This detailed book contains chapters using unbiased approaches such as chemical and transposon mutagenesis, as well as a protocol for allelic exchange, to make targeted mutants in Staphylococcus, as generating mutants of this bacteria can be accomplished in many ways, depending on the intended changes. Using these methods, the authors have been successful at making mutants that span individual single-nucleotide changes in the chromosome to whole genome mutant libraries. The latter strategy provides a useful tool for high-throughput screening, while single nucleotide changes are an elegant way of teasing apart the importance of single nucleotides in gene expression or to create specific amino acid substitutions to examine protein function. Written for the Methods in Molecular Biology series, 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. Practical and authoritative, The Genetic Manipulation of Staphylococci: Methods and Protocols serves as a vital resource and guide to scientists in the Staphylococcus community as they pursue their studies on these bacteria.

microRNAs (miRNAs) are small non-coding RNAs that regulate various biological phenomena, such as development and homeostasis. The dysregulation of miRNA leads to disease progression, particularly of cancer. In Circulating MicroRNAs: Methods and Protocols, expert researchers in the field detail recent advances in the isolation, purification and analysis of circulating miRNAs from a variety of sources for research. The book is divided into three main topics. The first section involves the study of secretory miRNAs in cell-cell communication, and the second, the study of circulating miRNAs in body fluids. The last describes the novel techniques used to study circulating miRNAs. 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, Circulating MicroRNAs: Methods and Protocols seeks to aid scientists in dealing with the recent advances of RNAi technology from the bench to the bedside.

Long non-coding RNAs (lnc)RNAs have emerged as a new paradigm in epigenetic regulation of the genome. Thousands of lncRNAs have been identified and observed in a wide range of organisms. Unlike mRNA, lncRNA have no protein-coding capacity. So, while their function is not entirely clear, they may serve as key organizers of protein complexes that allow for higher order regulatory events. Discovering these functions has been the result of intense research done of the last few years, and lncRNA research has had several critical developments during that time. This book will consolidate these ideas and models to better examine the most important issues in lncRNA biology. This will include critical studies that have led to the discovery and annotation of lncRNAs in numerous species, and the molecular mechanisms for a few lncRNA that have begun to emerge.

Quorum sensing (QS) describes a chemical communication behavior that is nearly universal among bacteria. Individual cells release a diffusible small molecule (an autoinducer) into their environment. A high concentration of this autoinducer serves as a signal of high population density, triggering new patterns of gene expression throughout the population. However QS is often much more complex than this simple census-taking behavior. Many QS bacteria produce and detect multiple autoinducers, which generate quorum signal cross talk with each other and with other bacterial species. QS gene regulatory networks respond to a range of physiological and environmental inputs in addition to autoinducer signals. While a host of individual QS systems have been characterized in great molecular and chemical detail, quorum communication raises many fundamental quantitative problems which are increasingly attracting the attention of physical scientists and mathematicians. Key questions include: What kinds of information can a bacterium gather about its environment through QS? What physical principles ultimately constrain the efficacy of diffusion-based communication? How do QS regulatory networks maximize information throughput while minimizing undesirable noise and cross talk? How does QS function in complex, spatially structured environments such as biofilms? Previous books and reviews have focused on the microbiology and biochemistry of QS. With contributions by leading scientists and mathematicians working in the field of physical biology, this volume examines the interplay of diffusion and signaling, collective and coupled dynamics of gene regulation, and spatiotemporal QS phenomena. Chapters will describe experimental studies of QS in natural and engineered or microfabricated bacterial environments, as well as modeling of QS on length scales spanning from the molecular to macroscopic. The book aims to educate physical scientists and quantitative-oriented biologists on the application of physics-based experiment and analysis, together with appropriate modeling, in the understanding and interpretation of the pervasive phenomenon of microbial quorum communication.

This volume focuses on modern computational and statistical tools for translational gene expression and regulation research to improve prognosis, diagnostics, prediction of severity, and therapies for human diseases. It introduces some of state of the art technologies as well as computational and statistical tools for translational bioinformatics in the areas of gene transcription and regulation, including the tools for next generation sequencing analyses, alternative spicing, the modeling of signaling pathways, network analyses in predicting disease genes, as well as protein and gene expression data integration in complex human diseases etc. The book is particularly useful for researchers and students in the field of molecular biology, clinical biology and bioinformatics, as well as physicians etc. Dr. Jiaqian Wu is assistant professor in the Vivian L. Smith Department of Neurosurgery and Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Centre, Houston, TX, USA.

