Showing papers in "Methods of Molecular Biology in 2013"

Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology.

Abstract: The use of primary mammalian neurons derived from embryonic central nervous system tissue is limited by the fact that once terminally differentiated into mature neurons, the cells can no longer be propagated. Transformed neuronal-like cell lines can be used in vitro to overcome this limitation. However, several caveats exist when utilizing cells derived from malignant tumors. In this context, the popular SH-SY5Y neuroblastoma cell line and its use in in vitro systems is described. Originally derived from a metastatic bone tumor biopsy, SH-SY5Y (ATCC® CRL-2266™) cells are a subline of the parental line SK-N-SH (ATCC® HTB-11™). SK-N-SH were subcloned three times; first to SH-SY, then to SH-SY5, and finally to SH-SY5Y. SH-SY5Y were deposited to the ATCC® in 1970 by June L. Biedler. Three important characteristics of SH-SY5Y cells should be considered when using these cells in in vitro studies. First, cultures include both adherent and floating cells, both types of which are viable. Few studies address the biological significance of the adherent versus floating phenotypes, but most reported studies utilize adherent populations and discard the floating cells during media changes. Second, early studies by Biedler's group indicated that the parental differentiated SK-N-SH cells contained two morphologically distinct phenotypes: neuroblast-like cells and epithelial-like cells (Ross et al., J Natl Cancer Inst 71(4):741-747, 1983). These two phenotypes may correspond to the "N" and "S" types described in later studies in SH-SY5Y by Encinas et al. (J Neurochem 75(3):991-1003, 2000). Cells with neuroblast-like morphology are positive for tyrosine hydroxylase (TH) and dopamine-β-hydroxylase characteristic of catecholaminergic neurons, whereas the epithelial-like counterpart cells lacked these enzymatic activities (Ross et al., J Natl Cancer Inst 71(4):741-747, 1983). Third, SH-SY5Y cells can be differentiated to a more mature neuron-like phenotype that is characterized by neuronal markers. There are several methods to differentiate SH-SY5Y cells and are mentioned below. Retinoic acid is the most commonly used means for differentiation and will be addressed in detail.

Detection of necrosis by release of lactate dehydrogenase activity.

Abstract: Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Although there are many assays for detection of apoptosis, relatively few assays are available for measuring necrosis. A key signature for necrotic cells is the permeabilization of the plasma membrane. This event can be quantified in tissue culture settings by measuring the release of the intracellular enzyme lactate dehydrogenase (LDH). When combined with other methods, measuring LDH release is a useful method for the detection of necrosis. In this chapter, we describe the step-by-step procedure for detection of LDH release from necrotic cells using a microtiter plate-based colorimetric absorbance assay.

What Is Synthetic Biology

Abstract: Synthetic biology is a rapidly developing field that aims to engineer new biological systems that do not already exist in Nature or redesign existing systems from scratch. The emergence of synthetic biology has been supported by a number of enabling technologies and what has developed is a broad field that currently encompasses many activities. The aim of this chapter is to introduce the field and discuss some key examples to date. What these examples have in common is a set of underlying molecular biology techniques including DNA assembly and combinatorial diversity generation, as well as computational modelling to assist in designing the new biological systems.

Immobilization of enzymes: a literature survey.

Abstract: The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.

A review of the methods for human iPSC derivation

Abstract: The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) offers an opportunity to generate pluripotent patient-specific cell lines that can help model human diseases. These iPSC lines could also be powerful tools for drug discovery and the development of cellular transplantation therapies. Many methods exist for generating iPSC lines but those best suited for use in studying human diseases and developing therapies must be of adequate efficiency to produce iPSCs from samples that may be of limited abundance, capable of reprogramming cells from both skin fibroblasts and blood, and footprint-free. Several reprogramming techniques meet these criteria and can be utilized to derive iPSCs in projects with both basic scientific and therapeutic goals. Combining these reprogramming methods with small molecule modulators of signaling pathways can lead to successful generation of iPSCs from even the most recalcitrant patient-derived somatic cells.

PharmGKB: The Pharmacogenomics Knowledge Base

Abstract: The Pharmacogenomics Knowledge Base, PharmGKB, is an interactive tool for researchers investigating how genetic variation affects drug response. The PharmGKB Web site, http://www.pharmgkb.org , displays genotype, molecular, and clinical knowledge integrated into pathway representations and Very Important Pharmacogene (VIP) summaries with links to additional external resources. Users can search and browse the knowledgebase by genes, variants, drugs, diseases, and pathways. Registration is free to the entire research community, but subject to agreement to use for research purposes only and not to redistribute. Registered users can access and download data to aid in the design of future pharmacogenetics and pharmacogenomics studies.

