Methods in molecular biology
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Human pluripotent stem cells (PSCs), which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs), represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such, it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. ⋯ Nevertheless, as the field develops, it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents, particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus, even for cell lines previously exposed to xenogeneic reagents, it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel, an animal-matrix, and CELLstart, an animal-free matrix, and these can be used to produce hESCs as part of a clinical manufacturing process.
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Septic syndromes represent a major, although largely under-recognized, healthcare problem worldwide accounting for thousands of deaths every year. Although flow cytometry (FCM) remains a relatively confidential diagnostic tool, it is useful at every step of intensive care unit (ICU) patients' management. This review will focus on biomarkers measurable by FCM on a routine standardized basis and usable for the diagnosis of sepsis and for prediction of adverse outcome, occurrence of secondary nosocomial infections or guidance of putative immunotherapy relative to innate and adaptive immune dysfunctions in ICU patients. ⋯ In the specific clinical context of ICU patients' monitoring, the increasing potential of FCM is further illustrated by the use of the biomarkers listed above as stratification tools in preliminary clinical studies. The next critical step is to use these standardized FCM protocols in large multicentric clinical trials testing individualized immunotherapy. Importantly, many other markers of immune dysfunction are currently under development that could further enable the administration of targeted individualized therapy in ICU patients.
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The posttranslational modification of proteins is important for the regulation of enzymatic activity, protein half-life, and interaction with other molecules. One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. ⋯ Furthermore, phosphoproteome analyses are incompatible with long organelle isolation procedures prior to analysis, because of the highly dynamic nature of regulatory phosphorylations. In this chapter, we provide a detailed step-by-step overview of the complex experimental setup required for successful chloroplast phosphoproteome analysis, report our experience with existing methods, and comment on their application in the field.
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Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.
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For certain applications, particularly experiments involving high-resolution imaging, it is necessary to culture cells on glass slides or cover glasses. This chapter describes techniques for successfully growing human embryonic stem cells (hESCs) on glass surfaces under three different conditions - serum-containing, serum-free, and following single-cell dissociation. It is anticipated that these techniques will extrapolate to other types of pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic germ cells (EGCs).