This volume includes comprehensive descriptions of miRNA biogenesis and their role in the development and progression of various human diseases. The first few chapters of MicroRNA Profiling: Methods and Protocols discuss the effects of over-expressing and repressing of a target miRNA and their effects on cell viability and proliferation. The next few chapters explore the protocols for total RNA isolation from cells and cell-derived product including formalin fixed paraffin embedded tissue and plant tissue. The last few chapters discuss isolation and characterization of exosomes from medium conditioned by cell lines, serum, and plasma specimens. This book also includes discussions of several software tools, such as miRandola, PicTar, DIANA, and miRWalk. 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. Comprehensive and cutting-edge, MicroRNA Profiling: Methods and Protocols is a valuable resource for anyone interested in the field of Micro RNAs.

This new volume reviews current progress on different approaches of in vivo reprogramming technology. Leaders in the field discuss how in vivo cell lineage reprogramming can be used for tissue repair and regeneration in different organs, including brain, spinal cord, pancreas, liver and heart. Recent studies on in vivo cell reprogramming towards pluripotency are reviewed; examples are given to show its potential in regenerative medicine. In each chapter, the regenerative potential of different in vivo reprogramming approaches is discussed in detail. More specifically, how different tissue failures or damages can be treated with this technology is explained. Examples from various animal models are given and the regenerative potential of in vivo reprogramming is compared to that of cell transplantation studies. The last chapter discusses current challenges of these preclinical studies and gives suggestions in order to improve the current strategies. Future directions are indicated for the transition of in vivo reprogramming technology to clinical settings. This is among the first books in the literature which specifically focuses on the in vivo reprogramming technology in regenerative medicine and these chapters collectively cover one of the most important and exciting topics of regenerative medicine.

Defining and understanding cellular and molecular mechanisms that are relevant to women's health has become a critical area of scientific pursuit. Until recently, very little effort has been place on defining or understanding critical differences between women and men that may be critical to the overall health of the woman. In 1990, the National Institutes of Health recognized this gap in knowledge resulting in the creation of the Office of Research on Women's Health. One of the purposes of this office was to advance the understanding of health issues from the women's perspective from both a basic and clinical scientific perspective. From a scientific evolution of understanding, the existence of this office is new and thus there has not been enough time for new information to integrate itself in our current scientific thought process. This book will seek to capture and disseminate our current understanding of scientific advancements relevant to women's health and provide the information to a broad audience. The purpose of this work is to discuss recent advancements in basic science across three areas of concern for women's health. In addition, the book will provide "translational" chapters that attempt to place the basic science work in context within our current understanding of the human. Although it is well acknowledge that gender differences exist across organ function which translates into differences in whole body function, until recently little effort has been made to define basic mechanisms within various tissues within the woman. This work will focus on recent scientific findings that are relevant to women's health and to provide novel and relevant information to interested scientists and clinicians.

This book serves as an introduction to targeted genome editing, beginning with the background of this rapidly developing field and methods for generation of engineered nucleases. Applications of genome editing tools are then described in detail, in iPS cells and diverse organisms such as mice, rats, marine invertebrates, fish, frogs, and plants. Tools that are mentioned include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, all of which have received much attention in recent years as breakthrough technologies. Genome editing with engineered nucleases allows us to precisely change the target genome of living cells and is a powerful way to control functional genes. It is feasible in almost all organisms ranging from bacteria to plants and animals, as well as in cultured cells such as ES and iPS cells. Various genome modifications have proven successful, including gene knockout and knock-in experiments with targeting vectors and chromosomal editing. Genome editing technologies hold great promise for the future, for example in biomedical research, clinical medicine, and generation of crops and livestock with desirable traits. A wide range of readers will find this book interesting, and with its focus on applications in a variety of organisms and cells, the book will be valuable for life scientists in all fields.

Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium has the ability to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature 30 years ago has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agric- tural development. Compared to physical transformation methods such as particle bomba- ment or electroporation, Agrobacterium-mediated DNA delivery has a number of advantages. One of the features is its propensity to generate single or a low copy number of integrated transgenes with defined ends. Integration of a single transgene copy into the plant genome is less likely to trigger "gene silencing" often associated with multiple gene insertions. When the first edition of Agrobacterium Protocols was published in 1995, only a handful of plants could be routinely transformed using Agrobacterium. Ag- bacterium-mediated transformation is now commonly used to introduce DNA into many plant species, including monocotyledon crop species that were previously considered non-hosts for Agrobacterium. Most remarkable are recent devel- ments indicating that Agrobacterium can also be used to deliver DNA to non-plant species including bacteria, fungi, and even mammalian cells.

