Methods in molecular biology (Clifton, N.J.) (Meth Mol Biol)

Publisher: Humana Press

Current impact factor: 1.29

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Other titles Methods in molecular biology (Clifton, N.J.), Methods in molecular biology
ISSN 1940-6029
OCLC 24839341
Material type Series
Document type Journal / Magazine / Newspaper

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Humana Press

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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: With its ability to perform rapid transcriptome profiling and profound transcriptomic analysis powered by high-throughput sequencing at a high resolution with deep coverage, the advent of RNA sequencing technology, RNA-Seq, outperforms other methods in the field, such as microarrays, and has changed our way of performing transcriptomic investigation. Protocols for preparing libraries for RNA-Seq using the Illumina and Roche 454 sequencing platforms are included in this chapter. Common steps for library preparation in both platforms include RNA fragmentation, cDNA synthesis, adaptor ligation, and PCR amplification of cDNA strands. Illumina adopts solid-phase bridge PCR amplification, while 454 uses water-in-oil emulsion-based PCR amplification. Despite differences in the PCR amplification step, both platforms employ the same sequencing-by-synthesis technology for the sequencing process. Application of the RNA-Seq technique in the context of dysregulation of the transcriptome in Alzheimer's disease is also discussed.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:327-47. DOI:10.1007/978-1-4939-2627-5_20
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    ABSTRACT: In the past 5 years, imaging network properties in the brain of patients with Alzheimer's disease (AD) has revolutionized our understanding of this disorder. Postmortem data had already suggested that the damage spreads along functional neural networks, but postmortem studies do not provide information on the temporal evolution of the damage in the same patient, essential to determine spreading. These data can be provided by functional and structural neuroimaging, which allow for the visualization over time of the progressive damage inflicted by AD. Functional networks can be mapped by determining the synchrony across brain regions of the blood oxygenation level dependence (BOLD) signal on functional magnetic resonance imaging (MRI) during quiet wakefulness. Other less extensively used techniques are also useful. For instance, amyloid deposition can be imaged and its progression mapped to determine whether it follows brain networks, and, if so, which are affected earliest. Network patterns of neurobiological changes, including tau deposition, may prove critical to our understanding of the neurobiology of AD and therefore open the way for therapeutic interventions.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:509-19. DOI:10.1007/978-1-4939-2627-5_31
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    ABSTRACT: To produce synchronized cell colonies, many cell cycle synchronization technologies have been developed, among which the baby machine may be considered the most artifact-free. Baby machines incubate "mother cells" under normal conditions and collects their "babies," producing cell cultures that are similar not only in cell cycle phase but also in age. Several macroscale and microfluidic baby machines have been applied to synchronized cell research. However, for rod-shaped cells like fission yeast (Schizosaccharomyces pombe), it is still a challenge to immobilize only the mother cells in a microfluidic device. Here, we present a new baby machine suitable for fission yeast. The device fixes one end of the cell and releases the free-end daughter cell every time the cell finishes cytokinesis. A variety of structures for cell immobilization were attempted to find the optimal design. For the convenience of collection and to enable further assays, we integrated a cell screener into the baby machine, which exploits the deformation of polymer material to switch between open and closed states. The device, producing synchronous populations of fission yeast cells, provides a new on-chip tool for cell biology studies.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1342:259-68. DOI:10.1007/978-1-4939-2957-3_15
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    ABSTRACT: The Signaling and Dynamic Regulatory Events Miner (SDREM) is a powerful computational approach for identifying which signaling pathways and transcription factors control the temporal cellular response to a stimulus. SDREM builds end-to-end response models by combining condition-independent protein-protein interactions and transcription factor binding data with two types of condition-specific data: source proteins that detect the stimulus and changes in gene expression over time. Here we describe how to apply SDREM to study human diseases, using epidermal growth factor (EGF) response impacting neurogenesis and Alzheimer's disease as an example.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:493-506. DOI:10.1007/978-1-4939-2627-5_30
