This thorough book covers the most recent proteomics techniques, databases, bioinformatics tools, and computational approaches that are used for the identification and functional annotation of proteins and their structure. The most recent proteomic resources widely used in the biomedical scientific community for storage and dissemination of data are discussed. In addition, specific MS/MS spectrum similarity scoring functions and their application in the field of proteomics, statistical evaluation of labeled comparative proteomics using permutation testing, and methods of phylogenetic analysis using MS data are also described in detail. Written for the highly successful Methods in Molecular Biology series, chapters contain the kind of detail and key implementation advice to ensure successful results. Authoritative and cutting-edge, Proteome Bioinformatics serves as a useful resource for researchers who are beginners as well as advanced investigators in the field of proteomics.

The polymerase chain reaction (PCR) is one of the most important molecular biological methods ever devised, with numerous applications to cli- cal molecular medicine. Since its description in 1985, PCR has undergone tremendous improvements, and many variations on the basic PCR theme have been published. With such a large volume of PCR-related literature, a clinical scientist wishing to use the technique will have a difficult task loc- ing the relevant information to implement it effectively. There is thus clearly a need for an up-to-date volume with detailed protocols to facilitate the setting up of those techniques most relevant to clinical applications. Unlike some other books on this topic, Clinical Applications of PCR includes only methods that are of direct relevance in clinical settings. The book is organized in three parts: an introductory section, a section on general methodology, and a final section with specific clinical applications. The first section covers the basic principles of PCR and is most useful to those new to molecular diagnosis. The next chapter includes useful tips for setting up a PCR laboratory. Section 2 then outlines some of the most commonly used PCR-based techniques in molecular diagnosis. Section 3 includes carefully chosen examples that represent typical applications of PCR in diverse clinical fields, encompassing hematology, oncology, genetics, and microbiology.

The enormous advances in molecular biology that have been witnessed in . Not recent years have had major impacts on many areas of the biological sciences least of these has been in the field of clinical bacteriology and infectious disease . Molecular Bacteriology: Protocols and ClinicalApplications aims to provide the reader with an insight into the role that molecular methodology has to play in modern medical bacteriology. The introductory chapter ofMolecular Bacteriology: ProtocolsandCli- cal Applications offers a personal overview by a Consultant Medical Microbio- gist of the impact and future potential offered by molecular methods. The next six chapters comprise detailed protocols for a range of such methods . We believe that the use of these protocols should allow the reader to establish the various methods described in his or her own laboratory. In selecting the methods to be included in this section, we have concentrated on those that, arguably, have greatest current relevance to reference clinical bacteriology laboratories; we have deliberately chosen not to give detailed protocols for certain methods, such as multilocus enzyme electrophoresis that, in our opinion, remain the preserve of specialist la- ratories and that are not currently suited for general use. We feel that the methods included in this section will find increasing use in diagnostic laboratories and that it is important that the concepts, advantages, and limitations of each are th- oughly understood by a wide range of workers in the field .

Completion of the sequence of the human genome represents an unpar- leled achievement in the history of biology. The project has produced nearly complete, highly accurate, and comprehensive sequences of genomes of s- eral organisms including human, mouse, drosophila, and yeast. Furthermore, the development of high-throughput technologies has led to an explosion of projects to sequence the genomes of additional organisms including rat, chimp, dog, bee, chicken, and the list is expanding. The nearly completed draft of genomic sequences from numerous species has opened a new era of research in biology and in biomedical sciences. In keeping with the interdisciplinary nature of the new scientific era, the chapters in Gene Mapping, Discovery, and Expression: Methods and Protocols recapitulate the necessity of integration of experimental and computational tools for solving - portant research problems. The general underlying theme of this volume is DNA sequence-based technologies. At one level, the book highlights the importance of databases, genome-browsers, and web-based tools for data access and ana- sis. More specifically, sequencing projects routinely deposit their data in p- licly available databases including GenBank, at the National Center of Biotechnology (NCBI) in the United States; EMBL, maintained by the European Bioinformatics Institute; and DDBJ, the DNA Data Bank of Japan. Currently, several browsers offer facile access to numerous genomic DNA sequences for gene mapping and data retrieval.

