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Benjamin Handel, Jonathan Kolstad
No abstract text is available yet for this article.
May 2017: American Economic Review
Valentine Svensson, Sarah A Teichmann, Oliver Stegle
Technological advances have made it possible to measure spatially resolved gene expression at high throughput. However, methods to analyze these data are not established. Here we describe SpatialDE, a statistical test to identify genes with spatial patterns of expression variation from multiplexed imaging or spatial RNA-sequencing data. SpatialDE also implements 'automatic expression histology', a spatial gene-clustering approach that enables expression-based tissue histology.
March 19, 2018: Nature Methods
Daniel Edsgärd, Per Johnsson, Rickard Sandberg
As methods for measuring spatial gene expression at single-cell resolution become available, there is a need for computational analysis strategies. We present trendsceek, a method based on marked point processes that identifies genes with statistically significant spatial expression trends. trendsceek finds these genes in spatial transcriptomic and sequential fluorescence in situ hybridization data, and also reveals significant gene expression gradients and hot spots in low-dimensional projections of dissociated single-cell RNA-seq data...
March 19, 2018: Nature Methods
Rekha Seshadri, Sinead C Leahy, Graeme T Attwood, Koon Hoong Teh, Suzanne C Lambie, Adrian L Cookson, Emiley A Eloe-Fadrosh, Georgios A Pavlopoulos, Michalis Hadjithomas, Neha J Varghese, David Paez-Espino, Rechelle Perry, Gemma Henderson, Christopher J Creevey, Nicolas Terrapon, Pascal Lapebie, Elodie Drula, Vincent Lombard, Edward Rubin, Nikos C Kyrpides, Bernard Henrissat, Tanja Woyke, Natalia N Ivanova, William J Kelly
Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions...
March 19, 2018: Nature Biotechnology
Miten Jain, Hugh E Olsen, Daniel J Turner, David Stoddart, Kira V Bulazel, Benedict Paten, David Haussler, Huntington F Willard, Mark Akeson, Karen H Miga
The human genome reference sequence remains incomplete owing to the challenge of assembling long tracts of near-identical tandem repeats in centromeres. We implemented a nanopore sequencing strategy to generate high-quality reads that span hundreds of kilobases of highly repetitive DNA in a human Y chromosome centromere. Combining these data with short-read variant validation, we assembled and characterized the centromeric region of a human Y chromosome.
March 19, 2018: Nature Biotechnology
Ryan Rosenberry, Susie Chung, Michael D Nelson
Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit...
February 20, 2018: Journal of Visualized Experiments: JoVE
Johannes A Eble
The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record...
February 15, 2018: Journal of Visualized Experiments: JoVE
Markus Köhler, Patrick Oßwald, Dominik Krueger, Ryan Whitside
This manuscript describes a high-temperature flow reactor experiment coupled to the powerful molecular beam mass spectrometry (MBMS) technique. This flexible tool offers a detailed observation of chemical gas-phase kinetics in reacting flows under well-controlled conditions. The vast range of operating conditions available in a laminar flow reactor enables access to extraordinary combustion applications that are typically not achievable by flame experiments. These include rich conditions at high temperatures relevant for gasification processes, the peroxy chemistry governing the low temperature oxidation regime or investigations of complex technical fuels...
February 19, 2018: Journal of Visualized Experiments: JoVE
Tara Nietzold, Bradley M West, Michael Stuckelberger, Barry Lai, Stefan Vogt, Mariana I Bertoni
The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale...
February 17, 2018: Journal of Visualized Experiments: JoVE
Hao-Hsiang Hsu, John-Kevin Kracht, Laura Elisabeth Harder, Kerstin Rudnik, Gerd Lindner, Katharina Schimek, Uwe Marx, Ralf Pörtner
In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP))...
February 23, 2018: Journal of Visualized Experiments: JoVE
José R Agudo, Jiwon Han, Jinyoung Park, Sinyoung Kwon, Soebiakto Loekman, Giovanni Luzi, Christoph Linderberger, Antonio Delgado, Andreas Wierschem
Two different experimental methods for determining the threshold of particle motion as a function of geometrical properties of the bed from laminar to turbulent flow conditions are presented. For that purpose, the incipient motion of a single bead is studied on regular substrates that consist of a monolayer of fixed spheres of uniform size that are regularly arranged in triangular and quadratic symmetries. The threshold is characterized by the critical Shields number. The criterion for the onset of motion is defined as the displacement from the original equilibrium position to the neighboring one...
February 22, 2018: Journal of Visualized Experiments: JoVE
Masahiko Mukaino, Kei Ohtsuka, Hiroki Tanikawa, Fumihiro Matsuda, Junya Yamada, Norihide Itoh, Eiichi Saitoh
Three-dimensional gait analysis (3DGA) is shown to be a useful clinical tool for the evaluation of gait abnormality due to movement disorders. However, the use of 3DGA in actual clinics remains uncommon. Possible reasons could include the time-consuming measurement process and difficulties in understanding measurement results, which are often presented using a large number of graphs. Here we present a clinician-friendly 3DGA method developed to facilitate the clinical use of 3DGA. This method consists of simplified preparation and measurement processes that can be performed in a short time period in clinical settings and intuitive results presentation to facilitate clinicians' understanding of results...
March 4, 2018: Journal of Visualized Experiments: JoVE
Sandra Claes, Thomas D'huys, Anneleen Van Hout, Dominique Schols, Tom Van Loy
G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor's function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12)...
February 20, 2018: Journal of Visualized Experiments: JoVE
Jing Huang, Peng Guo, Marsha A Moses
The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., proliferation, migration, and others) and in vivo models have been developed to investigate and characterize angiogenic and non-angiogenic cell phenotypes, these methods are time and labor intensive, and often require expensive reagents and instruments, as well as significant expertise. In a recent study, we used a novel quantitative phase imaging (QPI) technique to conduct time-lapse and labeling-free characterizations of angiogenic and non-angiogenic human osteosarcoma KHOS cells...
February 16, 2018: Journal of Visualized Experiments: JoVE
Chloé I Charendoff, Lisa Bouchier-Hayes
The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity...
March 5, 2018: Journal of Visualized Experiments: JoVE
Takuma Iguchi, Noriyo Niino, Satoshi Tamai, Ken Sakurai, Kazuhiko Mori
RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification...
February 12, 2018: Journal of Visualized Experiments: JoVE
Jessica C Stark, Euan Wallace, Rebecca Lim, Bryan Leaw
Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury...
February 16, 2018: Journal of Visualized Experiments: JoVE
Polina Zjablovskaja, Petr Danek, Miroslava Kardosova, Meritxell Alberich-Jorda
Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers...
February 21, 2018: Journal of Visualized Experiments: JoVE
Cedric Allier, Romaric Vincent, Fabrice Navarro, Mathilde Menneteau, Lamya Ghenim, Xavier Gidrol, Thomas Bordy, Lionel Hervé, Olivier Cioni, Sabine Bardin, Michel Bornens, Yves Usson, Sophie Morales
Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms...
February 23, 2018: Journal of Visualized Experiments: JoVE
Harmen B Gudde, Debra Griffiths, Kenny R Coventry
The memory game paradigm is a behavioral procedure to explore the relationship between language, spatial memory, and object knowledge. Using two different versions of the paradigm, spatial language use and memory for object location are tested under different, experimentally manipulated conditions. This allows us to tease apart proposed models explaining the influence of object knowledge on spatial language (e.g., spatial demonstratives), and spatial memory, as well as understanding the parameters that affect demonstrative choice and spatial memory more broadly...
February 19, 2018: Journal of Visualized Experiments: JoVE
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