The ovary is a suitable organ for studying the processes of cell death. Cell death was first described in the rabbit ovary (Graaffian follicles), the phenomenon being called 'chromatolysis'. To date, it is recognized that various forms of cell death (programmed cell death, apoptosis and autophagy) are essential components of ovarian development and function. Programmed cell death is responsable for the ovarian endowment of primordial follicles around birth; in the prepuberal and adult period, apoptosis is a basic mechanism by which oocytes are eliminated by cancer therapies and environmental toxicants; in the ovarian cycle, follicular atresia and luteal regression involve follicular cell apoptosis. Finally, abnormalities in cell death processes may lead to ovarian disease such as cancer and chemoresistance. In this book, after an introductory description of various forms of cell death and of the ovary development and function in mammals, the processes of cell death in ovarian somatic cells and oocytes are described at cytological, physiological and molecular levels and analyzed in the embryonic, prepuberal and adult ovary. A complex array of molecular pathways triggered by extrinsic and intrinsic signals able tor induce or suppress cell death in the same cell, according to cell type and ovary developmental stage, emerges. Physiological interactions with the axis hypothalamus-hypophysis as well as ovarian internal functional signal are also critically reviewed to explain the abortive development of follicles before the beginning of the ovarian cycle. The book conveys information useful to the updating of biologists and physicians who are interested to the ovary biology and functions. Hopefully it should provide also clues for stimulating novel experiments in the study of cell death in the mammalian ovary still at an early stage.

Studies related to recombinant gene expression have brought new advance such as the emergence of the "omics" technologies. While Escherichia coli, Sacharomyces cerevisiae and insect cells continue to be the dominant production platforms of recombinant proteins. In Recombinant Gene Expression: Review and Protocols, Third Edition, expert researchers in the field detail many of the methods now commonly used to study recombinant gene expression. These include methods and techniques for bacteria, lower eukaryotes, fungi, plants and plant cells, and animals and animal cells. 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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Recombinant Gene Expression: Review and Protocols, Third Edition seeks to aid scientists in the further study of this crucially important research into recombinant gene expression.

The purpose of these volumes is to provide a reference work for the methods of purifying many of the receptors we know about. This be comes increasingly important as full-length receptors are overexpressed in bacteria or in insect cell systems. A major problem for abundantly expressed proteins will be their purification. In addition to purification protocols, many other details can be found concerning an individual receptor that may not be available in standard texts or monographs. No book of this type is available as a compendium of purification procedures. Receptor Purification provides protocols for the purification of a wide variety of receptors. These include receptors that bind: neurotransmit ters, polypeptide hormones, steroid hormones, and ligands for related members of the steroid supergene family and others, including receptors involved in bacterial motion. The text of this information is substantial, so as to require its publication in two volumes. Consequently, a division was made by grouping receptors by the nature of their ligands. Thus, in Volume One there are contributions on serotonin receptors, adrenergic receptors, the purification of GTP-binding proteins, opioid receptors, neurotensin receptor, luteinizing hormone receptor, human chorionic gonadotropin receptor, follicle stimulating hormone receptor, thyro tropin receptor, prolactin receptor, epidermal growth factor receptor, platelet derived growth factor receptor, colony stimulating factor recep tor, insulin-like growth factor receptors, insulin receptor, fibronectin receptor, interferon receptor, and the cholecystokinin receptor."

Modern cell biology has brought improvements in therapy for advanced malignant diseases through immunomodulation, hematopoietic stem cell transplantation, and other advanced techniques. Collected here are selected papers from the Fifth International Symposium of Keio University for Life Sciences and Medicine on Cell Therapy.
All chapters include innovative basic research for clinical application: immunotherapy, cancer vaccination, molecular biology of hematopoietic stem cells, stem cell processing, and gene therapy.
The book is divided into three parts: Immunotherapy for malignant diseases; Hematopoietic stem cell biology and clinical application; and International collaboration in hematopoietic stem cell transplantation. Included in the third part is information on bone marrow registries from around the world.
The book thus presents up-to-date information on biological and clinical aspects of treatment, with insight into the future of cell therapy.

