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Journal of Visualized Experiments: JoVE

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https://www.readbyqxmd.com/read/28060341/in-vitro-differentiation-of-human-cd4-foxp3-induced-regulatory-t-cells-itregs-from-na%C3%A3-ve-cd4-t-cells-using-a-tgf-%C3%AE-containing-protocol
#1
Angelika Schmidt, Szabolcs Éliás, Rubin N Joshi, Jesper Tegnér
Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3...
December 30, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060332/production-crystallization-and-structure-determination-of-c-difficile-ppep-1-via-microseeding-and-zinc-sad
#2
Christian Pichlo, Angelika A Montada, Magdalena Schacherl, Ulrich Baumann
New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination...
December 30, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060330/hyperpolarized-13c-metabolic-magnetic-resonance-spectroscopy-and-imaging
#3
Eugen Kubala, Kim A Muñoz-Álvarez, Geoffrey Topping, Christian Hundshammer, Benedikt Feuerecker, Pedro A Gómez, Giorgio Pariani, Franz Schilling, Steffen J Glaser, Rolf F Schulte, Marion I Menzel, Markus Schwaiger
In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with (18)F-fluorodeoxyglucose ([(18)F]-FDG PET). (13)C magnetic resonance spectroscopic imaging ((13)CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on (13)C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of (13)C and its low natural abundance in biological samples...
December 30, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060327/led-thermo-flow-combining-optogenetics-with-flow-cytometry
#4
Kathrin Brenker, Kerstin Osthof, Jianying Yang, Michael Reth
Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics...
December 30, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060345/combining-solid-state-and-solution-based-techniques-synthesis-and-reactivity-of-chalcogenidoplumbates-ii-or-iv
#5
Günther Thiele, Carsten Donsbach, Isabell Nußbruch, Stefanie Dehnen
The phases of "PbCh2" (Ch = Se, Te) are obtained from solid-state syntheses (i.e., by the fusion of the elements under inert conditions in silica glass ampules). Reduction of such phases by elemental alkaline metals in amines affords crystalline chalcogenidoplumbate(II) salts comprised of [PbTe3](2)(-) or [Pb2Ch3](2)(-) anions, depending upon which sequestering agent for the cations is present: crown ethers, like 18-crown-6, or cryptands, like [2.2.2]crypt. Reactions of solutions of such anions with transition-metal compounds yield (poly-)chalcogenide anions or transition-metal chalcogenide clusters, including one with a µ-PbSe ligand (i...
December 29, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060321/imaging-of-cell-shape-alteration-and-cell-movement-in-drosophila-gastrulation-using-de-cadherin-reporter-transgenic-flies
#6
M Rezaul Karim, Tomohiro Haruta, Taro Matsumoto, Hiroki Oda, Hiroaki Taniguchi
Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis...
December 29, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060307/lateral-chronic-cranial-window-preparation-enables-in-vivo-observation-following-distal-middle-cerebral-artery-occlusion-in-mice
#7
Simon H Bayerl, Melina Nieminen-Kelhä, Thomas Broggini, Peter Vajkoczy, Vincent Prinz
Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide(1). Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis(2,3). Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process...
December 29, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060276/the-rodent-psychomotor-vigilance-test-rpvt-a-method-for-assessing-neurobehavioral-performance-in-rats-and-mice
#8
Catherine M Davis, Peter G Roma, Robert D Hienz
The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT-termed the "rPVT"-that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training...
December 29, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060349/co-localization-of-cell-lineage-markers-and-the-tomato-signal
#9
Yan Jing, Robert J Hinton, Kevin S Chan, Jian Q Feng
The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-Cre(ERT2) (Agg-Cre(ERT2)), crossed with Rosa26(tdTomato). Both Col10 and aggrecan are well-recognized markers for chondrocytes...
