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Methods in Molecular Biology

Alexander Golberg, Alexander Liberzon, Edward Vitkin, Zohar Yakhini
Displacing fossil fuels and their derivatives with renewables, and increasing sustainable food production are among the major challenges facing the world in the coming decades. A possible, sustainable direction for addressing this challenge is the production of biomass and the conversion of this biomass to the required products through a complex system coined biorefinery. Terrestrial biomass and microalgae are possible sources; however, concerns over net energy balance, potable water use, environmental hazards, and uncertainty in the processing technologies raise questions regarding their actual potential to meet the anticipated food, feed, and energy challenges in a sustainable way...
March 15, 2018: Methods in Molecular Biology
Nedime Serakinci, Huseyin Cagsin, Merdiye Mavis
Telomeres are repetitive genetic materials that protect the chromosomes by capping the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in the embryonic stem cells in high concentrations and declines with age. It is still unclear to what extend there is telomerase in adult stem cells, but considering these are the founder cells to the cells of the all tissues in a body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important...
March 15, 2018: Methods in Molecular Biology
Dan-Ya Wu, Xia Zhang, Yi-Liang Miao
Stem cells have the potential to differentiate into specialized cell types under specific conditions in vivo or in vitro, which are used to cure many diseases related to aging. Somatic cell nuclear transfer (SCNT) can reprogram differential somatic cells into cloned embryos and embryonic stem cells can be derived from these cloned embryos. Recipient oocytes have healthier mitochondria and can improve the metabolism competence, lessen the ROS damage, and rejuvenate mitochondrial function of aged cells during reprogramming...
March 7, 2018: Methods in Molecular Biology
Hui Gao, Chao Liu, Wei Li
Autophagy is an important intracellular degradation system which is implicated in many physiological and pathological processes. During autophagy, cytosolic constituents such as organelles and macromolecules are engulfed by autophagosome, and then they fuse with lysosomes for degradation and recycle of the engulfed components within the autolysosome to maintain cellular homeostasis. In male testis, the Leydig cells provide the major source of testosterone production. Autophagy is extremely active in Leydig cells and is involved in the steroid production...
February 27, 2018: Methods in Molecular Biology
Jacqueline Jerney, Kristian Spilling
There are two main approaches for cultivating microalgae on a large scale: open or closed cultivation. The main difference between open and closed systems is related to how they operate (e.g., cooling and gas exchange), vulnerability for outside influence (e.g., rainwater and introduction of unwanted species), and costs for building and operating the system. In this chapter we introduce the main cultivation technologies and discuss their main advantages and disadvantages when cultivating microalgae.
February 27, 2018: Methods in Molecular Biology
Hua Qian, Xue Leng, Jie Wen, Qian Zhou, Xin Xu, Xunwei Wu
Stem cells play a crucial role in maintaining and repairing tissues during homeostasis and following injury. The efficient procurement of high quantity and quality of skin stem cells is important for both laboratory studies and clinical applications. Here, we describe a one-step isolation procedure to efficiently obtain both epidermal and dermal cell population from human skin specimen, based on the different influence of the Rho kinase inhibitor Y27632 on the growth of epidermal and dermal cells during the initial culture...
February 25, 2018: Methods in Molecular Biology
Daniel Haensel, Melissa A McNeil, Xing Dai
Stem cells that reside in the bulge of adult mouse hair follicles are a leading model of tissue stem cell research. Ex vivo culturing, molecular and cell biological characterizations, as well as genetic manipulation of fluorescence-activated cell sorting-isolated bulge stem cells offer a useful experimental pipeline to complement in vivo studies. Here we describe detailed methods for culturing, immunostaining, live cell imaging, and adenoviral infection of bulge stem cells for downstream applications such as in vitro clonal and in vivo patch assays...
February 25, 2018: Methods in Molecular Biology
Georgios Konstantinidis, Sonja Sievers, Yao-Wen Wu
Autophagy is a fundamental cellular catabolic pathway mediating the recycling of cellular components. Autophagy has been implicated in pathogenesis of diverse diseases such as neurodegeneration and cancer. Due to the therapeutic potential, the autophagy-modulating agents have profoundly enriched the spectrum of tools used to investigate autophagy. However, many of these compounds have additional off-target effects that may confound elucidation of autophagy in certain contexts. There remains high demand for highly specific and novel chemotypes that can be used to study the regulation mechanism of autophagy and contribute novel pharmacophores for therapeutic purposes...
February 23, 2018: Methods in Molecular Biology
Ceren Aksoy, Feride Severcan
The effect of aging process on stem cell function is crucial because of their critical role in tissue regeneration and repair. The impact of aging on stem cells needs to be understood clearly for the success of clinical application and obtaining desired therapeutic outcome throughout the novel stem cell based therapies. The existing methods used to monitor and characterize the stem cells have some unwanted effects on the properties of stem cells and these methods also do not provide real-time information about cellular conditions...
February 21, 2018: Methods in Molecular Biology
Aurora Montali, Morena Casartelli, Daniele Bruno, Annalisa Grimaldi, Gianluca Tettamanti
In holometabolous insects, various larval organs are remodeled by autophagy during metamorphosis. Although moths and butterflies are among the first animal models in which this self-eating process was described, only in recent years autophagy has been analyzed in detail in these insects. In particular, the silkworm Bombyx mori, which represents a well-studied model among Lepidoptera, provides a wide repertoire of cellular and molecular tools useful for studying the occurrence of autophagy and for evaluating its role in postembryonic development...