Partial least squares methods: partial least squares correlation and partial least square regression

Abstract: Partial least square (PLS) methods (also sometimes called projection to latent structures) relate the information present in two data tables that collect measurements on the same set of observations. PLS methods proceed by deriving latent variables which are (optimal) linear combinations of the variables of a data table. When the goal is to find the shared information between two tables, the approach is equivalent to a correlation problem and the technique is then called partial least square correlation (PLSC) (also sometimes called PLS-SVD). In this case there are two sets of latent variables (one set per table), and these latent variables are required to have maximal covariance. When the goal is to predict one data table the other one, the technique is then called partial least square regression. In this case there is one set of latent variables (derived from the predictor table) and these latent variables are required to give the best possible prediction. In this paper we present and illustrate PLSC and PLSR and show how these descriptive multivariate analysis techniques can be extended to deal with inferential questions by using cross-validation techniques such as the bootstrap and permutation tests.

Human nasal and tracheo-bronchial respiratory epithelial cell culture

Abstract: Human airway epithelial (hAE) cell cultures are instrumental for studying basic and applied aspects of respiratory tract biology, disease, and therapy. When primary epithelial cells from the human nasal passages or tracheo-bronchial airways are grown on porous supports at an air-liquid interface (ALI) they undergo mucociliary differentiation, reproducing both the in vivo morphology and key physiologic processes. These cultures are useful for studying basic biology, disease pathogenesis, gene therapy and aerosol administration of drugs. This chapter gives detailed protocols for tissue procurement, cell isolation, production of complex media, and cell culture initiation and maintenance needed for hAE cell ALI cultures with non-proprietary reagents.

ASC speck formation as a readout for inflammasome activation.

Abstract: All inflammasomes require the adapter protein apoptosis associated speck-like protein containing a CARD (ASC) for the activation of caspase-1. After inflammasome activation, ASC assembles into a large protein complex, which is termed "speck". ASC specks can be observed as they reach a size of around 1 μm and in most cells only one speck forms upon inflammasome activation. Hence, ASC speck formation can be used as a simple upstream readout for inflammasome activation. Here, we describe a method for analyzing inflammasome activation by ASC speck visualization. First, we describe the generation of a clonal inflammasome reporter macrophage cell line overexpressing fluorescently tagged ASC. We then discuss stimulation conditions and the microscopic evaluation of ASC speck formation.

Primary Mouse Small Intestinal Epithelial Cell Cultures

Abstract: The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have been made to establish primary small intestinal culture systems. However, no defined, reproducible and robust culture system had been developed. To establish such a system, we screened for optimal growth factor combinations based on genetic evidence of self-renewal regulation, differentiation, and carcinogenesis of intestinal stem cells. Here, we describe methods that we have established for the isolation and culture of primary small intestinal epithelial stem cells. In this culture system, isolated crypts form "organoid structures" with a histological hierarchy recapitulating in vivo small intestinal epithelium. Single isolated Lgr5+ intestinal stem cells also form these organoid structures, in which stem cells are maintained by self-renewal and give rise to all lineages of the intestinal epithelium. This culture system is particularly useful for studying the regulation of intestinal stem cell self-renewal and differentiation.

Generation and characterization of murine alternatively activated macrophages.

Abstract: Macrophages play a key role in the innate immune response and help to direct the acquired immune response. Early in the innate immune response, they produce reactive oxygen species and pro-inflammatory cytokines and chemokines to drive inflammation and are referred to as "classically activated" or "killer" macrophages (M1). During the resolution phase of inflammation, they switch to what is known as an "alternatively activated" phenotype or "healer" macrophage (M2) and contribute to debris scavenging, angiogenesis, and wound healing. M1 macrophages are activated by treatment with IFNγ or LPS and M2 macrophages are activated by treatment with Th2 cytokines IL-4 or IL-13 and the M2 phenotype switch can be enhanced by IL-10. Macrophages can also be skewed during differentiation in vitro, and the resultant phenotype depends upon the cytokine provided to support their differentiation. In murine macrophages, MCSF promotes differentiation to an M1 phenotype, GM-CSF promotes differentiation to an M2 phenotype and IL-3 promotes differentiation into a profoundly M2 skewed phenotype. A defining feature of the phenotype of murine M1 versus M2 macrophages is how they metabolize L-arginine. In response to an inflammatory stimulus like LPS, M1 macrophages produce inducible nitric oxide synthase (iNOS) which uses L-arginine as a substrate to produce nitric oxide (NO). M2 macrophages constitutively produce the enzyme arginase I (argI), which sequesters L-arginine from iNOS and results in the production of ornithine and downstream polyamines and L-proline. M1 macrophages also produce relatively higher levels of pro-inflammatory IL-12 and lower levels of anti-inflammatory IL-10 relative to M2 macrophages. In this chapter, we describe in vitro derivation of polarized bone marrow macrophages and methods to analyze the resulting phenotype including Q-PCR, Western blotting, and enzyme assays to determine argI and iNOS expression and activity, as well as production of IL-12p40 and IL-10 and determination of IL-12/IL-10 ratios. Production of iNOS, NO, IL-12p40, and IL-10 are measured after treatment with LPS.