New concepts arise in science when apparently unrelated fields of knowledge are put together in a coherent way. The recent results in molecular biology allow to explain the emergence of body patterns in animals that before could not be understood by zoologists. There are no �fancy curiosities� in nature. Every pattern is a product of a molecular cascade originating in genes and a living organism arises from the collaboration of these genes with the outer physical environment. Tropical fishes are as startling in their colors and geometric circles as peacocks. Tortoises are covered with the most regular triangles, squares and concentric circles that can be green, brown or yellow. Parallel scarlet bands are placed side by side of black ones along the body of snakes. Zebras and giraffes have patterns which are lessons in geometry, with their transversal and longitudinal stripes, their circles and other geometric figures. Monkeys, like the mandrills, have a spectacularly colored face scarlet nose with blue parallel flanges and yellow beard. All this geometry turns out to be highly molecular. The genes are many and have been DNA sequenced. Besides they not only deal with the coloration of the body but with the development of the brain and the embryonic process. A precise scenario of molecular events unravels in the vertebrates. It may seem far-fetched, but the search for the origin of this geometry made it mandatory to study the evolution of matter and the origin of the brain. It turned out that matter from its onset is pervaded by geometry and that the brain is also a prisoner of this ordered construction. Moreover, the brain is capable of altering the body geometry and the geometry of the environment changes the brain. Nothing spectacular occurred when the brain arrived in evolution. Not only it came after the eye, which had already established itself long ago, but it had a modest origin. It started from sensory cells on the skin that later aggregated into clusters of neurons that formed ganglia. It also became evident that pigment cells, that decide the establishment of the body pattern, originate from the same cell population as neurons (the neural crest cells). This is a most revealing result because it throws light on the power that the brain has to rapidly redirect the coloration of the body and to change its pattern. Recent experiments demonstrate how the brain changes the body geometry at will and within seconds, an event that could be hardly conceived earlier. Moreover, this change is not accidental it is related to the surrounding environment and is also used as a mating strategy. Chameleons know how to do it as well as flat fishes and octopuses. No one would have dared to think that the brain had its own geometry. How could the external geometry of solids or other figures of our environment be apprehended by neurons if these had no architecture of their own? Astonishing was that the so called �simple cells�, in the neurons of the primary visual cortex, responded to a bar of light with an axis of orientation that corresponded to the axis of the cell’s receptive field. We tend to consider our brain a reliable organ. But how reliable is it? From the beginning the brain is obliged to transform reality. Brain imagery involves: form, color, motion and sleep. Unintentionally these results led to unexpected philosophical implications. Plato’s pivotal concept that �forms� exist independently of the material world is reversed. Atoms have been considered to be imaginary for 2,000 years but at present they can be photographed, one by one, with electron microscopes. The reason why geometry has led the way in this inquiry is due to the fact that where there is geometry there is utter simplicity coupled to rigorous order that underlies the phenomenon where it is recognized. Order allows variation but imposes at the same time a canalization that is patent in what we call evolution.

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.

Having experienced unprecedented growth since the turn of the millennium, the dramatic expansion of resources and techniques in fungal genomics is poised to fundamentally redefine the study of fungal biology. In Fungal Genomics: Methods and Protocols, expert researchers explore the three most likely fronts upon which the field will advance: the sequencing of more and more fungal genomes, the mining of sequenced genomes for useful information, and most importantly, the use of genomics sequences to provide a foundation for powerful techniques to explain biological processes. Much of the book is dedicated to explaining established and emerging genomics-based technologies in filamentous fungi, including gene expression profiling techniques, techniques for fungal proteomics as well as various case studies that could be adapted to a wide range of fungi. Written in the highly successful Methods in Molecular Biology (TM) series format, protocol chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and key unpublished tips, potential pitfalls, common mistakes, and special considerations based on the unique experiences of the contributors. Authoritative and cutting-edge, Fungal Genomics: Methods and Protocols provides fungal biologists at any stage of their careers a user-friendly resource for fungal genomics, especially as readers branch out into unfamiliar but exciting new areas of study.

Lung cancer and autoimmune diseases are complex entities in that they involve gene disturbance, gene polymorphism, and impaired gene repair mechanisms. The volume focuses on altered gene expression in tumor processes and in chronic autoimmune disorders. The chapters discuss the biological rationale for novel disease protein markers, present relevant clinical results, and give some diagnostic and therapeutic tips.