February 15, 2018: Methods in Molecular Biology
Jea-Young Lee, Roger Lin, Hung Nguyen, M Grant Liska, Trenton Lippert, Yuji Kaneko, Cesar V Borlongan
Traumatic brain injury (TBI) displays cognitive and motor symptoms following the initial injury which can be exacerbated by secondary cell death. Aging contributes significantly to the morbidity of TBI, with higher rates of negative neurological and behaviors outcomes. In the recent study, young and aged animals were injected intravenously with human adipose-derived mesenchymal stem cells (hADSCs) (Tx), conditioned media (CM), or vehicle (unconditioned media) following TBI. The beneficial effects of hADSCs were analyzed using various molecular and behavioral techniques...
February 15, 2018: Methods in Molecular Biology
Katariina Natunen
The widely applied Nile red (NR) method allows near real-time monitoring of microalgal neutral lipid accumulation. When added to a culture sample, optimally, the fluorescent dye NR penetrates the microalgal cell wall staining the intracellular neutral lipids, and the measured fluorescence is linearly correlated to the neutral lipid concentration. Here I describe an optimization protocol for determining the optimal staining parameters for each new microalgal species, followed by a basic NR staining protocol to be applied for monitoring of microalgal neutral lipid accumulation...
January 26, 2018: Methods in Molecular Biology
Azad Eshghi, Christoph H Borchers
Multiple reaction monitoring (MRM) is a technique used in tandem mass spectrometry where the first mass analyzer preselects parent ions for fragmentation and the second mass analyzer transmits selected product ions to the detector. This targeted technique has found widespread application in bottom-up proteomics for monitoring target peptides in a complex enzymatic digest. Quantitative MRM can be performed on enzymatically digested samples using spiked-in synthetic peptide standards, providing unsurpassed quantitative accuracy and a dynamic range of four orders of magnitude, often eliminating the need for prior depletion of high-abundance proteins...
December 19, 2017: Methods in Molecular Biology
Andrea J SantamarĂ­a, Juan P Solano, Francisco D Benavides, James D Guest
Cell transplant-mediated tissue repair of the damaged spinal cord is being tested in several clinical trials. The current candidates are neural stem cells, stromal cells, and autologous Schwann cells (aSC). Due to their peripheral origin and limited penetration of astrocytic regions, aSC are transplanted intralesionally as compared to neural stem cells that are transplanted into intact spinal cord. Injections into either location can cause iatrogenic injury, and thus technical precision is important in the therapeutic risk-benefit equation...
2018: Methods in Molecular Biology
Christine D Plant, Giles W Plant
Schwann cells are the primary inducers of regeneration of the peripheral nervous system. Schwann cells can be isolated from adult peripheral nerves, expanded in large numbers, and genetically transduced by viral vectors in vitro prior to their use in vivo. Here we describe how to use lentiviral vectors to transduce primary Schwann cells in vitro. We also describe how cultured Schwann cells can be used in conjunction with decellularized peripheral nerve sheaths prepared by multiple freeze thawing of peripheral nerve tissue...
2018: Methods in Molecular Biology
Christine D Plant, Giles W Plant
Biomaterials can be utilized to assist in the transplantation of Schwann cells to the central and peripheral nervous system. The biomaterials can be natural or man-made, and can have preformed shapes or injectable formats. Biomaterials can play multiple roles in cellular transplantation; for example, they can assist with cellular integration and protect Schwann cells from cell death initiated by the lack of a substrate, an occurrence known as "anoikis." In addition, biomaterials can be engineered to increase cell proliferation and differentiation by the addition of ligands bound to the substrate...
2018: Methods in Molecular Biology
Ying Dai, Caitlin E Hill
Adult Schwann cells (SCs) can provide both a permissive substrate for axonal growth and a source of cells to ensheath and myelinate axons when transplanted into the injured spinal cord. Multiple studies have demonstrated that SC transplants can be used as part of a combinatorial approach to repairing the injured spinal cord. Here, we describe the protocols for collection and transplantation of adult rat primary SCs into the injured spinal cord. Protocols are included for the tissue culture procedures necessary for collection, quantification, and suspension of the cells for transplantation and for the surgical procedures for spinal cord injury at thoracic level nine (T9), reexposure of the injury site for delayed transplantation, and injection of the cells into the spinal cord...
2018: Methods in Molecular Biology
Rebecca L Cunningham, Kelly R Monk
The optical transparency of zebrafish larvae enables live imaging. Here we describe the methodology for live imaging and detail how to mount larvae for live imaging of Schwann cell development.
2018: Methods in Molecular Biology
Rebecca L Cunningham, Kelly R Monk
Transmission electron microscopy (TEM) enables visualization of the ultrastructure of the myelin sheath. Schwann cells on the posterior lateral line nerves and motor nerves can be imaged by TEM. Here, we detail the multiday processing of larval trunks and dissected posterior lateral line for TEM, as well as how to trim embedded samples, section, and stain grids for imaging.
2018: Methods in Molecular Biology
Rebecca L Cunningham, Kelly R Monk
In situ hybridization enables visualization of mRNA localization, and immunohistochemistry enables visualization of protein localization within a tissue or organism. Both techniques have been extensively utilized in zebrafish (Thisse et al., Development 119:1203-1215, 1993; Dutton et al., Development 128:4113-4125, 2001; Gilmour et al., Neuron 34:577-588, 2002; Lyons et al., Curr Biol 15:513-524, 2005) including for visualization of mRNA localization in Schwann cells (Lyons et al., Curr Biol 15:513-524, 2005; Monk et al...
2018: Methods in Molecular Biology
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