Download or read book Microfluidic Fractionation and Analysis of Cytoplasmic Versus Nuclear Acids in Single Cells written by Denitsa Milanova and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell gene expression studies have matured and are now widely used to uncover cell-to-cell variability, transcript processing and inhomogeneous response to external stimuli. Despite great progress, significant challenges remain including understanding fundamental questions of human genome transcription, gene expression ranges, localization, and processing destinies of RNAs. Eukaryotic cells make different types of primary and processed transcripts, which are either exclusively found in particular sub-cellular compartments or dispersed throughout the whole cell. These sub-cellular localizations of RNAs are weakly understood at a single-cell level, and are important for fully realizing gene functions. Understanding sub-cellular transcript localization is particularly important for studying the process of splicing, a pathway during which introns of pre-messenger RNA's are excised and exons are stitched together to form mature mRNA transcript. We here introduce a single-cell-isotachophoresis (sc-ITP) method to analyze co-transcriptional and alternative splicing in reference lymphoblastoid cell line (LCL-Snyder), and two sub-lines of CML cells (K562-ATCC and K562-ENCODE). The method is unique in that we physically separate the contents of cell nucleus from those of the cytoplasm and analyze independently. It allows for rapid, electric-field-based selective lysis of cytoplasmic membrane (leaving nucleus intact); and then purification and simultaneous fractionation of total RNA in cytosol (cyt-RNA) and total RNA in the nucleus (nuc-RNA) from single cells with no intra-compartment cross-contamination. We then use our system and state-of-the-art NGS technologies to perform single-cell nuclear and cytoplasmic RNA-seq to study fundamental questions of human genome transcription, differential gene expression, localization, and processing destinies of RNAs for various cell types, disease, and differentiation states. We evaluate the distribution of whole transcriptome gene features and gene expression of precursor and processed transcripts and find that there are considerable differences in expression levels across sub-cellular compartments and among individual cells. We then evaluate cell-to-cell variability of the highly expressed GAPDH housekeeping gene in alternative splicing between the lymphoblastoid and leukaemia cells using sequencing-specific probes and RT-qPCR. The data suggest evidence of significant distinction in splicing patterns. By analyzing nuclear and cytoplasmic compartments of a single cell individually at a whole transcriptome level, we achieve an unprecedented precision in splicing quantification. Together, our results describe an experimental method for single-cell fractionation and an analytical tool for gene and isoform expression analysis in rare cell types, cell differentiation and disease states.

Download or read book Single Cell Assays written by Paul C.H. Li and published by Springer Nature. This book was released on 2023-07-10 with total page 227 pages. Available in PDF, EPUB and Kindle. Book excerpt: This detailed volume explores the use of single-cell assays in research for drug discovery, microfluidics, and more. The book delves into methodologies involving a variety of cell types and diseases, small molecules and biologics, as well as studies of the genome and transciptome. 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 and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Single-Cell Assays: Microfluidics, Genomics, and Drug Discovery serves to enable researchers to obtain a rapid overview in state-of-the-art microfluidic single-cell assays and an impression of what possibilities these assays offer to drug discovery.

Download or read book Enzyme Activity in Single Cells written by and published by Academic Press. This book was released on 2019-10-31 with total page 328 pages. Available in PDF, EPUB and Kindle. Book excerpt: Enzyme Activity in Single Cells, Volume 628, the latest release in the Methods of Enzymology series, discusses groundbreaking cellular physiology research that is taking place in the biological sciences. Chapters in this new release cover Spatial and temporal resolution of caspase waves in single Xenopus eggs during apoptosis, Spatial and temporal organization of metabolic complexes in cells, Measuring cellular efflux and biomolecular delivery: synthetic approaches to imaging and engineering cells, Slide-based, single-cell enzyme assays, Single-cell assays using integrated continuous-flow microfluidics, High-throughput screening of single-cell lysates, Microfluidic capture of single cells for drug resistance assays, and much more. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Methods in Enzymology series - Includes the latest information on Enzyme Activity in Single Cells

