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Methods in Molecular Biology

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https://www.readbyqxmd.com/read/28434167/analysis-of-s1p-receptor-expression-by-uterine-immune-cells-using-standardized-multi-parametric-flow-cytometry
#1
Jianhong Zhang, Annie Bang, Stephen J Lye
Flow cytometry is a powerful tool for phenotypic and functional analyses of single immune cells. The increasing capability of flow cytometry technology has driven a more detailed understanding of immune cell subsets and functions in complex cellular systems such as the developing human decidua/placenta. We propose a standardized procedure for the isolation and analysis of human decidual natural killer (dNK) cells and this method can be extended to investigation of other uterine lymphocytes. Here this platform is used to examine the expression of sphingosine-1-phosphate (S1P) receptor and functional growth factors by dNK cells...
April 23, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28434166/antibody-uptake-assay-in-the-embryonic-zebrafish-forebrain-to-study-notch-signaling-dynamics-in-neural-progenitor-cells-in-vivo
#2
Kai Tong, Mahendra Wagle, Su Guo
Stem cells can generate cell fate heterogeneity through asymmetric cell division (ACD). ACD derives from the asymmetric segregation of fate-determining molecules and/or organelles in the dividing cell. Radial glia in the embryonic zebrafish forebrain are an excellent model for studying the molecular mechanisms regulating ACD of stem cells in vertebrates, especially for live imaging concerning in vivo molecular and cellular dynamics. Due to the current difficulty in expressing fluorescent reporter-tagged proteins at physiological levels in zebrafish for live imaging, we have developed an antibody uptake assay to label proteins in live embryonic zebrafish forebrain with high specificity...
April 23, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28425080/monitoring-of-paternal-mitochondrial-degradation-in-caenorhabditis-elegans
#3
Miyuki Sato, Ken Sato
In Caenorhabditis elegans embryos, paternally inherited mitochondria and their mitochondrial DNA are degraded via selective autophagy called allophagy (allogeneic organelle autophagy). This is a developmentally programmed autophagy and combined with C. elegans genetics and in vivo imaging provides a unique opportunity to analyze selective autophagy under physiological conditions.
April 20, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361485/the-use-of-correlative-light-electron-microscopy-clem-to-study-pink1-parkin-mediated-mitophagy
#4
Chieko Kishi-Itakura, Folma Buss
In this chapter we describe the use of correlative light-electron microscopy (CLEM) to study, in cultured cells, the turnover of damaged mitochondria by PINK1/Parkin-dependent mitophagy. CLEM combines the advantages of light microscopy, which allows to image and rapidly screen a large number of the cells, while electron microscopy provides high-resolution imaging of these selected cells and a detailed structural analysis of their cellular organelles. We describe in detail how to prepare the cell cultures for optimum preservation of their cellular ultrastructure for CLEM using the most suitable buffers, fixatives, and embedding resins...
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361484/a-cleanup-method-for-mass-spectrometric-analysis-of-sphingosine-and-ceramide-1-phosphate-in-blood-and-solid-tissue-using-a-phosphate-capture-molecule
#5
Jun-Ichi Morishige, Ryouhei Yamashita, Tamotsu Tanaka, Kiyoshi Satouchi
Cleanup technology and mass spectrometric determination of sphingosine-1-phosphate (S1P) using a phosphate capture molecule are shown. The protocol is rapid, requires neither thin-layer chromatography nor liquid chromatography, and is applicable to both blood and solid tissue samples. The mass spectrometric method is also applicable to ceramide-1-phosphate.
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361483/immunocytochemical-monitoring-of-pink1-parkin-mediated-mitophagy-in-cultured-cells
#6
Motoki Fujimaki, Shinji Saiki, Yukiko Sasazawa, Kei-Ichi Ishikawa, Yoko Imamichi, Katsuhiko Sumiyoshi, Nobutaka Hattori
Both PINK1 and parkin are the responsible genes (PARK6 and PARK2, respectively) for familial early-onset Parkinson's disease (PD). Several lines of evidences have suggested that mitochondrial dysfunction would be associated with PD pathogenesis. Lewy body, one of PD pathological hallmarks, contains alpha-synuclein, a familial PD (PARK1/4)-gene product, which is eliminated by macroautophagy, while PINK1 and parkin coordinately mediate mitophagy (hereafter called as PINK1/parkin-mediated mitophagy) reported firstly by Youle's group...