This volume is a collection of chapters contributed by leading experts in the emerging field of analytical morphology. Its contents cover a wide range of techniques for morphologic research and diagnosis and it is intended for anyone who wants to keep abreast of the rapid development in this field. Analytical morphology is a contemporary science dealing with the analy- sis of shape, size and color arrangement of cell and tissue components by means of a variety of analytical maneuvers. It differs from conventional mor- phology and histopathology in that it employs methods beyond routine hema- toxylin and eosin or histochemical staining. To a great extent, the advance- ment of analytical morphology is based on new advances in other disciplines, such as immunology, molecular biology, laser technology, microwave tech- nology and computer science. Using these new methods, particular cellular components that would otherwise remain invisible, such as peptides, pro- teins, or nucleotide sequences, are highlighted by visible markers through chemical, physical, immunological or molecular biological reactions. These methods include immunocytochemistry, in situ hybridization, in situ poly- merase chain reaction, antigen retrieval, image analysis, and the like. Analytical morphology is the foundation of contemporary medicine. It provides concrete and visible evidence for many conceptual deductions of other life science disciplines.

Computers have revolutionized the analysis of sequencing data. It is unlikely that any sequencing projects have been performed in the last few years without the aid of computers. Recently their role has taken a further major step forward. Computers have become smaller and more powerful and the software has become simpler to use as it has grown in sophistication. This book reflects that change since the majority of packages described here are designed to be used on desktop computers. Computer software is now available that can run gels, collect data, and assess its accuracy. It can assemble, align, or compare multiple fragments, perform restriction analyses, identify coding regions and specific motifs, and even design the primers needed to extend the sequencing. Much of this soft ware may now be used on relatively inexpensive computers. It is now possible to progress from isolate d DNA to database submission without writing a single base down. To reflect this progression, the chapters in our Sequence Data Analysis Guidebook are arranged, not by software package, but by fimction. The early chapters deal with examining the data produced by modem automated sequenc ers, assessing its quality, and removing extraneous data. The following chap ters describe the process of aligning multiple sequences in order to assemble overlapping fragments into sequence contigs to compare similar sequences from different sources. Subsequent chapters describe procedures for compar ing the newly derived sequence to the massive amounts of information in the sequence databases."

Mitosis: Methods and Protocols provides state-of-the-art overviews on the most important approaches currently used in mitosis research spanning from the analysis of single molecules in isolation to their utilization within the complex environment of the cell. The volume is divided into four parts, each focused on methods pertaining to distinct aspects of mitosis research. Part I presents approaches for visualizing and analyzing the dynamic behaviors of the spindle apparatus, the microtubule based machine that drives chromosome segregation. Part II focuses more generally on methods for studying and manipulating the microtubule cytoskeleton in cells and complex cell free extracts. Part III provides state of the art biophysical and high resolution microscopy approaches for assessing complex interactions between microtubules and microtubule-associated proteins in isolation as well as microtubule structure in cells. Part IV provides methods for studying the effects of cell shape on cell division and methods for quantifying aneuploidy (aberrant chromosome number) which frequently results from mitotic defects and has been linked to human maladies ranging from birth defects to cancer. 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, Mitosis: Methods and Protocols seeks to provide diverse methods and new techniques to address new or old questions related to the mechanisms of mitosis.

Recently, important new findings in the polyamine field and a variety of new experimental systems have revolutionized the study of these ubiquitous cellular components, essential for normal growth and development. In "Polyamines: Methods and Protocols," leading researchers contribute an extensive collection of up-to-date laboratory techniques for the further pursuit of polyamine study. The volume delves into vital subjects such as neoplasia studies with animal models and human patients, therapeutic roles for polyamine inhibitors and analogs, polyamine metabolism and oxidative damage, polyamines as regulators of critical ion channels, as well as polyamine transport systems and polyamine-responsive genes. 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 expert notes on troubleshooting and avoiding known pitfalls.

Comprehensive and cutting-edge, "Polyamines: Methods and Protocols" provides a key resource for all scientists pursuing the study of this dynamic and significant aspect of cellular biology."

Signaling networks are composed of numerous signaling pathways and each has its own intricate component parts. Signaling outputs are dynamic, extraordinarily complex and yet highly specific. In, Computational Modeling of Signaling Networks: Methods and Protocols, expert researchers in the field provide key techniques to study signaling networks. Focusing on Systems of ODEs, parameterization of signaling models, signaling pathways, mass-action kinetics and ODEs, and how to use modeling to plan experiments. Written in the highly successful Methods in Molecular Biology (TM) series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Thorough and intuitive, Computational Modeling of Signaling Networks: Methods and Protocols aids scientists in continuing study of how signaling networks behave in space and time to generate specific biological responses and how those responses impact biology and medicine.

During the last decade, an increased interest in somatic stem cells has led to a flurry of research on one of the most accessible tissues of the body: skin. Much effort has focused on such topics as understanding the heterogeneity of stem cell pools within the epidermis and dermis, and their comparative utility in regenerative medicine applications. In Skin Stem Cells: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study skin stem cells. These include methods and techniques for the isolation, maintenance and characterization of stem cell populations from skin. 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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Skin Stem Cells: Methods and Protocols seeks to aid scientists in the further understanding of these diverse cell types and the translation of their biological potential to the in vivo setting.