Download or read book Microfluidics for Single Cell Analysis written by Jin-Ming Lin and published by Springer Nature. This book was released on 2019-08-28 with total page 263 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book summarizes the various microfluidic-based approaches for single-cell capture, isolation, manipulation, culture and observation, lysis, and analysis. Single-cell analysis reveals the heterogeneities in morphology, functions, composition, and genetic performance of seemingly identical cells, and advances in single-cell analysis can overcome the difficulties arising due to cell heterogeneity in the diagnostics for a targeted model of disease. This book provides a detailed review of the state-of-the-art techniques presenting the pros and cons of each of these methods. It also offers lessons learned and tips from front-line investigators to help researchers overcome bottlenecks in their own studies. Highlighting a number of techniques, such as microfluidic droplet techniques, combined microfluidics-mass-spectrometry systems, and nanochannel sampling, it describes in detail a new microfluidic chip-based live single-cell extractor (LSCE) developed in the editor’s laboratory, which opens up new avenues to use open microfluidics in single-cell extraction, single-cell mass spectrometric analysis, single-cell adhesion analysis and subcellular operations. Serving as both an elementary introduction and advanced guidebook, this book interests and inspires scholars and students who are currently studying or wish to study microfluidics-based cell analysis methods.

Download or read book The Individual Microbe Single Cell Analysis and Agent Based Modelling written by Johan H. J. Leveau and published by Frontiers Media SA. This book was released on 2019-02-19 with total page 187 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent technological advances in single-cell microbiology, using flow cytometry, microfluidics, x-ray fluorescence microprobes, and single-cell -omics, allow for the observation of individuals within populations. Simultaneously, individual-based models (or more generally agent-based models) allow for individual microbes to be simulated. Bridging these techniques forms the foundation of individual-based ecology of microbes (µIBE). µIBE has elucidated genetic and phenotypic heterogeneity that has important consequences for a number of human interests, including antibiotic or biocide resistance, the productivity and stability of industrial fermentations, the efficacy of food preservatives, and the potential of pathogens to cause disease. Individual-based models can help us to understand how these sets of traits of individual microbes influence the above. This eBook compiles all publications from a recent Research Topic in Frontiers in Microbiology. It features recent research where individual observational and/or modelling techniques are applied to gain unique insights into the ecology of microorganisms. The Research Topic “The Individual Microbe: Single-Cell Analysis and Agent-Based Modelling” arose from the 2016 @ASM conference of the same name hosted by the American Society for Microbiology at its headquarters in Washington, D.C. We are grateful to ASM for funding and hosting this conference.

Download or read book Flow Cytometry and Cell Sorting written by Andreas Radbruch and published by Springer Science & Business Media. This book was released on 2013-03-14 with total page 365 pages. Available in PDF, EPUB and Kindle. Book excerpt: The analysis and sorting of large numbers of cells with a fluorescence-activated cell sorter (FACS) was first achieved some 30 years ago. Since then, this technology has been rapidly developed and is used today in many laboratories. A Springer Lab Manual Review of the First Edition: "This is a most useful volume which will be a welcome addition for personal use and also for laboratories in a wide range of disciplines. Highly recommended." CYTOBIOS