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361482/induction-of-pink1-parkin-mediated-mitophagy
#7
Shigeto Sato, Norihiko Furuya
PINK1/Parkin mitophagy is a key mechanism to contribute mitochondrial quality control, and the defects are thought to be a cause of those Parkinson's disease onsets. Upon loss of mitochondrial membrane potential, PINK1 and Parkin are activated to promote the proteasomal degradation of mitochondrial outer membrane proteins and selective elimination of damaged mitochondria by autophagy. In this chapter, we describe the methods for induction of PINK1/Parkin-mediated mitophagy in tissue culture cell lines.
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361481/sphingosine-1-phosphate-s1p-signaling-in-neural-progenitors
#8
Phillip Callihan, Mohammed Alqinyah, Shelley B Hooks
Sphingosine-1-phosphate (S1P) and its receptors are important in nervous system development. Reliable in vitro human model systems are needed to further define specific roles for S1P signaling in neural development. We have described S1P-regulated signaling, survival, and differentiation in a human embryonic stem cell-derived neuroepithelial progenitor cell line (hNP1) that expresses functional S1P receptors. These cells can be further differentiated to a neuronal cell type and therefore represent a good model system to study the role of S1P signaling in human neural development...
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361480/human-intestinal-enteroids-new-models-to-study-gastrointestinal-virus-infections
#9
Winnie Y Zou, Sarah E Blutt, Sue E Crawford, Khalil Ettayebi, Xi-Lei Zeng, Kapil Saxena, Sasirekha Ramani, Umesh C Karandikar, Nicholas C Zachos, Mary K Estes
Human rotavirus (HRV) and human norovirus (HuNoV) infections are recognized as the most common causes of epidemic and sporadic cases of gastroenteritis worldwide. The study of these two human gastrointestinal viruses is important for understanding basic virus-host interactions and mechanisms of pathogenesis and to establish models to evaluate vaccines and treatments. Despite the introduction of live-attenuated vaccines to prevent life-threatening HRV-induced disease, the burden of HRV illness remains significant in low-income and less-industrialized countries, and small animal models or ex vivo models to study HRV infections efficiently are lacking...
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28361479/a-simple-method-of-generating-3d-brain-organoids-using-standard-laboratory-equipment
#10
Magdalena Sutcliffe, Madeline A Lancaster
3D brain organoids are a powerful tool with prospective application for the study of neural development and disease. Here we describe the growth factor-free method of generating cerebral organoids from feeder-dependent or feeder-free human pluripotent stem cells using standard laboratory equipment. The protocol outlined below allows generation of 3D tissues, which replicate human early in vivo brain development up to the end of the first trimester, both in terms of morphology and gene expression pattern.
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28349502/measuring-sphingosine-1-phosphate-protein-interactions-with-the-kinetic-exclusion-assay
#11
Jonathan K Fleming, Jonathan M Wojciak
By directly detecting the ligand-free binding sites in a sample, the kinetic exclusion assay (KinExA(®)) provides a compelling alternative to SPR-based techniques for determining equilibrium dissociation constants of protein-ligand interactions. It is especially useful for observing protein-lipid interactions, as binding of native lipids occurs entirely in solution, and monoclonal antibodies can be used to directly compete with a protein of interest for lipid binding. By measuring the antigen-free binding sites on the antibody and using competition affinity analysis, the K d for the lipid binding the protein and the antibody can be determined simultaneously...
March 28, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28337709/exploring-micrornas-on-nix-dependent-mitophagy
#12
Wen Li, Hao Chen, Shupeng Li, Guanghong Lin, Du Feng
The dysregulation of autophagy is implicated in many pathological disorders including infections, aging, neurodegenerative diseases, and cancer. Autophagy can be precisely controlled both transcriptionally and translationally. Accumulating evidences show that the autophagy response is regulated by microRNAs, which therefore becomes subject area of interest in recent years. Herein, we give a brief introduction of the recent advancement in the regulation of microRNA on autophagy, and then we focus on the microRNA regulation of the mitophagy receptor, NIX...
March 24, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28337708/intestinal-crypt-organoid-isolation-of-intestinal-stem-cells-in-vitro-culture-and-optical-observation
#13
Yun Chen, Chuan Li, Ya-Hui Tsai, Sheng-Hong Tseng
The isolation and culture of intestinal stem cells (ISCs) was first demonstrated in the very recent decade with the identification of ISC marker Lgr5. The growth of ISCs into crypt organoids provides an in vitro model for studying the mucosal physiology, intestinal cancer tumorigenesis, and intestinal regeneration. Here, we describe two different isolation protocols and demonstrate a fixation method that aids in the confocal observation of the organoids.