A DNA barcode in its simplest definition is one or more short gene sequences taken from a standardized portion of the genome that is used to identify species through reference to DNA sequence libraries or databases. In DNA Barcodes: Methods and Protocols expert researchers in the field detail many of the methods which are now commonly used with DNA barcodes. These methods include the latest information on techniques for generating, applying, and analyzing DNA barcodes across the Tree of Life including animals, fungi, protists, algae, and plants. 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, DNA Barcodes: Methods and Protocols aids scientists in continuing to study methods from wet-lab protocols, statistical, and ecological analyses along with guides to future, large-scale collections campaigns.

In DNA Electrophoresis: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study DNA using electrophoresis as the major approach. A powerful tool that allows separating DNA molecules according to their size and shape, this volume includes methods and techniques such as 2-dimentional gel electrophoresis as the major approach. These include methods and techniques such as 2-dimentional gel electrophoresis, DNA electrophoresis under conditions in which DNA molecules are completely or partially denatured during the runs, Pulse Field Gel Electrophoresis, electrophoresis coupled to fluorescence in situ hybridization, as well as protein-DNA interactions studied using electrophoreses. 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, DNA Electrophoresis: Methods and Protocols aids scientists in continuing to study DNA dynamics both in live cells and in test tubes.

This edited reflects the current state of knowledge about the role of microRNAs in the formation and progression of solid tumours. The main focus lies on computational methods and applications, together with cutting edge experimental techniques that are used to approach all aspects of microRNA regulation in cancer. We are sure that the emergence of high-throughput quantitative techniques will make this integrative approach absolutely necessary in the near future. This book will be a resource for researchers starting out with cancer microRNA research, but is also intended for the experienced researcher who wants to incorporate concepts and tools from systems biology and bioinformatics into his work. Bioinformaticians and modellers are provided with a general perspective on microRNA biology in cancer, and the state-of-the-art in computational microRNA biology.

Extensive studies have been conducted on the identification, biogenesis, and processing of microRNA (miRNA) as well as research on the exact mechanism by which miRNAs bring about translational silencing of their targets. In addition, numerous publications point to an important role of miRNAs in development, reprogramming, epigenetics, pathogenesis of cancer, oncogenes and tumor suppressors, biomarkers of various disease onset, and regulation of adipogenesis and obesity; yet many questions still remain. In MicroRNA Protocols, Second Edition, experts in the field provide up-to-date coverage of this diverse area of study. The specific chapters of this edition are related to the analysis of miRNA, targets and expression profiling, various methods to determine its regulation of gene expression, the preparation and isolation of miRNAs in specific tissues, its detection in the saliva, and potential application in cosmetics, wound healing, and prostate cancer. 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 known pitfalls. Fully updated and easy to use, MicroRNA Protocols, Second Edition aims to stimulate readers to explore diverse ways to understand the mechanisms in which miRNAs facilitate the molecular aspects of not only biomedical research but also a wide range of other research fields.

Stem Cell therapy for lysosomal diseases (LSDs) is developing rapidly. This volume discusses the history, current practice and future perspectives of stem cells in inborn errors of metabolism (IEM) and provides an international perspective on progress, limitations, and future directions (e.g. gene therapy, iPS, ES) in the field. Beginning with an overview of these diseases, the book covers the breadth of this topic from treatment options, bone marrow transplantation, and alternative treatment options, through long-term outcomes and future perspectives.

Undoubtedly, Drosophila melanogaster, fruit fly, has proved to be one of the most popular invertebrate model organisms, and the work horse for modern day biologists. Drosophila, a highly versatile model with a genetic legacy of more than a century, provides powerful genetic, cellular, biochemical and molecular biology tools to address many questions extending from basic biology to human diseases. One of the most important questions in biology focuses on how does a multi-cellular organism develop from a single-celled embryo. The discovery of the genes responsible for pattern formation has helped refine this question, and led to other questions, such as the role of various genetics and cell biological pathways in regulating the crucial process of pattern formation and growth during organogenesis. Drosophila eye model has been extensively used to study molecular genetic mechanisms involved in patterning and growth. Since the genetic machinery involved in the Drosophila eye is similar to humans, it has been used to model human diseases and homology to eyes in other taxa. This book will discuss molecular genetic mechanisms of pattern formation, mutations in axial patterning, Genetic regulation of growth in Drosophila eye, and more. There have been no titles in the past ten years covering this topic, thus an update is urgently needed.