Download or read book Introduction to Single Cell Omics written by Xinghua Pan and published by Frontiers Media SA. This book was released on 2019-09-19 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell omics is a progressing frontier that stems from the sequencing of the human genome and the development of omics technologies, particularly genomics, transcriptomics, epigenomics and proteomics, but the sensitivity is now improved to single-cell level. The new generation of methodologies, especially the next generation sequencing (NGS) technology, plays a leading role in genomics related fields; however, the conventional techniques of omics require number of cells to be large, usually on the order of millions of cells, which is hardly accessible in some cases. More importantly, harnessing the power of omics technologies and applying those at the single-cell level are crucial since every cell is specific and unique, and almost every cell population in every systems, derived in either vivo or in vitro, is heterogeneous. Deciphering the heterogeneity of the cell population hence becomes critical for recognizing the mechanism and significance of the system. However, without an extensive examination of individual cells, a massive analysis of cell population would only give an average output of the cells, but neglect the differences among cells. Single-cell omics seeks to study a number of individual cells in parallel for their different dimensions of molecular profile on genome-wide scale, providing unprecedented resolution for the interpretation of both the structure and function of an organ, tissue or other system, as well as the interaction (and communication) and dynamics of single cells or subpopulations of cells and their lineages. Importantly single-cell omics enables the identification of a minor subpopulation of cells that may play a critical role in biological process over a dominant subpolulation such as a cancer and a developing organ. It provides an ultra-sensitive tool for us to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. Besides, it also empowers the clinical investigation of patients when facing a very low quantity of cell available for analysis, such as noninvasive cancer screening with circulating tumor cells (CTC), noninvasive prenatal diagnostics (NIPD) and preimplantation genetic test (PGT) for in vitro fertilization. Single-cell omics greatly promotes the understanding of life at a more fundamental level, bring vast applications in medicine. Accordingly, single-cell omics is also called as single-cell analysis or single-cell biology. Within only a couple of years, single-cell omics, especially transcriptomic sequencing (scRNA-seq), whole genome and exome sequencing (scWGS, scWES), has become robust and broadly accessible. Besides the existing technologies, recently, multiplexing barcode design and combinatorial indexing technology, in combination with microfluidic platform exampled by Drop-seq, or even being independent of microfluidic platform but using a regular PCR-plate, enable us a greater capacity of single cell analysis, switching from one single cell to thousands of single cells in a single test. The unique molecular identifiers (UMIs) allow the amplification bias among the original molecules to be corrected faithfully, resulting in a reliable quantitative measurement of omics in single cells. Of late, a variety of single-cell epigenomics analyses are becoming sophisticated, particularly single cell chromatin accessibility (scATAC-seq) and CpG methylation profiling (scBS-seq, scRRBS-seq). High resolution single molecular Fluorescence in situ hybridization (smFISH) and its revolutionary versions (ex. seqFISH, MERFISH, and so on), in addition to the spatial transcriptome sequencing, make the native relationship of the individual cells of a tissue to be in 3D or 4D format visually and quantitatively clarified. On the other hand, CRISPR/cas9 editing-based In vivo lineage tracing methods enable dynamic profile of a whole developmental process to be accurately displayed. Multi-omics analysis facilitates the study of multi-dimensional regulation and relationship of different elements of the central dogma in a single cell, as well as permitting a clear dissection of the complicated omics heterogeneity of a system. Last but not the least, the technology, biological noise, sequence dropout, and batch effect bring a huge challenge to the bioinformatics of single cell omics. While significant progress in the data analysis has been made since then, revolutionary theory and algorithm logics for single cell omics are expected. Indeed, single-cell analysis exert considerable impacts on the fields of biological studies, particularly cancers, neuron and neural system, stem cells, embryo development and immune system; other than that, it also tremendously motivates pharmaceutic RD, clinical diagnosis and monitoring, as well as precision medicine. This book hereby summarizes the recent developments and general considerations of single-cell analysis, with a detailed presentation on selected technologies and applications. Starting with the experimental design on single-cell omics, the book then emphasizes the consideration on heterogeneity of cancer and other systems. It also gives an introduction of the basic methods and key facts for bioinformatics analysis. Secondary, this book provides a summary of two types of popular technologies, the fundamental tools on single-cell isolation, and the developments of single cell multi-omics, followed by descriptions of FISH technologies, though other popular technologies are not covered here due to the fact that they are intensively described here and there recently. Finally, the book illustrates an elastomer-based integrated fluidic circuit that allows a connection between single cell functional studies combining stimulation, response, imaging and measurement, and corresponding single cell sequencing. This is a model system for single cell functional genomics. In addition, it reports a pipeline for single-cell proteomics with an analysis of the early development of Xenopus embryo, a single-cell qRT-PCR application that defined the subpopulations related to cell cycling, and a new method for synergistic assembly of single cell genome with sequencing of amplification product by phi29 DNA polymerase. Due to the tremendous progresses of single-cell omics in recent years, the topics covered here are incomplete, but each individual topic is excellently addressed, significantly interesting and beneficial to scientists working in or affiliated with this field.

Download or read book Alternative Splicing and Disease written by Philippe Jeanteur and published by Springer. This book was released on 2010-11-30 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Splicing of primary RNA transcript is a quasi-systematic step of gene expression in higher organisms. This is the first book to highlight the medical implications, i.e. diseases, caused by alternative splicing. Alternative splicing not only vastly increases protein diversity but also offers numerous opportunities for aberrant splicing events with pathological consequences. The book also outlines possible targets for therapy.

Download or read book Isotachophoresis written by F.M. Everaerts and published by Elsevier. This book was released on 2011-09-22 with total page 433 pages. Available in PDF, EPUB and Kindle. Book excerpt: Isotachophoresis

Download or read book Gastrulation From Embryonic Pattern to Form written by and published by Academic Press. This book was released on 2020-01-18 with total page 480 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gastrulation: From Embryonic Pattern to Form Volume 136 in the Current Topics in Developmental Biology series highlights new advances in the field, with this new volume presenting interesting chapters on D. melanogaster, Zebrafish, Chick, Mouse and Human, C. elegans, D. melanogaster Internalization, Sea urchin, Ascidians, Xenopus Internalization, Xenopus Convergent Extension, Zebrafish Epiboly, Zebrafish Internalization, Zebrafish Convergence and Extension, Chick Primitive streak formation and mesendoderm internalization, Octavian Voiculescu, Mouse Primitive streak formation and internalization, Mouse Definitive endoderm morphogenesis, Conservation of movements, and more. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Current Topics in Developmental Biology series - Includes the latest information on gastrulation from embryonic pattern to form