This doctoral thesis reports on an innovative data repository offering adaptive metadata management to maximise information sharing and comprehension in multidisciplinary and geographically distributed collaborations. It approaches metadata as a fluid, loosely structured and dynamical process rather than a fixed product, and describes the development of a novel data management platform based on a schemaless JSON data model, which represents the first fully JSON-based metadata repository designed for the biomedical sciences. Results obtained in various application scenarios (e.g. integrated biobanking, functional genomics and computational neuroscience) and corresponding performance tests are reported on in detail. Last but not least, the book offers a systematic overview of data platforms commonly used in the biomedical sciences, together with a fresh perspective on the role of and tools for data sharing and heterogeneous data integration in contemporary biomedical research.

Plasmids are fascinating entities which can replicate autonomously in bacterial, archaeal, and eukaryotic cells. They profit from the cellular environment of the host but can also carry a rich diversity of genes which can be beneficial for the host. Plasmids confer the ability to degrade organic compounds and to fix nitrogen. In addition, plasmids carry antibiotic resistance genes and their spread in pathogenic bacteria is of great medical importance. Plasmids are used in molecular studies of various organisms with ramifications in synthetic biology, medicine, ecology, and evolution, as well as basic research in molecular and structural biology. Written by acknowledged experts in the field, this volume provides an up-to-date treatment of the structure, function, and application of plasmids, with a particular emphasis on current and future trends. The book is aimed primarily at research scientists, graduate students, and professional scientists, but will also be of great interest to all

This volume provides a comprehensive collection of protocols on molecular diagnostics of bacteria that will suit the needs of molecular biologists, clinical laboratorians, and physician scientists alike. Chapters detail common bacterial pathogens, protocols that can be applied to diverse or even unknown pathogens, digital PCR, next generation sequencing, and bioinformatic analyses. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Diagnostic Bacteriology: Methods and Protocols delivers a wide range of assay types all on the cutting edge of diagnostic bacteriology.

The study of inflammation has captured the interest of scholars since the earliest recorded history. Symbols identifying the cardinal signs of inflammation were uncovered in both Sanskrit and hieroglyphics (1). Since complete apprecia tion of the inflammatory process is underscored by the need for knowledge at both the cellular and molecular levels, academic inquiry in the area of inflammation has led, in many respects, the foray of current biomedical research. Molecular and Cellular Basis of Inflammation represents research from the cutting edge in the broad view of inflammation. The chapters are written by experts with a multidisciplinary approach to the study of inflammatory and cellular processes, and thus include contributions form the fields of molecular biology, biochemistry, pharmacology, immunology, and pathobiology. Molecular and Cellular Basis of Inflammation was first conceived during a mini symposium sponsored by the American Society for Investigative Pathology held at FASEB in 1995 entitled "The Role of Reactive Lipids, Oxygen and Nitro gen Metabolites in Inflammation," at which several of the contributing authors delivered lectures. This present, much-extended volume includes leading-front descriptions of both protein and lipid mediators. The chapter devoted to the comple ment cascade by Ward and colleagues, as well as Chapters 3-7 and 13, provide up to-date descriptions of the biosynthesis, molecular biology, chemistry, and actions of both protein and lipid mediators.

Protein engineering is a fascinating mixture of molecular biology, protein structure analysis, computation, and biochemistry, with the goal of developing useful or valuable proteins. Protein Engineering Protocols will consider the two general, but not mutually exclusive, strategies for protein engineering. The first is known as rational design, in which the scientist uses detailed knowledge of the structure and function of the protein to make desired changes. The s- ond strategy is known as directed evolution. In this case, random mutagenesis is applied to a protein, and selection or screening is used to pick out variants that have the desired qualities. By several rounds of mutation and selection, this method mimics natural evolution. An additional technique known as DNA shuffling mixes and matches pieces of successful variants to produce better results. This process mimics recombination that occurs naturally during sexual reproduction. The first section of Protein Engineering Protocols describes rational p- tein design strategies, including computational methods, the use of non-natural amino acids to expand the biological alphabet, as well as impressive examples for the generation of proteins with novel characteristics. Although procedures for the introduction of mutations have become routine, predicting and und- standing the effects of these mutations can be very challenging and requires profound knowledge of the system as well as protein structures in general.

As researchers continue to make enormous progress in mapping disease genes, exciting, novel, and complex analyses have emerged. In this book, scientists from around the world, who are leaders in this field, contribute their vast experience and expertise to produce a comprehensive and fascinating text for researchers and clinicians alike. They provide cutting-edge analysis of the most up-to-date and preeminent information available.