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    ABSTRACT: The cause of Alzheimer disease (AD) is not well understood and there is no cure. Our ability to understand the early events in the course of AD is severely limited by the difficulty of identifying individuals who are in the early, preclinical stage of this disease. Most individuals with Down's syndrome (DS, trisomy 21) will predictably develop AD and that they will do so at a young age makes them an ideal population in which to study the early stages of AD. Several recent studies have exploited induced pluripotent stem cells (iPSCs) generated from individuals with familial AD, spontaneous AD and DS to attempt to identify early events and discover novel biomarkers of disease progression in AD. Here, we summarize the progress and limitations of these iPSC studies with a focus on iPSC-derived neurons. Further, we outline the methodology and results for comparing gene expression between AD and DS iPSC-derived neurons. We highlight differences and commonalities in these data that may implicate underlying genes and pathways that are causative for AD.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:247-65. DOI:10.1007/978-1-4939-2627-5_15
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    ABSTRACT: The autophagosome-associated protein LC3-II is commonly used as a marker of autophagic activity within cells, but its levels are affected by both formation and degradation of autophagosomes. This can make the significance of altered LC3-II levels ambiguous. Here we describe the method of Bafilomycin A1 blotting, in which the degradation of autophagosomes is prevented in cultured cells, allowing the causes of altered LC3-II levels to be determined.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:161-70. DOI:10.1007/978-1-4939-2627-5_8
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    ABSTRACT: Systems biology has shown its potential in facilitating pathway-focused therapy development for central nervous system (CNS) diseases. An integrated network can be utilized to explore the multiple disease mechanisms and to discover repositioning opportunities. This review covers current therapeutic gaps for CNS diseases and the role of systems biology in pharmaceutical industry. We conclude with a Multiple Level Network Modeling (MLNM) example to illustrate the great potential of systems biology for CNS diseases. The system focuses on the benefit and practical applications in pathway centric therapy and drug repositioning.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:531-47. DOI:10.1007/978-1-4939-2627-5_33
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    ABSTRACT: T lymphocytes (T cells) are essential for proper adaptive immune responses. They perform a variety of functions in defenses against pathogens, and notably control, positively or negatively, other cells involved in immune responses. T cells develop in the thymus from bone marrow-derived precursors. These precursors (thymocytes) proliferate, rearrange the genes encoding subunits of the T cell antigen receptor, which endow them with their unique antigen specificity, and undergo various degrees of pre-programming for their functions in immune responses. Thus, analyzing T cell development in the thymus is essential for understanding their functions in immune responses. In addition, the thymus constitutes an attractive experimental model to analyze mechanisms of cell proliferation, differentiation and survival, all of which are involved in thymocyte development. This chapter presents a quick overview of the key events characterizing intrathymic T cell development, as an introduction for readers entering this field of study.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1323:3-21. DOI:10.1007/978-1-4939-2809-5_1
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    ABSTRACT: From the moment a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its ultimate fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. The identification of signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and the molecular associations of numerous proteins within the developing T cell. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1323:169-78. DOI:10.1007/978-1-4939-2809-5_15
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    ABSTRACT: Tightly controlled degradation of specific regulatory proteins is crucial for transitioning to the next cell cycle phase, ensuring precise DNA replication and an equal distribution of chromosomes to provide genomic stability and avoid tumorigenesis. To study mitotic control at the metaphase-to-anaphase transition, a histone H2-GFP-based reporter system was established, allowing simultaneous monitoring of the alignment of mitotic chromosomes and cyclin B proteolysis. To depict the proteolytic profile, a chimeric cyclin B-SNAP reporter molecule that can be labeled with a fluorochrome-carrying SNAP substrate was generated for measurement of the decline in fluorescence intensity via live-cell imaging. This reporter system can be adapted for other cell cycle oscillatory proteins.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1342:201-8. DOI:10.1007/978-1-4939-2957-3_11