In the last decade, several different optical imaging techniques, either based on various voltage or calcium dyes, or more recently on modified fluorescent or bioluminescent proteins (genetically encoded) that are sensitive to calcium, have been developed to study neuronal activity, and especially groups of neurons, with the goal of mapping and deciphering the neural code underlying major neurophysiological functions. Genetically Encoded Functional Indicators brings together expert contributors to present the development of recent genetic techniques that allow for generating genetically encoded activity sensors in order to investigate neuronal activity. Each chapter describes a specific sensor and its utilization to study neuronal activity in a particular way. Written in the Neuromethods series style, chapters contain the kind of key description and implementation advice that guarantees successful results. Helpful and easy to use, Genetically Encoded Functional Indicators aims to inspire students and researchers and to serve as a useful guide to those who wish to start using these different brain imaging techniques and require a bit of guidance in how best to choose a technique to match the goal of their study.

Over the last two decades advances in genotyping technology, and the development of quantitative genetic analytical techniques have made it possible to dissect complex traits and link quantitative variation in traits to allelic variation on chromosomes or quantitative trait loci (QTLs). In Quantitative Trait Loci (QTLs):Methods and Protocols, expert researchers in the field detail methods and techniques that focus on specific components of the entire process of quantitative train loci experiments. These include methods and techniques for the mapping populations, identifying quantitative trait loci, extending the power of quantitative trait locus analysis, and case studies. 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, Quantitative Trait Loci (QTLs):Methods and Protocols aids scientists in the further study of the links between phenotypic and genotypic variation in fields from medicine to agriculture, from molecular biology to evolution to ecology.

Together with early theoretical work in population genetics, the debate on sources of genetic makeup initiated by proponents of the neutral theory made a solid contribution to the spectacular growth in statistical methodologies for molecular evolution. Evolutionary Genomics: Statistical and Computational Methods is intended to bring together the more recent developments in the statistical methodology and the challenges that followed as a result of rapidly improving sequencing technologies. Presented by top scientists from a variety of disciplines, the collection includes a wide spectrum of articles encompassing theoretical works and hands-on tutorials, as well as many reviews with key biological insight. Volume 2 begins with phylogenomics and continues with in-depth coverage of natural selection, recombination, and genomic innovation. The remaining chapters treat topics of more recent interest, including population genomics, -omics studies, and computational issues related to the handling of large-scale genomic data. Written in the highly successful Methods in Molecular Biology (TM) series format, this work provides the kind of advice on methodology and implementation that is crucial for getting ahead in genomic data analyses. Comprehensive and cutting-edge, Evolutionary Genomics: Statistical and Computational Methods is a treasure chest of state-of the-art methods to study genomic and omics data, certain to inspire both young and experienced readers to join the interdisciplinary field of evolutionary genomics.

Together with early theoretical work in population genetics, the debate on sources of genetic makeup initiated by proponents of the neutral theory made a solid contribution to the spectacular growth in statistical methodologies for molecular evolution. Evolutionary Genomics: Statistical and Computational Methods is intended to bring together the more recent developments in the statistical methodology and the challenges that followed as a result of rapidly improving sequencing technologies. Presented by top scientists from a variety of disciplines, the collection includes a wide spectrum of articles encompassing theoretical works and hands-on tutorials, as well as many reviews with key biological insight. Volume 1 includes a helpful introductory section of bioinformatician primers followed by detailed chapters detailing genomic data assembly, alignment, and homology inference as well as insights into genome evolution from statistical analyses. Written in the highly successful Methods in Molecular Biology (TM) series format, this work provides the kind of advice on methodology and implementation that is crucial for getting ahead in genomic data analyses. Comprehensive and cutting-edge, Evolutionary Genomics: Statistical and Computational Methods is a treasure chest of state-of the-art methods to study genomic and omics data, certain to inspire both young and experienced readers to join the interdisciplinary field of evolutionary genomics.