Quantitative Proteomics by Mass Spectrometry, from the Methods in Molecular Biologya"[ series, is a compendium of cutting-edge protocols for quantitative proteomics, and presents the most significant methods used in the field today. The focus on mass spectrometry (MS) is integral, as MS has, and will continue to be, an essential tool in proteomics for studying complex biological systems and human diseases. This volume, written and compiled by leading quantitative proteomic experts, is an indispensable resource in the search for novel biomarkers.
Quantitative Proteomics by Mass Spectrometry presents several innovative MS quantitative procedures, including a variety of methods for introducing isotopic labels and quantifying post-translational modifications. Some of these methods include growing an organism in isotope-enriched media, performing trypsin proteolysis in the presence of 18O-water, reacting protein samples with isotopically labeled reagents, quantifying relative amount of proteins without the use of any isotopic labels. Attention is also given to state-of-the-art techniques for the characterization of the phosphoproteome and tandem MS for detection of inborn errors of metabolism. Specifically, the procedure for determinations of enzymatic activity could be used for large-scale screening of newborns. The protocols in this volume expand both the breadth and depth of readily available methods for quantitative proteomic researchers using MS.
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Hands-on experts in nanomaterial synthesis and application describe in detail the key experimental techniques currently employed in novel materials synthesis, dynamic cellular imaging, and biological assays. The author's emphasize diverse strategies to synthesize and functionalize the use of nanoparticles for biological applications. Additional chapters focus on the use of biological components (peptides, antibodies, and DNA) to synthesize and organize nanoparticles to be used a building block in larger assemblies. These new materials make it possible to image cellular processes for longer durations, leading to high throughput cellular-based screens for drug discovery, drug delivery, and diagnostic applications. Highlights include overview chapters on quantum dots and DNA nanotechnology, and cutting-edge techniques in the emerging nanobiotachnology arena.

It has been over 40 years since the original report by Salmon and Daughaday demon strating that the ability of GH to stimulate sulfation of cartilage was mediated by a "sulfation factor. " In the ensuing decades, it has become apparent that this "sulfation factor activity" encompasses a complex system ofligands (IGFs), receptors, and carrier proteins that are, in tum, responsible for a wide array of cellular actions. The IGF system has been demonstrated to be critically involved in both intrauterine and postnatal growth, and to have important implications in cancer biology as well, owing to the ability of the IGFs to function in endocrine, paracrine, and autocrine modes and given the wide distri bution of IGFs in virtually every organ system. The contributions to The /GF System reflect the wide span of interest in the IGF system and its implications for normal and abnormal growth and metabolism. The chapters have been divided into four broad sections: I. Molecular biology of the IGF system; II. Bio logical actions of the IGFs; III. IGF physiology; and IV. Clinical aspects of the IGFs. We have made every effort to highlight the major contemporary themes in IGF biology, but as is inevitable in such a fast-moving field, perspectives will continue to change as new information is accumulated."

Written by specialists in the field, this book provides an overview of the structural and molecular biology of cellular processes that occur at or near bacterial membranes.

After 20 years of intensive effort, novel neuropeptides continue to be discovered, and the field of neuropeptide research is still expanding. As new analytical techniques become available, their applicability to the study of neu ropeptides brings fresh insights into the properties and functions of these ubiq uitous chemical messengers. Presented in this single volume, Neuropeptide Protocols, are 33 chapters covering these new techniques, together with more established methods. Each contributor is actively engaged in neuropeptide research and so brings to his or her description an awareness of the practical problems inher ent in the method, and provides sound advice on how to overcome them. The format conforms to the style of previous books in the Methods in Molecular Biology series. Each chapter provides an instruction to the technique, and item ized list of equipment and reagents, and a step-by-step set of instructions to enable practitioners to reproduce the method. The Notes section gives insights into pitfalls or critical stages, tips to overcome these obstacles, and sugges tions for extensions or modifications of the basic protocol. Neuropeptide Protocols is intended as a benchtop manual providing the entire gamut of techniques that form the essential tool kit of the practicing neuropeptide researcher. It will be useful for those new to the field, as well as for established workers who wish to try a new technique for the first time."

