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High throughput microscopy

Sean Yu, Pranav Joshi, Yi Ju Park, Kyeong-Nam Yu, Moo-Yeal Lee
Layer-by-layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D-printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries...
December 14, 2017: Biotechnology Progress
Joanna Hołówka, Małgorzata Płachetka
According to recent reports, bacterial chromosomes exhibit a hierarchical organization. The number of proteins that bind DNA are responsible for local and global organization of the DNA ensuring proper chromosome compaction. Advanced molecular biology techniques combined with high-throughput DNA sequencing methods allow a precise analysis of bacterial chromosome structures on a local and global scale. Methods such as in vivo footprinting and ChIP-seq allow to map binding sites of analyzed proteins in certain chromosomal regions or along the whole chromosome while analysis of the spatial interactions on global scale could be performed by 3C techniques...
December 8, 2017: Postȩpy Higieny i Medycyny Doświadczalnej
Xusi Han, Qing Wei, Daisuke Kihara
With the rapid growth in the number of solved protein structures stored in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB), it is essential to develop tools to perform real-time structure similarity searches against the entire structure database. Since conventional structure alignment methods need to sample different orientations of proteins in the three-dimensional space, they are time consuming and unsuitable for rapid, real-time database searches. To this end, we have developed 3D-SURFER and EM-SURFER, which utilize 3D Zernike descriptors (3DZD) to conduct high-throughput protein structure comparison, visualization, and analysis...
December 8, 2017: Current Protocols in Bioinformatics
Namrata Gautam, Shvetha Sankaran, John A Yason, Kevin S W Tan, Nicholas R J Gascoigne
Mitochondria, the powerhouse of the cell, are known to remodel their membrane structures through the process of fusion or fission. Studies have indicated that T cells adopt different energy metabolic phenotypes, namely oxidative phosphorylation and glycolysis depending on whether they are naïve, effector and memory T cells. It has recently been shown that changes in mitochondrial morphology dictate T cell fate via regulation of their metabolism. Our keen interest in T cell function and metabolism led us to explore and establish a method to study mitochondria in live T cells through a novel high content approach called Imaging Flow Cytometry (IFC)...
November 30, 2017: Methods: a Companion to Methods in Enzymology
Sarthak Gupta, Diana W Chan, Kristien J Zaal, Mariana J Kaplan
Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by various mechanisms, including formation of neutrophil extracellular traps through a recently described distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in an unbiased, reproducible, and efficient way are lacking, considerably limiting the advancement of research in this field. We optimized and validated, a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using the IncuCyte ZOOM imaging platform and the membrane-permeability properties of two DNA dyes...
December 1, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
Nico Salmaso, Davide Albanese, Camilla Capelli, Adriano Boscaini, Massimo Pindo, Claudio Donati
High-throughput sequencing (HTS) was used to analyze the seasonal variations in the bacterioplankton community composition (BCC) in the euphotic layer of a large and deep lake south of the Alps (Lake Garda). The BCC was analyzed throughout two annual cycles by monthly samplings using the amplification and sequencing of the V3-V4 hypervariable region of the 16S rRNA gene by the MiSeq Illumina platform. The dominant and most diverse bacterioplankton phyla were among the more frequently reported in freshwater ecosystems, including the Proteobacteria, Cyanobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Planctomycetes...
December 1, 2017: Microbial Ecology
Yuri Lysov, Victor Barsky, Dmitriy Urasov, Roman Urasov, Alecksey Cherepanov, Dmitryi Mamaev, Yegor Yegorov, Alexander Chudinov, Sergey Surzhikov, Alla Rubina, Olga Smoldovskaya, Alexander Zasedatelev
A microarray analyzer was developed to obtain images and measure the fluorescence intensity of microarrays at three wavelengths from 380 nm to 850 nm. The analyzer contains lasers to excite fluorescence, barrier filters, optics to project images on an image detector, and a device for suppressing laser speckles on the microarray support. The speckle suppression device contains a fibre-optic bundle and a rotating mirror positioned in a way to change the distance between the bundle butt and mirror surface during each mirror revolution...
