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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
Xiaosa Li, Ying Wang, Yajing Liu, Bei Yang, Xiao Wang, Jia Wei, Zongyang Lu, Yuxi Zhang, Jing Wu, Xingxu Huang, Li Yang, Jia Chen
The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich protospacer-adjacent motif (PAM) sequences. To overcome this limitation, we developed a CRISPR-Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and catalyzes C-to-T conversion in human cells, while inducing low levels of indels, non-C-to-T substitutions and off-target editing.
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
Joseph B DeShields, Rachel A Bomberger, James W Woodhall, David L Wheeler, Natalia Moroz, Dennis A Johnson, Kiwamu Tanaka
On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection...
February 23, 2018: Journal of Visualized Experiments: JoVE
Xing Chen, Detlef Wolf, Juliane Siebourg-Polster, Christian Czech, Ulrike Bonati, Dirk Fischer, Omar Khwaja, Martin Strahm
Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people's movement in many medical and research fields...
February 12, 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
Barun Das, Jashdeep Bhattacharjee, Preeti, Alaknanda Mishra, Kshama Jain, Srikanth Iyer, Ashwani Kesarwani, Parul Sahu, Prakriti Sinha, Perumal Nagarajan, Pramod Upadhyay
Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD...
February 10, 2018: Journal of Visualized Experiments: JoVE
Youn Young Shim, Rana Mustafa, Jianheng Shen, Kornsulee Ratanapariyanuch, Martin J T Reaney
Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce stable foams and emulsions, and can act as a thickener. Recently interest in this product has been enhanced through the internet where it is proposed that this solution, now called aquafaba by a growing community, can be used a replacement for egg and milk protein. As aquafaba is both new and being developed by an internet based community little is known of its composition or properties...
February 10, 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
Michael D Guthrie, Donald L Gilbert, David A Huddleston, Ernest V Pedapati, Paul S Horn, Stewart H Mostofsky, Steve W Wu
We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution...
February 8, 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
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
Gizem Ertürk, Rolf Lood
The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study...
February 16, 2018: Journal of Visualized Experiments: JoVE
Holger Fehlauer, Adam L Nekimken, Anna A Kim, Beth L Pruitt, Miriam B Goodman, Michael Krieg
One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C...
February 19, 2018: Journal of Visualized Experiments: JoVE
Federica Cavalera, Mario Zanoni, Valeria Merico, Thi Thu Hien Bui, Martina Belli, Lorenzo Fassina, Silvia Garagna, Maurizio Zuccotti
Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure...
March 3, 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
Jianhua Ren, Yadwinder S Mann, Yuntao Zhang, Michael D Browne
Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques...
February 21, 2018: Journal of Visualized Experiments: JoVE
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