The revolution in biological research initiated by the demonstration that particular DNA molecules could be isolated, recombined in novel ways, and conveniently replicated to high copy number in vivo for further study, that is, the recombinant DNA era, has spawned many additional advances, both methodological and intellectual, that have enhanced our understanding of cellular processes to an astonishing degree. As part of the subsequent outpouring of information, research exploring the mechanisms of gene regulation, both in prokaryotes and eukaryotes (but particularly the latter), has been particularly well represented. Although no one technical approach can be said to have brought the filed to its current level of sophistication, the ability to map the interactions of trans-acting factors with their DNA recognition sequences to a high level of precision has certainly been one of the more important advances. This "footprinting" approach has become almost ubiquitous in gene regulatory studies; however, it is in its ""in vivo"" application that ambiguities, confusions, and inconsistencies that may arise from a purely ""in vitro""-based approach can often be resolved and placed in their proper perspective. Put more simply, that an interaction can be demonstrated to occur between purified factors and a particular piece of DNA in a test tube does not, of course, say anything regarding whether such interactions are occurring "in vivo." The ability to probe for such interactions as they occur inside cells, with due attention paid to the relevant developmental stage, or to the tissue specificity of the interaction being probed, has made "in vivo" footprinting approach an invaluable adjunct to the "gene jockey's" arsenal of weapons.

Every cell in our bodies contains a great variety and number of permeability pathways for various organic and inorganic ions, water, metabolites, nutrients, and signalling molecules. Maintenance and precise control of gating within these pathways are fundamental principles of life as these underlie basic cellular functions such as communication, contractility, and metabolism. In Ion Channels: Methods and Protocols, Second Edition, experts in the field contribute chapters that focus on the strategies, approaches, methods, and protocols for studying a large family of proteins that form ionic channels in the plasma membrane and intracellular membranes of cells. Using practical examples from the cutting-edge current research, this volume will take a look back at the major methods and approaches that aided the progress toward the current understanding of ion channel function, structural design, and biological roles. The volume also aims to look forward and identify approaches that will lead us to future discoveries. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Ion Channels: Methods and Protocols, Second Edition will greatly assist researchers searching for specific methodology in studying ion channels on the path toward a greater understanding of these key biological features.

Systems biology can now be considered an established and fundamental field in life sciences. It has moved from the identification of molecular 'parts lists' for living organisms towards synthesising information from different 'omics'-based approaches to generate and test new hypotheses about how biological systems work. In In Silico Systems Biology: Methods and Protocols, expert researchers in the field detail a practical set of chapters based often on actual materials used and develop for face-to-face training with examples and case studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, step-by-step workflows, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, In Silico Systems Biology: Methods and Protocols seeks to aid scientists in the further study of network biology and mathematical models of biological systems.

Senior scientists Marilena Aquino de Muro and Ralph Rapley have brought together an outstanding collection of time-tested protocols for designing and using genes probes in a wide variety of applications. The applications covered range from searching for specific genes in the human chromosome to the detection of microorganisms and their toxins in the environment and in food samples, as well as in the diagnosis of human disease. Helpful tutorials explain the principles of gene probe design, labeling, detection, target formation, and hybridization conditions.

Radicals for Life: the Various Forms of Nitric Oxide provides an up-to-date overview of the role of nitrosocompounds and nitrosyl-iron complexes in physiology. Nitrosocompounds can be considered as stabilised forms of nitric oxide, one of the most important regulatory molecules in physiology today. Many nitrosocompounds share some of the physiological functions of nitric oxide, and may be formed inside living organisms. This is the first book to be published that is dedicated to the role of such nitrosocompounds in physiology, with particular emphasis on the nitrosocompounds that are endogenously formed in higher organisms and humans.
Points of discussion include: physical and chemical properties of the compounds, the main chemical pathways in vivo, as well as the physiological effects that have been recognised to date. Each of the nineteen chapters is written by distinguished specialists in the field, well known for their original and important contributions to the subject. Also included are results from a wide range of studies in vitro, in cell cultures, animal models and human volunteers.
Examples of alternative forms of nitric oxide, with special emphasis on their protective role against widespread human diseases like atherosclerosis, Alzheimer's disease, diabetes, sexual dysfunction, and renal insufficiency to stroke and ischemia are also included.
* First monograph to consider and provide an overview of endogenous nitrosocompounds and nitrosyl-iron complexes
* Extensive bibliographic references, written by specialists of human physiology
* Providing high scientific quality with a focus on implications for human diseases