November 1, 2017: Biomedical Optics Express
Prabhakar R Gudla, Koh Nakayama, Gianluca Pegoraro, Tom Misteli
DNA fluorescence in situ hybridization (FISH) is the technique of choice to map the position of genomic loci in three-dimensional (3D) space at the single allele level in the cell nucleus. High-throughput DNA FISH methods have recently been developed using complex libraries of fluorescently labeled synthetic oligonucleotides and automated fluorescence microscopy, enabling large-scale interrogation of genomic organization. Although the FISH signals generated by high-throughput methods can, in principle, be analyzed by traditional spot-detection algorithms, these approaches require user intervention to optimize each interrogated genomic locus, making analysis of tens or hundreds of genomic loci in a single experiment prohibitive...
November 28, 2017: Cold Spring Harbor Symposia on Quantitative Biology
Xing-Ru Chen, Xiao-Ting Wang, Mei-Qi Hao, Yong-Hui Zhou, Wen-Qiang Cui, Xiao-Xu Xing, Chang-Geng Xu, Jing-Wen Bai, Yan-Hua Li
The imidazole glycerophosphate dehydratase (IGPD) protein is a therapeutic target for herbicide discovery. It is also regarded as a possible target in Staphylococcus xylosus (S. xylosus) for solving mastitis in the dairy cow. The 3D structure of IGPD protein is essential for discovering novel inhibitors during high-throughput virtual screening. However, to date, the 3D structure of IGPD protein of S. xylosus has not been solved. In this study, a series of computational techniques including homology modeling, Ramachandran Plots, and Verify 3D were performed in order to construct an appropriate 3D model of IGPD protein of S...
2017: Frontiers in Chemistry
Paola Roncaglia, Teunis J P van Dam, Karen R Christie, Lora Nacheva, Grischa Toedt, Martijn A Huynen, Rachael P Huntley, Toby J Gibson, Jane Lomax
Background: Recent research into ciliary structure and function provides important insights into inherited diseases termed ciliopathies and other cilia-related disorders. This wealth of knowledge needs to be translated into a computational representation to be fully exploitable by the research community. To this end, members of the Gene Ontology (GO) and SYSCILIA Consortia have worked together to improve representation of ciliary substructures and processes in GO. Methods: Members of the SYSCILIA and Gene Ontology Consortia suggested additions and changes to GO, to reflect new knowledge in the field...
2017: Cilia
Miguel García-Tecedor, Smagul Karazhanov, Geraldo Cristian Vásquez, Halvard Haug, David Maestre, A Cremades, María Taeño, Julio Ramirez-Castellanos, José María González-Calbet, Javier Piqueras, Chang Chuan You, Erik Stensrud Marstein
In this paper, we present a study of the silicon surface passivation based on the use of spin coated hybrid composite layers. We investigate both undoped poly(3,4-ethylenedioxythiophene)/poly-(styrenesulfonate) (PEDOT:PSS), as well as PEDOT:PSS functionalized with semiconducting oxide nanomaterials (TiO2 and SnO2). The hybrid compound was deposited at room temperature by spin coating, a potentially lower cost, process time and higher throughput alternative compared with the commonly used vacuum-based techniques...
November 23, 2017: Nanotechnology
Bastiaan Spanjaard, Jan Philipp Junker
Determining the lineage origin of cell types is a major goal in developmental biology. Furthermore, lineage tracing is a powerful approach for understanding the origin of developmental defects as well as the origin of diseases such as cancer. There is now a variety of complementary approaches for identifying lineage relationships, ranging from direct observation of cell divisions by light microscopy to genetic labeling of cells using inducible recombinases and fluorescent reporters. A recent development, and the main topic of this review article, is the use of high-throughput sequencing data for lineage analysis...