Download or read book Microsystems for Pharmatechnology written by Andreas Dietzel and published by Springer. This book was released on 2016-01-22 with total page 353 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a comprehensive, state-of-the-art review of microfluidic approaches and applications in pharmatechnology. It is appropriate for students with an interdisciplinary interest in both the pharmaceutical and engineering fields, as well as process developers and scientists in the pharmaceutical industry. The authors cover new and advanced technologies for screening, production by micro reaction technology and micro bioreactors, small-scale processing of drug formulations, and drug delivery that will meet the need for fast and effective screening methods for drugs in different formulations, as well as the production of drugs in very small volumes. Readers will find detailed chapters on the materials and techniques for fabrication of microfluidic devices, microbioreactors, microsystems for emulsification, on-chip fabrication of drug delivery systems, respiratory drug delivery and delivery through microneedles, organs-on-chip, and more.

Download or read book Andrology written by Eberhard Nieschlag and published by Springer Science & Business Media. This book was released on 2010-01-13 with total page 630 pages. Available in PDF, EPUB and Kindle. Book excerpt: The decade that has passed since publication of the second edition of this textbook has not only witnessed a tremendous increase in knowledge within the ? eld of and- logy, but also seen the ? eld itself achieve a newfound status within the medical p- fession. Knowledge and status have been of mutual bene? t to the ? eld and the growing critical mass of diagnostic and therapeutic possibilities have caused andrology to be recognized as a medical subspecialty in some countries such as Germany, Poland, and Estonia. The European Academy of Andrology (EAA) served as a pacemaker for this development and continues to strive for establishment of andrology as a clinical ? eld. Well-designed curricula and qualifying examinations have contributed to the of? cial recognition of andrology as a speciality. This recognition of the ? eld helps patients with andrological problems to ? nd the specialist they seek. This textbook summarizes the current state of knowledge in the ? eld of andrology. It is a source of knowledge to all those who are or want to become andrologists. In addition, as andrology is clearly an interdisciplinary ? eld, this book may serve as a compendium and source of reference for all those physicians and biologists active in neighboring areas, who want to obtain an overview of andrology and who require information on special problems. The extensive references are timely and up to date.

Download or read book Micro Nano Devices for Blood Analysis written by Rui A. Lima and published by MDPI. This book was released on 2019-12-03 with total page 174 pages. Available in PDF, EPUB and Kindle. Book excerpt: The development of micro- and nanodevices for blood analysis is an interdisciplinary subject that demands the integration of several research fields, such as biotechnology, medicine, chemistry, informatics, optics, electronics, mechanics, and micro/nanotechnologies. Over the last few decades, there has been a notably fast development in the miniaturization of mechanical microdevices, later known as microelectromechanical systems (MEMS), which combine electrical and mechanical components at a microscale level. The integration of microflow and optical components in MEMS microdevices, as well as the development of micropumps and microvalves, have promoted the interest of several research fields dealing with fluid flow and transport phenomena happening in microscale devices. Microfluidic systems have many advantages over their macroscale counterparts, offering the ability to work with small sample volumes, providing good manipulation and control of samples, decreasing reaction times, and allowing parallel operations in one single step. As a consequence, microdevices offer great potential for the development of portable and point-of-care diagnostic devices, particularly for blood analysis. Moreover, the recent progress in nanotechnology has contributed to its increasing popularity, and has expanded the areas of application of microfluidic devices, including in the manipulation and analysis of flows on the scale of DNA, proteins, and nanoparticles (nanoflows). In this Special Issue, we invited contributions (original research papers, review articles, and brief communications) that focus on the latest advances and challenges in micro- and nanodevices for diagnostics and blood analysis, micro- and nanofluidics, technologies for flow visualization, MEMS, biochips, and lab-on-a-chip devices and their application to research and industry. We hope to provide an opportunity to the engineering and biomedical community to exchange knowledge and information and to bring together researchers who are interested in the general field of MEMS and micro/nanofluidics and, especially, in its applications to biomedical areas.