Genome-wide complex trait analysis (GCTA): methods, data analyses, and interpretations.

Abstract: Estimating genetic variance is traditionally performed using pedigree analysis. Using high-throughput DNA marker data measured across the entire genome it is now possible to estimate and partition genetic variation from population samples. In this chapter, we introduce methods and a software tool called Genome-wide Complex Trait Analysis (GCTA) to estimate genomic relationships between pairs of conventionally unrelated individuals using genome-wide single nucleotide polymorphism (SNP) data, to estimate variance explained by all SNPs simultaneously on genomic or chromosomal segments or over the whole genome, and to perform a joint and conditional multiple SNPs association analysis using summary statistics from a meta-analysis of genome-wide association studies and linkage disequilibrium between SNPs estimated from a reference sample.

On the Development and Validation of QSAR Models

Abstract: The fundamental and more critical steps that are necessary for the development and validation of QSAR models are presented in this chapter as best practices in the field. These procedures are discussed in the context of predictive QSAR modelling that is focused on achieving models of the highest statistical quality and with external predictive power. The most important and most used statistical parameters needed to verify the real performances of QSAR models (of both linear regression and classification) are presented. Special emphasis is placed on the validation of models, both internally and externally, as well as on the need to define model applicability domains, which should be done when models are employed for the prediction of new external compounds.

The Martini Coarse-Grained Force Field

Abstract: The Martini force field is a coarse-grained force field suited for molecular dynamics simulations of biomolecular systems. The force field has been parameterized in a systematic way, based on the reproduction of partitioning free energies between polar and apolar phases of a large number of chemical compounds. In this chapter the methodology underlying the force field is presented together with details of its parameterization and limitations. Then currently available topologies are described with a short overview of the key elements of their parameterization. These include the new polarizable Martini water model. A set of three selected ongoing studies using the Martini force field is presented. Finally the latest lines of development are discussed.

Three-dimensional culture of human breast epithelial cells: the how and the why.

Abstract: Organs are made of the organized assembly of different cell types that contribute to the architecture necessary for functional differentiation. In those with exocrine function, such as the breast, cell-cell and cell-extracellular matrix (ECM) interactions establish mechanistic constraints and a complex biochemical signaling network essential for differentiation and homeostasis of the glandular epithelium. Such knowledge has been elegantly acquired for the mammary gland by placing epithelial cells under three-dimensional (3D) culture conditions.Three-dimensional cell culture aims at recapitulating normal and pathological tissue architectures, hence providing physiologically relevant models to study normal development and disease. The specific architecture of the breast epithelium consists of glandular structures (acini) connected to a branched ductal system. A single layer of basoapically polarized luminal cells delineates ductal or acinar lumena at the apical pole. Luminal cells make contact with myoepithelial cells and, in certain areas at the basal pole, also with basement membrane (BM) components. In this chapter, we describe how this exquisite organization as well as stages of disorganization pertaining to cancer progression can be reproduced in 3D cultures. Advantages and limitations of different culture settings are discussed. Technical designs for induction of phenotypic modulations, biochemical analyses, and state-of-the-art imaging are presented. We also explain how signaling is regulated differently in 3D cultures compared to traditional two-dimensional (2D) cultures. We believe that using 3D cultures is an indispensable method to unravel the intricacies of human mammary functions and would best serve the fight against breast cancer.

Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells

Abstract: Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing, and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.

