www.medworm.com/index.php?rid=2823599&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_7 Lignocellulosic biomass is an abundant and renewable resource for fuel ethanol production. However, the lignocellulose is recalcitrant to enzymatic hydrolysis because of its structural complexity. Controlled-pH liquid hot water (LHW) pretreatment of cellulosic feedstock improves its enzymatic digestibility by removing hemicellulose and making the cellulose more accessible to cellulase enzymes. The removed hemicellulose is solubilized in the liquid phase of the pretreated feedstock as oligosaccharides. Formation of monomeric sugars during the LHW pretreatment is minimal. The LHW pretreatment is carried out by cooking the feedstock in process water at temperatures between 160 and 190°C and at a pH of 4–7. No additional chemicals are needed. This chapter presents the detailed proced... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823599 Wed, 23 Sep 2009 18:01:11 +0100 2823599 http://www.medworm.com/index.php?rid=2823598&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_6 The methods of pretreatment of lignocellulosic biomass using aqueous ammonia are described. The main effect of ammonia treatment of biomass is delignification without significantly affecting the carbohydrate contents. It is a very effective pretreatment method especially for substrates that have low lignin contents such as agricultural residues and herbaceous feedstock. The ammonia-based pretreatment is well suited for simultaneous saccharification and co-fermentation (SSCF) because the treated biomass retains cellulose as well as hemicellulose. It has been demonstrated that overall ethanol yield above 75% of the theoretical maximum on the basis of total carbohydrate is achievable from corn stover pretreated with aqueous ammonia by way of SSCF. There are two different types of pretreatment... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823598 Wed, 23 Sep 2009 18:01:11 +0100 2823598 http://www.medworm.com/index.php?rid=2823597&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_5 Although cellulose is the most abundant organic molecule, its susceptibility to hydrolysis is restricted due to the rigid lignin and hemicellulose protection surrounding the cellulose micro fibrils. Therefore, an effective pretreatment is necessary to liberate the cellulose from the lignin–hemicellulose seal and also reduce cellulosic crystallinity. Some of the available pretreatment techniques include acid hydrolysis, steam explosion, ammonia fiber expansion (AFEX), alkaline wet oxidation, and hot water pretreatment. Besides reducing lignocellulosic recalcitrance, an ideal pretreatment must also minimize formation of degradation products that inhibit subsequent hydrolysis and fermentation. AFEX is an important pretreatment technology that utilizes both physical (high temperature and... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823597 Wed, 23 Sep 2009 18:01:11 +0100 2823597 http://www.medworm.com/index.php?rid=2823596&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_4 Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5–6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation. (Source: Springer protocols feed by Biotechnolog... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823596 Wed, 23 Sep 2009 18:01:11 +0100 2823596 http://www.medworm.com/index.php?rid=2823595&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_3 Since 2002, researchers at the University of Illinois, Urbana-Champaign, Illinois, have been studying the perennial warm-season grass Miscanthus × giganteus (M. × g.) to determine its potential as a biomass feedstock. M. × g. originated in Japan and is a hybrid believed to have M. sinensis and M. sacchariflorus as its parents. Until recently, it was used as a landscape plant in the United States, but it is now the subject of research interest because of its potentially great biomass production. In central Illinois, M. × g. begins growth in April, typically reaches 2 m by the end of May, and is normally greater than 3 m by the end of September. The grass is sterile and propagated asexually using plantlets produced in tissue culture or by rhizome divisions. Following ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823595 Wed, 23 Sep 2009 18:01:11 +0100 2823595 http://www.medworm.com/index.php?rid=2823594&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_2 Switchgrass is being widely considered as a feedstock for biofuel production. Much remains to be learned about ideal feedstock characteristics, but switchgrass offers many advantages already and can perhaps be manipulated to offer more. When planning to grow switchgrass, select a cultivar that is well adapted to the location – generally a lowland cultivar for the southern United States and an upland cultivar at higher latitudes. Plant non-dormant seed after soils are well warmed, preferably with no-till methods and always with good weed control. Except for weeds, few pests appear to be widespread; but disease and insect pests could become more important as acreages increase. Fertilization requirements are relatively low, with 50 kg N/ha/year being a good “generic” recomme... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823594 Wed, 23 Sep 2009 18:01:11 +0100 2823594 http://www.medworm.com/index.php?rid=2823593&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_1 Feedstock supply system encompasses numerous unit operations necessary to move lignocellulosic feedstock from the place where it is produced (in the field or on the stump) to the start of the conversion process (reactor throat) of the biorefinery. These unit operations, which include collection, storage, preprocessing, handling, and transportation, represent one of the largest technical and logistics challenges to the emerging lignocellulosic biorefining industry. This chapter briefly reviews the methods of estimating the quantities of biomass, followed by harvesting and collection processes based on current practices on handling wet and dry forage materials. Storage and queuing are used to deal with seasonal harvest times, variable yields, and delivery schedules. Preprocessing can be as s... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823593 Wed, 23 Sep 2009 18:01:11 +0100 2823593 http://www.medworm.com/index.php?rid=2823592&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_18 Biodiesel is produced by reacting vegetable oils or animal fats with alcohol in the presence of an alkaline catalyst. The resulting methyl esters, which are the biodiesel fuel, are separated from the by-product glycerin, and then washed with water and dehydrated to produce fuel that must meet standardized specifications. Degraded oils containing high levels of free fatty acids can also be converted to biodiesel, but pretreatment with acid-catalyzed esterification is required. The resulting fuel is suitable for use as a neat fuel in diesel engines or blended with conventional diesel fuel. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823592 Wed, 23 Sep 2009 18:01:11 +0100 2823592 http://www.medworm.com/index.php?rid=2823591&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_17 Ethanol production by fermentation of lignocellulosic biomass-derived sugars involves a fairly ancient art and an ever-evolving science. Production of ethanol from lignocellulosic biomass is not avant-garde, and wood ethanol plants have been in existence since at least 1915. Most current ethanol production relies on starch- and sugar-based crops as the substrate; however, limitations of these materials and competing value for human and animal feeds is renewing interest in lignocellulose conversion. Herein, we describe methods for both simultaneous saccharification and fermentation (SSF) and a similar but separate process for partial saccharification and cofermentation (PSCF) of lignocellulosic biomass for ethanol production using yeasts or pentose-fermenting engineered bacteria. These meth... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823591 Wed, 23 Sep 2009 18:01:11 +0100 2823591 http://www.medworm.com/index.php?rid=2823590&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_16 Oxygen is either limiting or absent in many ecosystems. Anaerobic bacteria are often key players in such environments and these organisms have important roles in geo-elemental cycling, agriculture, and medicine. The metabolic versatility of anaerobes is exploited in a variety of industrial processes including fermented food production, biochemical synthesis, and bioremediation. There has been recent considerable interest in developing and enhancing technologies that employ anaerobes as biocatalysts. The study of anaerobic bacteria requires specialized techniques, and specific methods are described for the culture and manipulation of these microbes. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823590 Wed, 23 Sep 2009 18:01:11 +0100 2823590 http://www.medworm.com/index.php?rid=2823589&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_15 We describe two procedures that use washed pretreated cellulosic material to measure the efficacy of cellulase enzymes. First, a saccharification assay that measures glucose yield as a function of the amount of cellulase used in the process. And second, the simultaneous saccharification and fermentation (SSF) assay measures cellulase performance by the amount of ethanol produced from enzymatic hydrolysis of the cellulosic material. You can use both assays to screen cellulases under a variety of substrate types, loadings, and process conditions. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823589 Wed, 23 Sep 2009 18:01:11 +0100 2823589 http://www.medworm.com/index.php?rid=2823588&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_14 Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and β-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture. (Source: Springer protocols feed by Biotechnology) Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823588 Wed, 23 Sep 2009 18:01:11 +0100 2823588 http://www.medworm.com/index.php?rid=2823587&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_13 Lignin, comprised primarily of three randomly polymerized phenylpropenyl monomers, is, arguably, the second most common organic molecule on earth. In current biorefinery applications, lignin is burned, usually in concentrated pulping or hydrolysis liquor, as a source of process steam and both internal and exported electricity. The aromatic content of lignin makes it a potentially attractive feedstock for high-value aromatic chemicals, polymers, and carbon products (graphite, activated carbon, and carbon fiber). Revenue from production of lignin-based chemicals could play a major role in biorefinery profitability if cost-effective methods for lignin separation and purification can be developed. This chapter presents descriptions of methods for assessing and purifying biorefinery lignins so ... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823587 Wed, 23 Sep 2009 18:01:10 +0100 2823587 http://www.medworm.com/index.php?rid=2823586&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_12 We describe a high-throughput method for estimating cell-wall chemistry traits using analytical pyrolysis. The instrument used to perform the high-throughput cell-wall chemistry analysis consists of a commercially available pyrolysis unit and autosampler coupled to a custom-built molecular beam mass spectrometer. The system is capable of analyzing approximately 42 biomass samples per hour. Lignin content and syringyl to guaiacol (S/G) ratios can be estimated directly from the spectra and differences in cell wall chemistry in large groups of samples can easily be identified using multivariate statistical data analysis methods. The utility of the system is demonstrated on a set of 800 greenhouse-grown poplar trees grown under two contrasting nitrogen treatments. High-throughput analytical py... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823586 Wed, 23 Sep 2009 18:01:10 +0100 2823586 http://www.medworm.com/index.php?rid=2823585&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_11 In its broadest definition, biomass can be described as all material that was or is a part of a living organism. For renewable energy applications, however, the definition of biomass is usually limited to include only materials that are plant-derived such as agricultural residues (e.g., wheat straw, corn stover) by-products of industrial processes (e.g., sawdust, sugar cane bagasse, pulp residues, distillers grains), or dedicated energy crops (e.g., switchgrass, sorghum, Miscanthus, short-rotation woody crops). This chapter describes analytical methods developed to measure plant components with an emphasis on the measurement of components that are important for biomass conversion. The methods described here can be viewed as a portfolio of analytical methods, with consistent assumptions and... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823585 Wed, 23 Sep 2009 18:01:10 +0100 2823585 http://www.medworm.com/index.php?rid=2823584&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-214-8_10 A variety of chemicals are produced upon pretreatment of lignocellulosic biomass. Aliphatic acids, aromatic acids, aldehydes, and phenolic compounds are of particular interest due to their presumed inhibitory influence on downstream enzymatic or microbial steps in biomass-to-ethanol conversion. Herein, we describe a series of analytical protocols that collectively enable quantitative monitoring of 40 potential fermentation inhibitors in biomass pretreatment samples. Solid samples are accommodated by first employing pressurized fluid extraction to generate an aqueous “wash stream.” Sample preparation for liquids involves an initial precipitation-filtration step, followed by liquid–liquid extraction and reconstitution of extracts in water. Samples are analyzed using high&nd... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2823584 Wed, 23 Sep 2009 18:01:10 +0100 2823584 http://www.medworm.com/index.php?rid=2629509&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-202-1_6 The objective of this chapter is to describe methods developed in this and other laboratories for microinjection and calcium imaging in the unfertilized zebrafish egg. Methods of immobilizing the oocyte include a holding chamber and a holding pipette. The holding chamber allows imaging of three or four oocytes simultaneously, while the holding pipette facilitates imaging of localized regions in the oocyte. Injection of calcium green dextran via holding chambers allowed detection of global changes in Ca2+ release following fertilization and development through early blastula stages. Injection and imaging with the holding pipette method allowed discrimination of calcium changes in the egg cortex from that in the central regions of the cell. The results demonstrate the highly localized nature... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2629509 Mon, 01 Dec 2008 00:00:00 +0100 2629509 http://www.medworm.com/index.php?rid=2629508&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-202-1_7 A major goal in cell biology is to understand the molecular mechanisms of the biological process under study, which requires functional information about the roles of individual proteins in the cell. For many non-genetic model organisms researchers have relied on the use of inhibitory reagents, such as antibodies that can be microinjected into cells. More recently, the advent of RNA-mediated interference (RNAi) has allowed scientists to knockdown individual proteins and to examine the consequences of the knockdown. In this chapter we present a comparison between microinjection of inhibitory reagents and RNAi for the analysis of protein function in mammalian tissue culture cells, providing both a description of the techniques as well as a discussion of the benefits and drawbacks of each app... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2629508 Mon, 01 Dec 2008 00:00:00 +0100 2629508 http://www.medworm.com/index.php?rid=2629507&cid=s_37119_70_f&fid=37119&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-202-1_11 Quantitative microinjection is used to introduce known quantities of molecules or probes into single cells to examine cellular function. The relatively large mammalian oocyte or egg is easily manipulated and can be injected with impermeant reagents including a variety of signaling molecules and fluorescent probes. Techniques have been developed to inject picoliter quantities of solution into oocytes and eggs with precision and reliability. The methods described here outline the quantitative injection procedures as they are used to inject mouse oocytes and eggs in a culture dish on the stage on an inverted microscope. The techniques are applicable to the oocytes, eggs, and early embryos of most mammalian species. Included are some general instructions on fabrication of transfer pipettes, ho... Springer protocols feed by Biotechnology news http://www.medworm.com/rss/comments.php?id=2629507 Mon, 01 Dec 2008 00:00:00 +0100 2629507