The process whereby a single cell, the fertilized egg, develops into an adult has fascinated for centuries. Great progress in understanding that process, h- ever, has been made in the last two decades, when the techniques of molecular biology have become available to developmental biologists. By applying these techniques, the exact nature of many of the interactions responsible for forming the body pattern are now being revealed in detail. Such studies are a large, and it seems ever-expanding, part of most life-science groups. It is at newcomers to this field that this book is primarily aimed. A number of different plants and animals serve as common model org- isms for developmental studies. In Molecular Methods in Developmental Bi- ogy: Xenopus and Zebrafish, a range of the molecular methods applicable to two of these organisms are described, these are the South African clawed frog, Xenopus laevis, and the zebrafish, Brachydanio rerio. The embryos of both of these species develop rapidly and externally, making them particularly suited to investigations of early vertebrate development. However, both Xenopus and zebrafish have their own advantages and disadvantages. Xenopus have large, robust embryos that can be manipulated surgically with ease, but their pseudotetraploidy and long generation time make them unsuitable candidates for genetics. This disadvantage may soon be overcome by using the diploid Xenopus tropicalis, and early experiments are already underway. The transp- ent embryos of zebrafish render them well-suited for in situ hybridization and immunohistochemistry, and good for observing mutations in genetic screens.

Ribonucleic acids are central to cellular and molecular processes and perform vital functions in both structural and functional roles. RNA molecules form the bridge between the stable genetic information contained within DNA and enzymes and proteins that carry out much of the metabolism within the cell. Many of the sites of protein synthesis, the ribosomes within the cell, are composed of these ribonucleic acids as are the tRNA molecules that deliver the amino acid building blocks to the ribosomes. Of all the RNA species, the nucleic acid intermediate, messenger RNA, is a desirable source of material to biologists, since this reflects much of, what ultimately, is translated into enzymes and proteins. In order to determine the qualitative and quantitative changes in mRNA expression, a vast number of molecular biological techniques have been developed. Key molecular methods that provide the means to initially isolate and analyze RNA molecules are the focus of this volume. In putting together this collection of protocols, we have tried to provide techniques that are most applicable and widely used. In particular, there are a number of iso- tion techniques included that have been developed, modified, or adapted to enable extraction from a variety of cell types, organisms, or subcellular organelles. Successful isolation of intact RNA is an essential starting point for any sub- quent analysis. This is why we have aimed to make this section comprehensive. The analysis of RNA is the focus of the following chapters.

In miRNomics: MicroRNA Biology and Computational Analysis, expert researchers in the field present an overview of the current state of the art and aim to put the respective areas of research into a larger perspective. These include methods and techniques ranging from miRNA biogenesis, their biological function, computational analyses to their medical implications and applications. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, miRNomics: MicroRNA Biology and Computational Analysis seeks to aid scientists in the further study into miRNA research and statistics.

The papers in this volume arose out of the workshop Membrane Transport and Renal Physiology, which was conducted as part of the IMA 1998-1999 program year, Mathematics in Biology. The workshop brought together physiologists, biophysicists, and applied mathematicians who share a common interest in solute and water transport in biological systems, especially in the integrated function of the kidney. Solute and water transport through cells involves fluxes across two cell membranes, usually via specialized proteins that are integral membrane components. By means of mathematical representations, transport fluxes can be related to transmembrane solute concentrations and electrochemical driving forces. At the next level of functional integration, these representations can serve as key components for models of renal transcellular transport. Ultimately, simulations can be developed for transport-dependent aspects of overall renal function. Workshop topics included solute fluxes through ion channels, cotransporters, and metabolically-driven ion pumps; transport across fiber-matrix and capillary membranes; coordinated transport by renal epithelia; the urine concetrating mechanism; and intra-renal hemodynamic control. This volume will be of interest to biological and mathematical scientists who would like a view of recent mathematical efforts to represent membrane transport and its role in renal function.

Gaining an understanding of the mechanisms by which cells process and respond to extracellular cues has become a major goal in many areas of bi- ogy and has attracted the attentions of almost every traditional discipline within the biological sciences. At the heart of these divergent endeavors are common methods that can aid biochemists, physiologists, and pharmacologists in ta- ling the specific questions addressed by their research. In Receptor Signal Transduction Protocols, a diverse array of meth- ologies employed to interrogate ligand--receptor and receptor-effector int- actions are described by authors who have devised and successfully applied them. The authors blend excellent descriptions and applications of fairly well established methodologies with new technologies at the cutting-edge of signal transduction research and as such I hope the present volume will complement and extend a previous excellent volume in this series edited by David Kendall and Stephen Hill (Methods Molecular Biology, vol. 41, Signal Transd- tion Protocols).