General Overview of Neuronal Cell Culture

Abstract: In this introductory chapter, we provide a general overview of neuronal cell culture. This is a rapidly evolving area of research and we provide an outline and contextual framework for the different chapters of this book. These chapters were all contributed by scientists actively working in the field who are currently using state-of-the-art techniques to advance our understanding of the molecular and cellular biology of the central nervous system. Each chapter provides detailed descriptions and experimental protocols for a variety of techniques ranging in scope from basic neuronal cell line culturing to advanced and specialized methods.

Overview of Statistical Methods for Genome-Wide Association Studies (GWAS).

Abstract: This chapter provides an overview of statistical methods for genome-wide association studies (GWAS) in animals, plants, and humans The simplest form of GWAS, a marker-by-marker analysis, is illustrated with a simple example The problem of selecting a significance threshold that accounts for the large amount of multiple testing that occurs in GWAS is discussed Population structure causes false positive associations in GWAS if not accounted for, and methods to deal with this are presented Methodology for more complex models for GWAS, including haplotype-based approaches, accounting for identical by descent versus identical by state, and fitting all markers simultaneously are described and illustrated with examples

Multisystemic functions of alkaline phosphatases.

Abstract: Human and mouse alkaline phosphatases (AP) are encoded by a multigene family expressed ubiquitously in multiple tissues. Gene knockout (KO) findings have helped define some of the precise exocytic functions of individual isozymes in bone, teeth, the central nervous system, and in the gut. For instance, deficiency in tissue-nonspecific alkaline phosphatase (TNAP) in mice (Alpl (-/-) mice) and humans leads to hypophosphatasia (HPP), an inborn error of metabolism characterized by epileptic seizures in the most severe cases, caused by abnormal metabolism of pyridoxal-5'-phosphate (the predominant form of vitamin B6) and by hypomineralization of the skeleton and teeth featuring rickets and early loss of teeth in children or osteomalacia and dental problems in adults caused by accumulation of inorganic pyrophosphate (PPi). Enzyme replacement therapy with mineral-targeting TNAP prevented all the manifestations of HPP in mice, and clinical trials with this protein therapeutic are showing promising results in rescuing life-threatening HPP in infants. Conversely, TNAP induction in the vasculature during generalized arterial calcification of infancy (GACI), type II diabetes, obesity, and aging can cause medial vascular calcification. TNAP inhibitors, discussed extensively in this book, are in development to prevent pathological arterial calcification. The brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in fatty acid (FA) absorption, in protecting gut barrier function, and in determining the composition of the gut microbiota via its ability to dephosphorylate lipopolysaccharide (LPS). Knockout mice (Akp3 (-/-)) deficient in duodenal-specific IAP (dIAP) become obese, and develop hyperlipidemia and hepatic steatosis when fed a high-fat diet (HFD). These changes are accompanied by upregulation in the jejunal-ileal expression of the Akp6 IAP isozyme (global IAP, or gIAP) and concomitant upregulation of FAT/CD36, a phosphorylated fatty acid translocase thought to play a role in facilitating the transport of long-chain fatty acids into cells. gIAP, but not dIAP, is able to modulate the phosphorylation status of FAT/CD36. dIAP, even though it is expressed in the duodenum, is shed into the gut lumen and is active in LPS dephosphorylation throughout the gut lumen and in the feces. Akp3 (-/-) mice display gut dysbiosis and are more prone to dextran sodium sulfate-induced colitis than wild-type mice. Of relevance, oral administration of recombinant calf IAP prevents the dysbiosis and protects the gut from chronic colitis. Analogous to the role of IAP in the gut, TNAP expression in the liver may have a proactive role from bacterial endotoxin insult. Finally, more recent studies suggest that neuronal death in Alzheimer's disease may also be associated with TNAP function on certain brain-specific phosphoproteins. This review recounts the established roles of TNAP and IAP and briefly discusses new areas of investigation related to multisystemic functions of these isozymes.

Antibody-drug conjugate (ADC) clinical pipeline: a review.