Download or read book Nematode Models of Development and Disease written by and published by Academic Press. This book was released on 2021-05-13 with total page 452 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nematode Models of Development and Disease, Volume 144 in the Current Topics in Developmental Biology series highlights new advances in the field, with this new volume presenting interesting chapters surrounding Transgenerational inheritance, Oscillatory expression and function, Concepts and functions of small RNA pathways in C. elegans, Exploring the nuclear lamina in health and pathology using C. elegans, Cellular Plasticity, Morphogenesis, Tubulogenesis, Organogenesis forces, Programmed cell fusion in development and homeostasis, One template, two outcomes: how does the sex-shared nervous system generate sex-specific behaviors?, Metabolic Cellular Coordination of Gene-Environment Interactions, and much more. Other chapters cover Chemical and physical cues and micro-evolution in early embryogenesis, Innate immunity, Sex and Death, Dendrites maturation, axonal growth and extracellular glycoproteins, Autophagocytosis, Spermatogenesis, and the developmental and physiological roles of phagocytosis in Caenorhabditis elegans. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Current Topics in Developmental Biology

Download or read book Molecular Epidemiology written by Paul A. Schulte and published by Academic Press. This book was released on 2012-12-02 with total page 609 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book will serve as a primer for both laboratory and field scientists who are shaping the emerging field of molecular epidemiology. Molecular epidemiology utilizes the same paradigm as traditional epidemiology but uses biological markers to identify exposure, disease or susceptibility. Schulte and Perera present the epidemiologic methods pertinent to biological markers. The book is also designed to enumerate the considerations necessary for valid field research and provide a resource on the salient and subtle features of biological indicators.

Download or read book Cancer Biology and the Nuclear Envelope written by Eric C. Schirmer and published by Springer Science & Business Media. This book was released on 2014-02-22 with total page 610 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Nuclear envelope (NE) defects have been linked to cancer biology since the mid-1800s, but it was not until the last few years that we have begun to understand these historical links and to realize that there are myriad ways that the NE impacts on tumorigenesis. The NE is a complex double membrane system that encloses the genome while providing structural support through the intermediate filament lamin polymer and regulating protein/ mRNA trafficking and signaling between the nucleus and cytoplasm via the nuclear pore complexes (NPCs). These functions already provide some mechanisms for NE influences on cancer biology but work in the past few years has elucidated many others. Lamins and many recently identified NE transmembrane proteins (NETs) have been now shown to function in DNA repair, regulation of cell cycle and signaling, apoptosis, cell migration in metastasis and nuclear architecture and morphology. This volume presents a comprehensive overview of the wide range of functions recently identified for NE proteins and their relevance in cancer biology, providing molecular mechanisms and evidence of their value as prognostic and diagnostic markers and suggesting new avenues for the treatment of cancer. Indeed some of these recent links are already yielding promising therapies, such as the current clinical trial of selective inhibitors of the nuclear export factor exportin in certain types of leukemia, melanoma and kidney cancer."

Download or read book The Perfect Slime written by Hans-Curt Flemming and published by IWA Publishing. This book was released on 2016-09-15 with total page 336 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Perfect Slime presents the latest state of knowledge and all aspects of the Extracellular Polymeric Substances, (EPS) matrix – from the ecological and health to the antifouling perspectives. The book brings together all the current material in order to expand our understanding of the functions, properties and characteristics of the matrix as well as the possibilities to strengthen or weaken it. The EPS matrix represents the immediate environment in which biofilm organisms live. From their point of view, this matrix has paramount advantages. It allows them to stay together for extended periods and form synergistic microconsortia, it retains extracellular enzymes and turns the matrix into an external digestion system and it is a universal recycling yard, it protects them against desiccation, it allows for intense communication and represents a huge genetic archive. They can remodel their matrix, break free and eventually, they can use it as a nutrient source. The EPS matrix can be considered as one of the emergent properties of biofilms and are a major reason for the success of this form of life. Nevertheless, they have been termed the “black matter of biofilms” for good reasons. First of all: the isolation methods define the results. In most cases, only water soluble EPS components are investigated; insoluble ones such as cellulose or amyloids are much less included. In particular in environmental biofilms with many species, it is difficult to impossible isolate, separate the various EPS molecules they are encased in and to define which species produced which EPS. The regulation and the factors which trigger or inhibit EPS production are still very poorly understood. Furthermore: bacteria are not the only microorganisms to produce EPS. Archaea, Fungi and algae can also form EPS. This book investigates the questions, What is their composition, function, dynamics and regulation? What do they all have in common?