Methods in molecular biology
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Human embryonic stem cells (hESCs) have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues. HESCs can potentially provide an extraordinary source of cells for tissue engineering and great insight into early embryonic development. Much attention has been given to the possibility that hESCs and their derivatives may someday play major roles in the study of the development, disease therapeutics, and repair of injuries to the central and peripheral nervous systems. ⋯ Using reduced numbers of mouse embryonic fibroblasts as feeder substrates, these markers of pluripotency are lost quickly and replaced by primarily neuroglial phenotypes with only a few cells representing other embryonic germ layer types remaining. Within the first 2 weeks of co-culture with reduced MEFs, the undifferentiated hESCs show progression from neuroectodermal to neural stem cell to maturing and migrating neurons to mature neurons in a stepwise fashion that is dependent on both the type of hESCs and the density of MEFs. In this chapter, we provide the methods for culturing pluripotent hESCs and MEFs, differentiating hESCs using reduced density MEFs, and phenotypic analyses of this culture system.
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Inflammation is a complex, multiscale biological response to threats - both internal and external - to the body, which is also required for proper healing of injured tissue. In turn, damaged or dysfunctional tissue stimulates further inflammation. ⋯ We have suggested the concept of translational systems biology, defined as a focused application of computational modeling and engineering principles to pathophysiology primarily in order to revise clinical practice. This chapter reviews the existing, translational applications of computational simulations and related approaches as applied to inflammation.
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The principles of fluorescence resonance energy transfer have been utilized to develop a high-throughput assay which detects compounds that interfere with interaction between retinol-binding protein (RBP) and transthyretin (TTR). In this assay, the intrinsic fluorescence from the RBP-retinol complex excites a probe molecule which is covalently coupled to TTR. ⋯ Thus, compounds which bind to RBP must compete with retinol in order to affect RBP-TTR interaction. This feature of the assay will be useful to identify test compounds which are more likely to have an effect in vivo.
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Methylation of cytosines is a very important epigenetic modification of genomic DNA in many different eukaryotes, and it is frequently involved in transcriptional regulation of genes. In plants, DNA methylation is regulated by a complex interplay between several methylating and demethylating enzymes. ⋯ Subsequent PCR and sequence analysis of individual amplicons displays the degree, position, and sequence context of methylation of every cytosine residue in individual genomic sequences. We describe the application of bisulfite sequencing for the analysis of DNA methylation at defined individual sequences of plant genomic DNA.
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Although a number of animal models such as endotoxic shock and bacteremia have been used to study the pathogenesis of sepsis, cecal ligation and puncture (CLP) represents a peritonitis model with clinical features of polymicrobial infection comparable with those of peritonitis in humans. The CLP consists in the surgical perforation of the legated cecum of mice that results in immediate and constant drainage of cecal bacteria into the peritoneal cavity. The severity of the diseases depends on the diameter of the needle used for the perforation as well as on the number of cecal punctures. The CLP model of sepsis in mice is the most commonly used for studying the process of septic peritonitis and can be used as a preclinical model to test the efficacy of pharmacological agents for the treatment of sepsis.