Abstract: Biological therapies play an increasing role in cancer treatment, although the number of naked antibodies showing clinical efficacy as single agent remains limited. One way to enhance therapeutic potential of antibodies is to conjugate them to small molecule drugs. This combination is expected to bring together the benefits of highly potent drugs on the one hand and selective binders of specific tumor antigens on the other hand. However, designing an ADC is more complex than a simple meccano game, requiring thoughtful combination of antibody, linker, and drugs in the context of a target and a defined cancer indication. Lessons learned from the first-generation antibody-drug conjugate (ADC) and improvement of the technology guided the design of improved compounds which are now in clinical trials. Brentuximab vedotin (Adcetris(®)), an anti-CD30 antibody conjugated to a potent microtubule inhibitor for the treatment of Hodgkin's lymphoma and anaplastic large cell lymphomas, is the only marketed ADC today. A total of 27 ADC are currently undergoing clinical trials in both hematological malignancies and solid tumor indications. Among them, T-DM1 (trastuzumab emtansine), an ADC comprised of trastuzumab conjugated to DM1, via a non-cleavable linker, is showing very promising results in phase III for the treatment of HER2-positive refractory/relapsed metastatic breast cancer. Other compounds, such as CMC-544, SAR3419, CDX-011, PSMA-ADC, BT-062, and IMGN901 currently in clinical trials, targeting varied antigens and bearing different linker and drugs, contribute to the learning curve of ADC, as do the discontinued ADC. Current challenges include improvement of the therapeutic index, linked to a careful selection of the targets, a better understanding of ADC mechanism of action, the management and understanding of ADC off-target toxicities, as well as the selection of appropriate clinical settings (patient selection, dosing regimen) where these molecules can bring highest clinical benefit.

Nanodiscs as a new tool to examine lipid-protein interactions

Abstract: Nanodiscs are self-assembled discoidal fragments of lipid bilayers 8 – 16 nm in diameter, stabilized in solution by amphipathic helical scaffold protein. As stable and highly soluble membrane mimetics with controlled lipid composition and ability to add affinity tags to the scaffold protein, Nanodiscs represent an attractive model system for solubilization, isolation, purification, and biophysical and biochemical studies of membrane proteins. We overview various approaches to the structural and functional studies of different classes of integral membrane proteins such as ion channels, transporters, GPCR and other receptors, membrane enzymes, blood coagulation cascade proteins, et cetera incorporated into Nanodiscs with the special focus on the advantages provided by homogeneity, ability to control oligomerization state of the target protein and lipid composition of the bilayer. Special attention is paid to the opportunities provided by Nanodisc system for the detailed studies of the role of different lipid properties and protein – lipid interactions in the functional behavior of membrane proteins.

Bayesian methods applied to GWAS.

Abstract: Bayesian multiple-regression methods are being successfully used for genomic prediction and selection. These regression models simultaneously fit many more markers than the number of observations available for the analysis. Thus, the Bayes theorem is used to combine prior beliefs of marker effects, which are expressed in terms of prior distributions, with information from data for inference. Often, the analyses are too complex for closed-form solutions and Markov chain Monte Carlo (MCMC) sampling is used to draw inferences from posterior distributions. This chapter describes how these Bayesian multiple-regression analyses can be used for GWAS. In most GWAS, false positives are controlled by limiting the genome-wise error rate, which is the probability of one or more false-positive results, to a small value. As the number of test in GWAS is very large, this results in very low power. Here we show how in Bayesian GWAS false positives can be controlled by limiting the proportion of false-positive results among all positives to some small value. The advantage of this approach is that the power of detecting associations is not inversely related to the number of markers.

Gene Targeting in Mice: a Review

Abstract: The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (1) delete or insert mutations into genes of interest to determine their function, (2) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g., mice susceptible to infections by human-specific pathogens of interest, (3) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (4) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse's lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice.

One stop shop for everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012.

Abstract: dictyBase (http:// dictybase.org), the model organism database for Dictyostelium discoideum, includes the complete genome sequence and expression data for this organism. Relevant literature is integrated into the database, and gene models and functional annotation are manually curated from experimental results and comparative multigenome analyses. dictyBase has recently expanded to include the genome sequences of three additional Dictyostelids, and has added new software tools to facilitate multigenome comparisons. The Dicty Stock Center, a strain and plasmid repository for Dictyostelium research has relocated to Northwestern University in 2009. This allowed us integrating all Dictyostelium resources to better serve the research community. In this chapter, we will describe how to navigate the website and highlight some of our newer improvements.

Assessment of spermatogenesis through staging of seminiferous tubules

Abstract: Male germ cells in all mammals are arranged within the seminiferous epithelium of the testicular tubules in a set of well-defined cell associations called stages. The cellular associations found in these stages and characteristics of the cells used to identify the stages have been well described. Here we present a binary decision key roadmap for identifying stages and present several examples of how staging tubules can be used to better assess the developmental profile of gene expression during spermatogenesis and defects in spermatogenesis arising in pathological conditions resulting from genetic mutations in mice. In particular, when one or more cells of a cellular association cannot be clearly identified or are missing, the cell types that should be present can be precisely identified by knowledge of the approximate or exact stage of the tubule cross section.