March 24, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28337707/investigation-of-yeast-mitophagy-with-fluorescence-microscopy-and-western-blotting
#14
Sachiyo Nagumo, Koji Okamoto
Selective clearance of superfluous or dysfunctional mitochondria is a fundamental process that depends on the autophagic membrane trafficking pathways found in many cell types. This catabolic event, called mitophagy, is conserved from yeast to humans and serves to control mitochondrial quality and quantity. In budding yeast, degradation of mitochondria occurs under various physiological conditions, such as respiration at stationary phase, or starvation in a prolonged period. During these events, the transmembrane protein Atg32 localizes to the mitochondrial surface and plays a specific and essential role in yeast mitophagy...
March 24, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324492/detection-of-hypoxia-induced-and-iron-depletion-induced-mitophagy-in-mammalian-cells
#15
Shun-Ichi Yamashita, Tomotake Kanki
Mitochondrial quality and quantity are not only regulated by mitochondrial fusion and fission but also by mitochondria degradation. Mitophagy, an autophagy specific for damaged or unnecessary mitochondria, is believed to be an important pathway for mitochondrial homeostasis. To date, several stimuli are known to induce mitophagy. Some of these stimuli, however, including hypoxia, iron depletion, and nitrogen starvation, induce mild mitophagy, which is difficult to detect through decreased mitochondrial mass...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324491/monitoring-mitophagy-during-aging-in-caenorhabditis-elegans
#16
Nikolaos Charmpilas, Konstantinos Kounakis, Nektarios Tavernarakis
Mitochondria constitute the main energy-producing centers of eukaryotic cells. In addition, they are involved in several crucial cellular processes, such as lipid metabolism, calcium buffering, and apoptosis. As such, their malfunction can be detrimental for proper cellular physiology and homeostasis. Mitophagy is a mechanism that protects and maintains cellular function by sequestering harmful or dysfunctional mitochondria to lysosomes for degradation. In this report, we present experimental procedures for quantitative, in vivo monitoring of mitophagy events in the nematode Caenorhabditis elegans...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324490/assessment-of-mitophagy-in-ips-cell-derived-neurons
#17
Kei-Ichi Ishikawa, Akihiro Yamaguchi, Hideyuki Okano, Wado Akamatsu
Aberrant mitochondrial function is associated with many neurological diseases. Mitophagy is a key mechanism for the elimination of damaged mitochondria and maintenance of mitochondrial homeostasis. Induced pluripotent stem (iPS) cell technologies developed over the last decade have allowed us to analyze functions of the human neuron. Here we describe an efficient induction method from human iPS cells to neurons, followed by an image-based mitophagy assay.
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324489/monitoring-mitochondrial-changes-by-alteration-of-the-pink1-parkin-signaling-in-drosophila
#18
Tsuyoshi Inoshita, Kahori Shiba-Fukushima, Hongrui Meng, Nobutaka Hattori, Yuzuru Imai
Mitochondrial quality control is a key process in tissues with high energy demands, such as the brain and muscles. Recent studies using Drosophila have revealed that the genes responsible for familial forms of juvenile Parkinson's disease (PD), PINK1 and Parkin regulate mitochondrial function and motility. Cell biological analysis using mammalian cultured cells suggests that the dysregulation of mitophagy by PINK1 and Parkin leads to neurodegeneration in PD. In this chapter, we describe the methods to monitor mitochondrial morphology in the indirect flight muscles of adult Drosophila and Drosophila primary cultured neurons and the methods to analyze the motility of mitochondria in the axonal transport of living larval motor neurons...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324488/flow-cytometer-monitoring-of-bnip3-and-bnip3l-nix-dependent-mitophagy
#19
Matilda Šprung, Ivan Dikic, Ivana Novak
Mitochondria are organelles with numerous vital roles in cellular metabolism. Impaired or damaged mitochondria are degraded in autophagolysosomes in a process known as mitophagy. Given the fundamental role of mitophagy in maintenance of cellular homeostasis, methods and techniques with which to study this process are constantly evolving and emerging. So far, mitophagy flux was mostly monitored using fluorescently labeled LC3 protein on autophagosomal membrane and any of the labeled outer mitochondrial membrane proteins...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324487/mitophagy-in-yeast-a-screen-of-mitophagy-deficient-mutants
#20
Kentaro Furukawa, Tomotake Kanki
Mitochondrial autophagy (mitophagy) is a process that selectively degrades mitochondria via autophagy. Recent studies have shown that mitophagy plays an important role in mitochondrial homeostasis by degrading damaged or excess mitochondria. The budding yeast Saccharomyces cerevisiae is a powerful model organism that has been employed to study several biological phenomena. Recently, there has been significant progress in the understanding of mitophagy in yeast following the identification of Atg32, a mitochondrial outer membrane receptor protein for mitophagy...
March 22, 2017: Methods in Molecular Biology
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