Quantitative Real-Time PCR: Methods and Protocols focuses on different applications of qPCR ranging from microbiological detections (both viral and bacterial) to pathological applications. Several chapters deal with quality issues which regard the quality of starting material, the knowledge of the minimal information required to both perform an assay and to set the experimental plan, while the others focus on translational medicine applications that are ordered following an approximate logical order of their medical application. The last part of the book gives you an idea of an emerging digital PCR technique that is a unique qPCR approach for measuring nucleic acid, particularly suited for low level detection and to develop non-invasive diagnosis. Written for the Methods in Molecular Biology series, most chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Practical and authoritative, Quantitative Real-Time PCR: Methods and Protocols aims to aid researchers seeking to devise new qPCR-based approaches related to his or her area of investigation.

Significant advancements have been made in the study of chromatin structure and function over the past fifty years but none as spectacular as those made in the last decade due to the development of novel techniques and the ability to sequence large stretches of DNA. In Chromatin Protocols, Second Edition, expert researchers delineate these cutting-edge techniques via step-by-step laboratory methods and protocols, which encompass a wide array of topics from the isolation of nucleosomes, assembly of nucleosomes and study of the basic chromatin structure to detailed analysis of histone modifications and chromatin function. Written in the highly successful Methods in Molecular Biology (TM) series style, chapters include brief introductions to the subjects, lists of the necessary materials and reagents, readily reproducible protocols, and Notes sections which highlight tips on troubleshooting and avoiding known pitfalls. Comprehensive and up-to-date, Chromatin Protocols, Second Edition is a valuable tool for scientists studying various aspects of chromatin function and an ideal guide to aid in the development of new techniques as well as new ideas in the field of chromatin biology.

Pancreatic ductal adenocarcinoma is the fifth leading cause of cancer death in the USA. Pancreatic cancer develops as a result of the accumulation of genetic and epigenetic alterations in cancer-causing genes such as oncogenes and tumor-suppressor genes. The second edition of Pancreatic Cancer: Methods and Protocols provides a broad range of protocols for molecular, cellular, pathological, and statistical analyses of sporadic and familial pancreatic cancer. It covers topics from in-vitro cell cultures to in-vivo mouse models, DNA to protein manipulation, and genetic and epigenetic analyses to treatment development. 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, written by highly renowned investigators with expertise in pancreatic cancer, Pancreatic Cancer: Methods and Protocols, Second Edition is an invaluable source of proven protocols to those who are interested in joining the fight against pancreatic cancer.

The ribosome is a macromolecular machine that synthesizes proteins with a high degree of speed and accuracy. Our present understanding of its structure, function and dynamics is the result of six decades of research. This book collects over 40 articles based on the talks presented at the 2010 Ribosome Meeting, held in Orvieto, Italy, covering all facets of the structure and function of the ribosome. New high-resolution crystal structures of functional ribosome complexes and cryo-EM structures of translating ribosomes are presented, while partial reactions of translation are examined in structural and mechanistic detail, featuring translocation as a most dynamic process. Mechanisms of initiation, both in bacterial and eukaryotic systems, translation termination, and novel details of the functions of the respective factors are described. Structure and interactions of the nascent peptide within, and emerging from, the ribosomal peptide exit tunnel are addressed in several articles. Structural and single-molecule studies reveal a picture of the ribosome exhibiting the energy landscape of a processive Brownian machine. The collection provides up-to-date reviews which will serve as a source of essential information for years to come.

The generation of genetically modified mice is absolutely crucial to gene function studies today, primarily because mice are genetically similar to man and because gene function studies in mice are in the context of a whole organism, making them particularly useful. In Transgenic Mouse Methods and Protocols, Second Edition, expert research explore current advances in the field through detailed laboratory protocols. Chapters provide a general introduction outlining how to deal with mice and how to generate transgenic mouse models, explore the generation of conditional and induced knockout and transgenic mice, and offer alternative routes to studying gene function in mice. Composed in the highly successful Methods in Molecular Biology (TM) series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls. Comprehensive and state of the art, Transgenic Mouse Methods and Protocols, second Edition is the ideal guide for all researchers interested in the latest information about the production and analysis of transgenic and knockout mice.

Despite the best efforts of many and despite landmark discoveries and experimental ingenuity, challenges in the pursuit of research related to olfactory receptors (ORs) continue to exist. In Olfactory Receptors: Methods and Protocols, experts in the field contribute chapters that serve to address these challenges. The volume consists of several sections: knowledge dissemination of ORs, theoretical assessments of OR structure and function, as well as development and use of expression systems and experimental functional analysis. 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. Concise and practical, Olfactory Receptors: Methods and Protocols seeks to aid researchers in furthering the knowledge of olfaction and moving us ever closer to the thrilling discoveries that will follow.