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    ABSTRACT: Recreating the thymic microenvironment in vitro poses a great challenge to immunologists. Until recently, the only approach was to utilize the thymic tissue in its three-dimensional form and to transfer the hematopoietic progenitors into this tissue to generate de novo T cells. With the advent of OP9-DL cells (bone marrow-derived cells that are transduced to express Notch ligand, Delta-like), hematopoietic stem cells (HSC) could be induced to differentiate into T cells in culture for the first time outside of the thymic tissue on a monolayer. We, as well as others, asked whether the ability to support T cell development in vitro in a monolayer is unique to BM-derived OP9 cells, and showed that provision of Delta-like expression to thymic epithelial cells and fibroblasts also allowed for T cell development. This provides the opportunity to design an autologous coculture system where the supportive stromal and the hematopoietic components are both derived from the same individual, which has obvious clinical implications. In this chapter, we describe methods for establishing a primary murine dermal fibroblast cell population that is transduced to express Delta-like 4, and describe the conditions for its coculture with HSCs to support T cell lineage initiation and expansion, while comparing it to the now classic OP9-DL coculture.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1323:159-67. DOI:10.1007/978-1-4939-2809-5_14
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    ABSTRACT: The 99-amino-acid-long APP-carboxy-terminal fragment, named C99, is a membrane-bound peptide generated from the amyloid precursor protein (APP) by β-secretase cleavage and is the direct precursor of amyloid beta (Aβ). Here we describe a method for the quantification of C99. The amount of C99 is an indicative value of the amyloid pathology in an Alzheimer's disease (AD) model, and could be used as a marker to study AD progression in comprehensive experiments, including screening for new compounds and repurposing of drugs to treat AD.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:117-23. DOI:10.1007/978-1-4939-2627-5_5
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    ABSTRACT: The cell cycle is the sequence of events through which a cell duplicates its genome, grows, and divides. Key cell cycle transitions are driven by oscillators comprising cyclin-dependent kinases and other kinases. Different cell cycle oscillators are inextricably linked to ensure orderly activation of oscillators. A recurring theme in their regulation is the abundance of auto-amplifying loops that ensure switch-like and unidirectional cell cycle transitions. The periodicity of many cell cycle oscillators is choreographed by inherent mechanisms that promote automatic inactivation, often involving dephosphorylation and ubiquitin-mediated protein degradation. These inhibitory signals are subsequently suppressed to enable the next cell cycle to occur. Although the activation and inactivation of cell cycle oscillators are in essence autonomous during the unperturbed cell cycle, a number of checkpoint mechanisms are able to halt the cell cycle until defects are addressed. Together, these mechanisms orchestrate orderly progression of the cell cycle to produce more cells and to safeguard genome integrity.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1342:3-19. DOI:10.1007/978-1-4939-2957-3_1
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    ABSTRACT: The complexity of human genetic variation has been extended by the observation of abundant and widespread variation in the copy number of submicroscopic DNA segments. The discovery of this novel level of genome organization opened new possibilities concerning the genetic variation that may confer susceptibility to or cause disease. Copy number variants (CNVs) influence gene expression, phenotypic variation and adaptation by altering gene dosage and genome organization. Concordant with the common disease common variant hypothesis these structural variants are now subject to interrogation for disease association. Alzheimer's disease (AD) is a progressive neurodegenerative disease with an estimated heritability of 60-80 %. Large scale genome-wide association studies (GWAS) using high frequency single nucleotide polymorphism (SNP) variants identified ten loci which do not account for the measured heritability. To find the missing heritability systematic assessment of all mutational mechanisms needs to be performed. Between the powerful SNP-GWAS studies and the planned Whole Genome Sequencing projects the contribution of copy number variation (CNV) to the genetic architecture of AD needs to be studied fully.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:315-26. DOI:10.1007/978-1-4939-2627-5_19
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    ABSTRACT: Cyclin-dependent kinases (CDKs) have been traditionally associated with the cell cycle. However, it is now known that CDK7 and CDK9 regulate transcriptional activity via phosphorylation of RNA polymerase II and subsequent synthesis of, for example, inflammatory mediators and factors that influence the apoptotic process; including apoptosis of granulocytes such as neutrophils and eosinophils. Successful resolution of inflammation and restoration of normal tissue homeostasis requires apoptosis of these inflammatory cells and subsequent clearance of apoptotic bodies by phagocytes such as macrophages. It is believed that CDK7 and CDK9 influence resolution of inflammation since they are involved in the transcription of anti-apoptotic proteins such as Mcl-1 which is especially important in granulocyte survival.This chapter describes various in vitro and in vivo models used to investigate CDKs and their inhibitors in granulocytes and particularly the role of CDKs in the apoptosis pathway. This can be performed in vitro by isolation and use of primary granulocytes and in vivo using animal models of inflammatory disease in rodents and zebrafish. Some of the methods described here to assess the role of CDKs in inflammation and apoptosis include flow cytometry and western blotting, together with imaging and quantification of apoptosis in fixed tissue, as well as in vivo models of inflammation.