Senescence regulation by the p53 protein family.

Abstract: p53, a guardian of the genome, exerts its tumor suppression activity by regulating a large number of downstream targets involved in cell cycle arrest, DNA repair, apoptosis, and cellular senescence. Although p53-mediated apoptosis is able to kill cancer cells, a role for cellular senescence in p53-dependent tumor suppression is becoming clear. Mouse studies showed that activation of p53-induced premature senescence promotes tumor regression in vivo. However, p53-mediated cellular senescence also leads to aging-related phenotypes, such as tissue atrophy, stem cell depletion, and impaired wound healing. In addition, several p53 isoforms and two p53 homologs, p63 and p73, have been shown to play a role in cellular senescence and/or aging. Importantly, p53, p63, and p73 are necessary for the maintenance of adult stem cells. Therefore, understanding the dual role the p53 protein family in cancer and aging is critical to solve cancer and longevity in the future. In this chapter, we provide an overview on how p53, p63, p73, and their isoforms regulate cellular senescence and aging.

Detection of mycoplasma contaminations.

Abstract: Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The -specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma control and should be an established method in every cell culture laboratory. New cell lines as well as cell lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory and can also be used to determine the contaminating species.The described assay can be performed in less than 3 hours, including sample preparation, DNA extraction, PCR run, and analysis of the PCR products. Special emphasis is given to steps taken to avoid false-negative results due to the presence of inhibitors of the Taq polymerase in the crude samples and the interpretation of the results. The technique can also be adapted to the requirements of the pharmacopoeia.

Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay

Abstract: MicroRNAs are post-transcriptional regulators that control mRNA stability and the translation efficiency of their target genes. Mature microRNAs are approximately 22-nucleotide in length. They mediate post-transcriptional gene regulation by binding to the imperfect complementary sequences (a.k.a. microRNA regulatory elements, MRE) in the target mRNAs. It is estimated that more than one-third of the protein-coding genes in the human genome are regulated by microRNAs. The experimental methods to examine the interaction between the microRNA and its targeting site(s) in the mRNA are important for understanding microRNA functions. The luciferase reporter gene assay has recently been adapted to test the effect of microRNAs. In this chapter, we use a previously identified miR-138 targeting site in the 3'-untranslated region (3'-UTR) of the RhoC mRNA as an example to describe a quick method for testing the interaction of microRNA and mRNA.

BioModels Database: a repository of mathematical models of biological processes.

Abstract: BioModels Database is a public online resource that allows storing and sharing of published, peer-reviewed quantitative, dynamic models of biological processes. The model components and behaviour are thoroughly checked to correspond the original publication and manually curated to ensure reliability. Furthermore, the model elements are annotated with terms from controlled vocabularies as well as linked to relevant external data resources. This greatly helps in model interpretation and reuse. Models are stored in SBML format, accepted in SBML and CellML formats, and are available for download in various other common formats such as BioPAX, Octave, SciLab, VCML, XPP and PDF, in addition to SBML. The reaction network diagram of the models is also available in several formats. BioModels Database features a search engine, which provides simple and more advanced searches. Features such as online simulation and creation of smaller models (submodels) from the selected model elements of a larger one are provided. BioModels Database can be accessed both via a web interface and programmatically via web services. New models are available in BioModels Database at regular releases, about every 4 months.

Automated genome annotation and metabolic model reconstruction in the SEED and Model SEED.

Abstract: Over the past decade, genome-scale metabolic models have proven to be a crucial resource for predicting organism phenotypes from genotypes. These models provide a means of rapidly translating detailed knowledge of thousands of enzymatic processes into quantitative predictions of whole-cell behavior. Until recently, the pace of new metabolic model development was eclipsed by the pace at which new genomes were being sequenced. To address this problem, the RAST and the Model SEED framework were developed as a means of automatically producing annotations and draft genome-scale metabolic models. In this chapter, we describe the automated model reconstruction process in detail, starting from a new genome sequence and finishing on a functioning genome-scale metabolic model. We break down the model reconstruction process into eight steps: submitting a genome sequence to RAST, annotating the genome, curating the annotation, submitting the annotation to Model SEED, reconstructing the core model, generating the draft biomass reaction, auto-completing the model, and curating the model. Each of these eight steps is documented in detail.