Methods in molecular biology
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Experimental spinal cord injury (SCI) can maintain the continuity of the spinal cord, as in the contusion (e.g., weight-fall) or compression models, or not, when there is a partial or a complete transection. The majority of acute human SCI is not followed by complete transection, but there is a combination of contusion, compression, and possibly partial transection. ⋯ This lesion was established by our group and represents a simple, reliable, and inexpensive clip compression model with functional and morphological reproducibility. In this chapter we describe, step by step, the protocol of this experimental SCI.
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In this first, introductory chapter, it is intended to summarize from a methodological point of view the state of the art in plant proteomics, focusing on mass spectrometry-based strategies. Thus, this chapter is mainly directed at beginners or at those trying to get into the field, rather than at those with real experience or a long trajectory in plant proteomics research. The different alternative workflows, methods, techniques, and protocols from the experimental design to the data analysis will be briefly commented, with cross references to previous monographs and reviews, as well as to the rest of the book chapters. ⋯ Furthermore, and even more important, we should move to data validation through other -omics or classical biochemical strategies, in an attempt to get a deeper, real, and more accurate view and understanding of cell biology. In the modern Systems Biology concept, proteomics must be considered as a part of a global, multidisciplinary approach. Making biological sense of a proteomics experiment requires a proper experimental design, data validation, interpretation, and publication policy.
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In animals, environmental exposure such as toxic chemicals and microorganisms or pathophysiological conditions in respiratory system could result in inflammatory response in their lungs. Bronchoalveolar lavage (BAL) is a procedure that can be used to collect samples from animal lungs to efficiently evaluate the immune response by examining both the compositions of cells and fluid from lavage. The profile of inflammatory cells in BAL provides a qualitative description of inflammatory response and the secretion in BAL fluid contains proteins of inflammatory mediators and albumin as a quantitative measurement of inflammation and tissue injury in the lungs. A consistent experimental approach on how to lavage mouse lungs and collect samples is important for a reproducible evaluation of pathological and physiological changes in mouse lung especially for the analysis of inflammation.
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The liver is a very complex organ with a large variety of functions, making it an attractive organ for gene replacement therapy. Many genetic disorders can be corrected by delivering gene products directly into the liver using viral vectors. In this chapter, we will describe gene delivery via portal vein administration in mice and dogs to correct the blood coagulation disorder hemophilia B. ⋯ Complete correction of murine hemophilia B and multi-year near-correction of canine hemophilia B have been achieved following portal vein delivery of adeno-associated viral (AAV) vectors expressing factor IX from hepatocyte-specific promoters. Peripheral vein injection can lead to increased vector dissemination to off-target organ such as the lung and spleen. Below, we will describe portal vein injection delivery route via laparotomy.
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The blood-brain barrier (BBB) proper is composed of endothelial cells (ECs) of the cerebral microvasculature, which are interconnected by tight junctions (TJs) that in turn form a physical barrier restricting paracellular flux. Tight control of vascular permeability is essential for the homeostasis and functionality of the central nervous system (CNS). In vitro BBB models have been in use for decades and have been of great benefit in the process of investigating and understanding the cellular and molecular mechanisms underlying BBB establishment. ⋯ Additionally, this chapter provides guidance through subsequent experiments such as permeability analysis (Pe, flux), expression analysis (qRT-PCR and Western blotting), and localization analysis of BBB junction proteins (immunocytochemistry) using the same inserts subjected earlier to impedance analysis. As numerous diseases are associated with BBB breakdown, researchers aim to continuously improve and refine in vitro BBB models to mimic in vivo conditions as closely as possible. This chapter summarizes protocols with the intention to provide a collection of BBB in vitro assays that generate reproducible results not only with primary brain ECs but also with EC lines to open up the field for a broader spectrum of researchers who intend to investigate the BBB in vitro particularly aiming at therapeutic aspects.