    Methods in Molecular Biology, 01/2016: chapter Cyclin Dependent Kinase Inhbitors (Methods & Protocols): pages 179-209; Springer New York., ISBN: 978-1-4939-2926-9
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    ABSTRACT: During their development, human T cells undergo similar genomic changes and pass through the same developmental checkpoints as developing thymocytes in the mouse. The difference between both species, however, is that some of these developmental stages are characterized by different phenotypic markers and as a result, evidence emerges that the molecular regulation of human T cell development subtly differs from the mouse [1-4]. In this chapter, we describe in detail how the different stages of human T cell development can be characterized and isolated using specific surface markers.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1323:221-37. DOI:10.1007/978-1-4939-2809-5_19
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    ABSTRACT: Intrathymic injection is used in several T cell-associated immunological studies to deliver cells or other substances directly into the thymus. Here, we describe the intrathymic injection procedure involving surgical incision of the mouse with or without a thoracotomy. Though this procedure can result in poor recovery, postsurgical complications, and distress to the animal, it is actually a simple procedure that can be carried out relatively easily and quickly with experience.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1323:203-9. DOI:10.1007/978-1-4939-2809-5_17
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    ABSTRACT: Alzheimer's disease (AD), and many neurodegenerative disorders, are multifactorial in nature. They involve a combination of genomic, epigenomic, interactomic and environmental factors. Progress is being made, and these complex diseases are beginning to be understood as having their origin in altered states of biological networks at the cellular level. In the case of AD, genomic susceptibility and mechanisms leading to (or accompanying) the impairment of the central Amyloid Precursor Protein (APP) processing and tau networks are widely accepted as major contributors to the diseased state. The derangement of these networks may result in both the gain and loss of functions, increased generation of toxic species (e.g., toxic soluble oligomers and aggregates) and imbalances, whose effects can propagate to supra-cellular levels. Although well sustained by empirical data and widely accepted, this global perspective often overlooks the essential roles played by the main counteracting homeostatic networks (e.g., protein quality control/proteostasis, unfolded protein response, protein folding chaperone networks, disaggregases, ER-associated degradation/ubiquitin proteasome system, endolysosomal network, autophagy, and other stress-protective and clearance networks), whose relevance to AD is just beginning to be fully realized. In this chapter, an integrative perspective is presented. Alzheimer's disease is characterized to be a result of: (a) intrinsic genomic/epigenomic susceptibility and, (b) a continued dynamic interplay between the deranged networks and the central homeostatic networks of nerve cells. This interplay of networks will underlie both the onset and rate of progression of the disease in each individual. Integrative Systems Biology approaches are required to effect its elucidation. Comprehensive Systems Biology experiments at different 'omics levels in simple model organisms, engineered to recapitulate the basic features of AD may illuminate the onset and sequence of events underlying AD. Indeed, studies of models of AD in simple organisms, differentiated cells in culture and rodents are beginning to offer hope that the onset and progression of AD, if detected at an early stage, may be stopped, delayed, or even reversed, by activating or modulating networks involved in proteostasis and the clearance of toxic species. In practice, the incorporation of next-generation neuroimaging, high-throughput and computational approaches are opening the way towards early diagnosis well before irreversible cell death. Thus, the presence or co-occurrence of: (a) accumulation of toxic Aβ oligomers and tau species; (b) altered splicing and transcriptome patterns; (c) impaired redox, proteostatic, and metabolic networks together with, (d) compromised homeostatic capacities may constitute relevant 'AD hallmarks at the cellular level' towards reliable and early diagnosis. From here, preventive lifestyle changes and tailored therapies may be investigated, such as combined strategies aimed at both lowering the production of toxic species and potentiating homeostatic responses, in order to prevent or delay the onset, and arrest, alleviate, or even reverse the progression of the disease.
    Methods in molecular biology (Clifton, N.J.) 01/2016; 1303:3-48. DOI:10.1007/978-1-4939-2627-5_1