Methods in molecular biology
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Normal and tumor stem cells are present in rare quantities in tissues and this has historically represented a major hurdle to in-depth investigations of their biology. In the case of the mammary gland, the relative promiscuity of the immunophenotypical markers described in several studies for the isolation of human and mouse mammary stem cells limits their usefulness, in particular when highly purified mammary stem cell fractions are required for an in-depth molecular and functional characterization (Stingl et al. Nature 439:993-997, 2006; Shackleton et al. ⋯ Cell 138:1083-1095, 2009). Following mammosphere dissociation, the differential degree of PKH26 epifluorescence displayed by stem cells compared to precursor cells is exploited for their purification by FACS sorting. As a result, the scarcely represented PKH26-labeled mammary stem cells are purified to near homogeneity and can be used for further molecular and biological studies.
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Many genetic mutations result in the disruption of (alternative) splicing. Prime examples are the SMN1 and SMN2 genes: a silent mutation in SMN2 leads to the skipping of the constitutive exon 7 in the majority of SMN2 transcripts, while this exon is generally included in SMN1 transcripts. ⋯ There are proteins and drugs that can chance alternative splicing events, e.g. increase the inclusion of exon 7 in SMN2. This chapter describes mini-genes and methods that can be employed to screen for candidate proteins and drugs.
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Computational methods now play an integral role in modern drug discovery, and include the design and management of small molecule libraries, initial hit identification through virtual screening, optimization of the affinity and selectivity of hits, and improving the physicochemical properties of the lead compounds. In this chapter, we survey the most important data sources for the discovery of new molecular entities, and discuss the key considerations and guidelines for virtual chemical library design.
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The field of psychoneuroimmunology (PNI) aims to uncover the processes and consequences of nervous, immune, and endocrine system relationships. Behavior is a consequence of such interactions and manifests from a complex interweave of factors including immune-to-neural and neural-to-immune communication. Often the signaling molecules involved during a particular episode of neuroimmune activation are not known but behavioral response provides evidence that bioactives such as neurotransmitters and cytokines are perturbed. ⋯ Immunobehaviors include lethargy, loss of appetite, and disinterest in social activity and the surrounding environment. In addition, neuroimmune activation can precipitate feelings of depression and anxiety while negatively impacting cognitive function and physical activity. Provided is a detailed overview of behavioral tests frequently used to examine neuroimmune activation in mice with a special emphasis on preexperimental conditions that can confound or prevent successful immunobehavioral experimentation.
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Spinal cord injury-induced pain is a common clinical problem affecting adversely the quality of daily lives of spinal cord injured patients. Management with current pain medications can only lead to partial pain relief in some spinal cord injured patients, which is usually associated with unfavorable side effects. ⋯ We describe here the generation of a spinal cord contusion injury model that mimics the etiology and phenotypes of chronic pain states in spinal cord injured patients. Therefore, this model can be a useful tool for studying spinal cord injury mechanisms, functional recovery, research, and development of new medications for better functional and symptomatic improvements, including pain management.