Myc controls multiple cellular functions, including cell proliferation, growth, differentiation and death, both directly and indirectly, through its modulation of downstream transcriptional programs. In The Myc Gene: Methods and Protocols, experts in the field summarize the standard and novel techniques that allow the studying of Myc mechanism of action in normal and cancer cells, in vitro and in vivo, in one succinct manual. 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.

Protein glycosylation is now acknowledged as a major posttranslational modification with significant effects on protein folding, conformation distri- bution, stability, and activity. The added oligosaccharide chains are large and diverse and have specific recognition motifs important in many aspects of cell interactions and regulation. As such, there is a growing need to communicate the analytical methods of the specialist carbohydrate chemist, biochemist, and physicochemist to protein experts and the pharmaceutical industry. Other areas that come under the influence of the glycosciences are DNA interactions with ubiquitous saccharide-containing antibiotics and antitumor drugs; inhibitors of viral infection; bacterial, mycobacterial, and parasite antigens; glycolipids; glycophosphatidylinositol protein membrane anchors; and (glyco)protein- proteoglycan interactions. Compared to the first edition of this book, Glycopro- tein Analysis in Biomedicine, less emphasis is given to biomedical aspects, but these chapters are still pertinent today. The significant differences in the con- tent relate to advances in analysis relevant to biotechnology; for example, the production of recombinant glycoproteins and other therapeutics. It must also not be forgotten that the methods here described in Glycoanalysis Protocols are relevant to exploiting the commercial potential of carbohydrates in fields related to agriculture, food, and the domestic and chemical industries. The emphasis of the book remains in bringing the glycosciences into mainstream biochemistry. The analytical methods covered in Glycoanalysis Protocols are the re- sult of experts translating their life's works into easy-to-follow recipes.

Award-winning researchers review of key aspects of DNA repair in a wide variety of organisms, including all-important model systems. The book focuses on DNA damage and repair in prokaryotic and model eukaryotic systems, emphasizing the significant progress that has been made in the past five years. Each chapter has undergone a rigorous peer-review cycle to ensure definitive and comprehensive treatment. Major topics include UV and X-Ray repair, repair of chemical damage, recombinational repair, mismatch repair, transcription-repair coupling, and the role of DNA repair in cell cycle regulation.

Initially believed to be inactive molecules, glycans are now considered essential for life, both under normal and pathological conditions.

This volume of the series "Biology of Extracellular Matrix " reviews the most recent findings on the role of glycans in the development of diseases and the possible therapeutic use of this class of molecules. It shows how the interaction of glycans with growth factors, growth factor binding proteins, extracellular proteases, protease inhibitors, chemokines, morphogens, and adhesive proteins regulates inflammation, infection, cancer, atherosclerosis, thrombosis and embryonic stem cell biology. Furthermore, an extensive survey about the structure and pharmacological effects of unique marine glycosaminoglycans is discussed as well as the possibility of using these glycans as therapeutic agents.

Cytokines are pleiotropic regulatory proteins involved in essentially all biological processes and associated with a wide variety of diseases, including inflammatory disorders as well as many types of cancer and leukemia. Knowledge about the quantitative and qualitative nature of cytokine production is critical in the understanding of normal and pathological processes. The cytokine detection in biological and clinical samples faces many challenges including their low abundance, the need to distinguish between active and latent cytokine forms, and the need to measure multiple cytokines in a single assay. This volume will provide a comprehensive collection of classic and cutting-edge methodologies that are currently used to analyze and quantify cytokines and their biological activities in complex biological and clinical samples. The chapters are divided into four main categories. The first group focuses on the immunodetection of released cytokines in tissue culture supernatants, plasma, serum, and whole blood samples by immunoassays.These immunoassays measure the total concentrations of released cytokines regardless of their biological activity, and include ELISA, flow cytometry, ELISPOT, and the antibody-based proximity ligation. . The second group will focus on the analysis of biologically active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-induced cell degranulation assay, cell proliferation and differentiation, cytokine-induced cytokine production, and the radioreceptor cytokine assay. The third group focuses on the analysis of intracellular cytokines by flow cytometry, western blotting and fluorescence and confocal microscopy. In addition, this category includes protocols for quantitative analysis of cytokine gene expression by real time RT-PCR and analysis of the cytokine promoter occupancy by chromatin immunoprecipitation. The fourth group focuses on the recently developed multiplex arrays that can measure multiple cytokines in the same sample at the same time.This group includes quantification of multiple cytokines using cytometric bead arrays, ELISPOT assays, proteomics cytokine evaluation, multiplexed proximity ligation assays for high-throughput cytokine analysis, and finally, cytokine gene expression analysis by gene arrays. The protocols will be written by experienced basic and clinical researchers with hands-on knowledge of the described protocols. By covering a broad variety of methods used in cytokine detection and analysis, this book will be of interest not only to biochemists, molecular biologists and immunologists but also to physician-scientists working in the field of cytokine research.