December 28, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060338/a-submerged-filter-paper-sandwich-for-long-term-ex-ovo-time-lapse-imaging-of-early-chick-embryos
#10
Manuel Schmitz, Ben K A Nelemans, Theodoor H Smit
Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation(2), neurulation(3)(-)(5), somitogenesis(6), heart bending(7,8), and brain formation(9)(-)(13), during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo(14)...
December 28, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060304/rearing-and-double-stranded-rna-mediated-gene-knockdown-in-the-hide-beetle-dermestes-maculatus
#11
Jie Xiang, Katie Reding, Leslie Pick
Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries...
December 28, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060265/analysis-of-cap-binding-proteins-in-human-cells-exposed-to-physiological-oxygen-conditions
#12
Sara Timpano, Gaelan Melanson, Sonia L Evagelou, Brianna D Guild, Erin J Specker, James Uniacke
Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m(7)GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation...
December 28, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060354/rapid-quantification-of-mitogen-induced-blastogenesis-in-t-lymphocytes-for-identifying-immunomodulatory-drugs
#13
Jennifer N Gibson, Pavani Beesetty, Courtney Sulentic, J Ashot Kozak
Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter...
December 27, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060335/identification-of-intracellular-signaling-events-induced-in-viable-cells-by-interaction-with-neighboring-cells-undergoing-apoptotic-cell-death
#14
Snezana Vujicic, Lanfei Feng, Angelika Antoni, Joyce Rauch, Jerrold S Levine
Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome...
December 27, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060302/a-platform-of-anti-biofilm-assays-suited-to-the-exploration-of-natural-compound-libraries
#15
Malena E Skogman, Pia M Vuorela, Adyary Fallarero
Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms...
December 27, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060278/nuclear-magnetic-resonance-spectroscopy-for-the-identification-of-multiple-phosphorylations-of-intrinsically-disordered-proteins
#16
Clément Danis, Clément Despres, Luiza M Bessa, Idir Malki, Hamida Merzougui, Isabelle Huvent, Haoling Qi, Guy Lippens, François-Xavier Cantrelle, Robert Schneider, Xavier Hanoulle, Caroline Smet-Nocca, Isabelle Landrieu
Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described...
December 27, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060350/2-methacryloyloxyethyl-phosphorylcholine-polymer-treatment-of-complete-dentures-to-inhibit-denture-plaque-deposition
#17
Kenji Ikeya, Miya Fukunishi, Fuminori Iwasa, Yuuki Inoue, Kazuhiko Ishihara, Kazuyoshi Baba
Removable dentures made of poly (methyl methacrylate) (PMMA) are prone to bacterial adherence and dental plaque formation, which is called denture plaque. Denture plaque-associated infection is a source of serious dental and medical complications in the elderly. 2-Methacryloyloxyethyl phosphorylcholine (MPC) is a well-known biomedical material that exhibits marked antithrombogenicity and tissue compatibility because of its high resistance to protein adsorption and cell adhesion. Therefore, MPC polymer coatings are suggested to have the potential to inhibit plaque deposition on the surface of PMMA dentures...
December 26, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060292/a-portal-vein-injection-model-to-study-liver-metastasis-of-breast-cancer
#18
Erica T Goddard, Jacob Fischer, Pepper Schedin
Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site...
December 26, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060279/quantitative-3d-in-silico-modeling-q3dism-of-cerebral-amyloid-beta-phagocytosis-in-rodent-models-of-alzheimer-s-disease
#19
Marie-Victoire Guillot-Sestier, Tara M Weitz, Terrence Town
Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis...
December 26, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28060271/utilizing-functional-genomics-screening-to-identify-potentially-novel-drug-targets-in-cancer-cell-spheroid-cultures
#20
Eamonn Morrison, Patty Wai, Andri Leonidou, Philip Bland, Saira Khalique, Gillian Farnie, Frances Daley, Barrie Peck, Rachael Natrajan
The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials. Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening...
December 26, 2016: Journal of Visualized Experiments: JoVE
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