November 22, 2017: Current Opinion in Cell Biology
Kazuma Yasuhara, Jin Arakida, Thirupathi Ravula, Sudheer Kumar Ramadugu, Bikash Sahoo, Jun-Ichi Kikuchi, Ayyalusamy Ramamoorthy
There is a growing interest in the use of lipid bilayer nanodiscs for various biochemical and biomedical applications. Among the different types of nanodiscs, the unique features of synthetic polymer-based nanodiscs have attracted additional interest. A styrene-maleic acid (SMA) copolymer demonstrated to form lipid nanodiscs have been used for structural biology related studies on membrane proteins. However, the application of SMA polymer based lipid nanodiscs is limited because of the strong absorption of the aromatic group interfering with various experimental measurements...
November 24, 2017: Journal of the American Chemical Society
Bruce E Deagle, Laurence J Clarke, John A Kitchener, Andrea M Polanowski, Andrew T Davidson
DNA metabarcoding is an efficient method for measuring biodiversity, but the process of initiating long-term DNA-based monitoring programs, or integrating with conventional programs, is only starting. In marine ecosystems, plankton surveys using the continuous plankton recorder (CPR) have characterised biodiversity along transects covering millions of kilometres with time-series spanning decades. We investigated the potential for use of metabarcoding in CPR surveys. Samples (n= 53) were collected in two Southern Ocean transects and metazoans identified using standard microscopic methods and by high-throughput sequencing of a cytochrome c oxidase subunit I marker...
November 23, 2017: Molecular Ecology Resources
Henry Hui, Kathryn A Fuller, Hun Chuah, James Liang, Hasib Sidiqi, Dejan Radeski, Wendy N Erber
Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells...
November 15, 2017: Methods: a Companion to Methods in Enzymology
Joana R Lérias, Madalena C Pinto, Hugo M Botelho, Nikhil T Awatade, Margarida C Quaresma, Iris A L Silva, Podchanart Wanitchakool, Rainer Schreiber, Rainer Pepperkok, Karl Kunzelmann, Margarida D Amaral
An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca(2+)-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization...
November 15, 2017: Biochimica et Biophysica Acta
E Huttunen-Saarivirta, P Rajala, M Marja-Aho, J Maukonen, E Sohlberg, L Carpén
In this work, six common stainless steel grades were compared with respect to ennoblement characteristics, corrosion performance and tendency to biofouling in brackish sea water in a pilot-scale cooling water circuit. Two tests were performed, each employing three test materials, until differences between the materials were detected. Open circuit potential (OCP) was measured continuously in situ. Potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) measurements were conducted before and after the tests...
November 5, 2017: Bioelectrochemistry
E Fréalle, V Bex, G Reboux, S Roussel, S Bretagne
INTRODUCTION: To study the impact of the constant and inevitable inhalation of moulds, it is necessary to sample, identify and count the spores. BACKGROUND: Environmental sampling methods can be separated into three categories: surface sampling is easy to perform but non quantitative, air sampling is easy to calibrate but provides time limited information, and dust sampling which is more representative of long term exposure to moulds. The sampling strategy depends on the objectives (evaluation of the risk of exposure for individuals; quantification of the household contamination; evaluation of the efficacy of remediation)...
November 15, 2017: Revue des Maladies Respiratoires
Sirong Zhou, Jiarong Zhu, Yaping Li, Liheng Feng
Conjugated polymer nanoparticles (CPNs) with high brightness in long wavelength region were prepared by the nano-precipitation method. Based on fluorescence resonance energy transfer (FRET) mechanism, the high brightness property of the CPNs was realized by four different emission polymers. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) displayed that the CPNs possessed a spherical structure and an average diameter of ~75nm. Analysis assays showed that the CPNs had excellent biocompatibility, good photostability and low cytotoxicity...
November 11, 2017: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
Jason Q Core, Mehrsa Mehrabi, Zachery R Robinson, Alexander R Ochs, Linda A McCarthy, Michael V Zaragoza, Anna Grosberg
Nuclear shape defects are a distinguishing characteristic in laminopathies, cancers, and other pathologies. Correlating these defects to the symptoms, mechanisms, and progression of disease requires unbiased, quantitative, and high-throughput means of quantifying nuclear morphology. To accomplish this, we developed a method of automatically segmenting fluorescently stained nuclei in 2D microscopy images and then classifying them as normal or dysmorphic based on three geometric features of the nucleus using a package of Matlab codes...
2017: PloS One
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