The Tohoku University Graduate School of Dentistry first introduced the concept of "Interface Oral Health Science", designed to establish and maintain healthy oral cavities, which are home to a number of mixed systems. Included in those systems are: (1) host tissues such as teeth, mucosa, muscle and bone, (2) parasites and microorganisms cohabiting the surfaces of the oral cavity and (3) biomaterials that are used for the rehabilitation of oral functions. In addition, (4) these systems are subject to severe and complex mechanical forces. Therefore, it is critical to promote dental studies that integrate a wide range of interdisciplinary research as medicine, agriculture, material science, engineering, and pharmacology. With this incentive, international symposiums for interface oral health science have been held several times in the past. The concept has since refined and expanded, the result being the "Biosis-Abiosis Intelligent Interface," and projects aiming at the creation of highly functional and autonomic intelligent interfaces are ongoing. This book brings together a number of studies on incentives and projects by leading authors. Topics include biosis-abiosis interface of dental implants, biomaterials in interface science, biomedical engineering interface and cell manipulation and tissue regeneration. Readers not only from the field of dentistry but also many related areas will find this book a valuable resource.

Advances in Applied Microbiology, Volume 123 continues the comprehensive reach of this widely read and authoritative review source in microbiology. Users will find invaluable references and information on a variety of areas relating to the topics of microbiology.

The concept of hormonal regulation using intercellular peptide messengers dates back to the discovery of secretin in 1902. The concept was simple: A peptide is released from specific hormone producing cells, endocrine cells, into circulation upon stimulation of the cells. The peptide hormone travels via blood to its target, the cells of which are equipped with specific receptors for high-affinity binding of the particular peptide hormone. Receptor binding subsequently elicits action of the target cells. This concept has been seriously challenged by modern biochemistry and cell biology. Thus, it is now well established that the gene of a specific peptide hormone may be expressed in different types of endocrine cells, in neurons, and in some instances also in adipocytes, myocytes, osteoblasts, and immune cells. Today, only a few hormones - including the old master hormone insulin - represent the original endocrine paradigm. Instead, the widespread cellular synthesis now raises the qu- tion of how the body maintains the regulation of its functions by peptide hormones when a hormone may originate from a variety of cells.

The discovery of adult neurogenesis caused a paradigm shift in the neurosciences. For more than 100 years, it was believed that adult neurons do not regenerate. Joseph Altman and Fernando Nottebohm found proof to the contrary and changed the course of history. Their research, included here, provides the foundations of the field. Today, adult neurogenesis is a rapidly expanding discipline applicable to the study of brain development and diseases, learning and memory, aging, and neuropsychiatric disorders. With multiple authors, the 27 chapters of this book contain the latest work in two volumes. The first presents the basic biology of adult neurogenesis in non-mammalian vertebrates and in the mammalian hippocampus and olfactory bulb, and the second discusses clinical implications and delves into adult neurogenesis and brain injury as well as neurodegenerative and neuropsychiatric pathologies. With details of the anatomy, physiology, and molecular biology of the two neurogenic brain regions, this book provides indispensable knowledge for many areas of neuroscience and for experimental and clinical applications of adult neurogenesis to brain therapy.

Soil salinity is a key abiotic-stress and poses serious threats to crop yields and quality of produce. Owing to the underlying complexity, conventional breeding programs have met with limited success. Even genetic engineering approaches, via transferring/overexpressing a single 'direct action gene' per event did not yield optimal results. Nevertheless, the biotechnological advents in last decade coupled with the availability of genomic sequences of major crops and model plants have opened new vistas for understanding salinity-responses and improving salinity tolerance in important glycophytic crops. Our goal is to summarize these findings for those who wish to understand and target the molecular mechanisms for producing salt-tolerant and high-yielding crops. Through this 2-volume book series, we critically assess the potential venues for imparting salt stress tolerance to major crops in the post-genomic era. Accordingly, perspectives on improving crop salinity tolerance by targeting the sensory, ion-transport and signaling mechanisms were presented in Volume 1. Volume 2 now focuses on the potency of post-genomic era tools that include RNAi, genomic intervention, genome editing and systems biology approaches for producing salt tolerant crops.