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https://www.readbyqxmd.com/read/28650509/a-co-expression-strategy-to-achieve-labeling-of-individual-subunits-within-a-dimeric-protein-for-single-molecule-analysis
#1
Fei Lou, Jie Yang, Si Wu, Sarah Perrett
A generic co-expression strategy for site-specific incorporation of a single donor-acceptor dye pair into any position in a dimeric protein, allowing single molecule FRET study of proteins previously inaccessible to this technique, such as the intrinsically disordered prion N-domain of Ure2 in the context of its globular dimeric C-domain.
June 26, 2017: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/28650478/a-dna-origami-platform-for-quantifying-protein-copy-number-in-super-resolution
#2
Francesca Cella Zanacchi, Carlo Manzo, Angel S Alvarez, Nathan D Derr, Maria F Garcia-Parajo, Melike Lakadamyali
Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy...
June 26, 2017: Nature Methods
https://www.readbyqxmd.com/read/28650176/the-role-of-through-space-interactions-in-modulating-constructive-and-destructive-interference-effects-in-benzene
#3
Anders Borges, Jianlong Xia, Sheng-Hua Liu, Latha Venkataraman, Gemma C Solomon
Quantum interference effects, be they constructive or destructive, are key to predicting and understanding the electrical conductance of single molecules. Here, through theory and experiment, we investigate a family of benzene-like molecules that exhibit both constructive and destructive interference effects arising due to more than one contact between the molecule and each electrode. In particular, we demonstrate that the π-system of meta-coupled benzene can exhibit constructive interference and its para-coupled analog can exhibit destructive interference - and vice versa - depending on the specific through-space interactions...
June 26, 2017: Nano Letters
https://www.readbyqxmd.com/read/28648629/single-molecule-fret-studies-of-ion-channels
#4
REVIEW
Boris Martinac
Different types of fluorescence spectroscopy approaches have over the last two decades become important techniques in studies of ion channel structure and dynamics. Many fluorescence methods have been used to examine a huge variety of ion channels. Any fluorescence study of ion channels requires the presence of fluorophores, which may be intrinsic to the channel protein, attached either extrinsically to the protein, or be simply located nearby the channel to monitor local conditions such as for many of the ion-sensitive dyes...
June 22, 2017: Progress in Biophysics and Molecular Biology
https://www.readbyqxmd.com/read/28646215/single-molecule-and-multiple-bond-characterization-of-catch-bond-associated-cytoadhesion-in-malaria
#5
Ying Bena Lim, Juzar Thingna, Jianshu Cao, Chwee Teck Lim
The adhesion of malaria infected red blood cells (iRBCs) to host endothelial receptors in the microvasculature, or cytoadhesion, is associated with severe disease pathology such as multiple organ failure and cerebral malaria. Malaria iRBCs have been shown to bind to several receptors, of which intercellular adhesion molecule 1 (ICAM-1) upregulation in brain microvasculature is the only one correlated to cerebral malaria. We utilize a biophysical approach to study the interactions between iRBCs and ICAM-1. At the single molecule level, force spectroscopy experiments reveal that ICAM-1 forms catch bond interactions with Plasmodium falciparum parasite iRBCs...
June 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28645642/ngs-and-blood-group-systems-state-of-the-art-and-perspectives
#6
Y Fichou, C Férec
Molecular analysis, or genotyping, of genes involved in the expression of blood group antigens has been a standard strategy used in immunohaematology laboratories routinely. For the past ten years, next-generation sequencing (NGS), or second-generation sequencing, has become the reference method in genetics. Extensive study of distinct targets, large genomic regions, and even whole genome is henceforth possible by this approach at minimal cost. Blood group genotyping has thus taken advantage of this technological advent...
June 20, 2017: Transfusion Clinique et Biologique: Journal de la Société Française de Transfusion Sanguine
https://www.readbyqxmd.com/read/28645380/comet-fish-for-ultrasensitive-strand-specific-detection-of-dna-damage-in-single-cells
#7
Manas Mondal, Jia Guo
The genome integrity of living organisms is constantly threatened by endogenous cellular metabolic processes and environmental agents. To quantify these low, physiologically relevant levels of DNA damage, a single-cell gel electrophoresis (comet) combined with strand-specific fluorescence in situ hybridization (FISH)-based approach has been developed. This approach enables the quantification of low levels of specific DNA lesions in each strand of the selected sequence at the single-molecule sensitivity, as well as in the genome overall in single cells...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28645379/dna-fiber-analysis-mind-the-gap
#8
Annabel Quinet, Denisse Carvajal-Maldonado, Delphine Lemacon, Alessandro Vindigni
Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28645371/ensemble-and-single-molecule-analysis-of-non-homologous-end-joining-in-frog-egg-extracts
#9
Thomas G W Graham, Johannes C Walter, Joseph J Loparo
Non-homologous end joining (NHEJ) repairs the majority of DNA double-strand breaks in human cells, yet the detailed order of events in this process has remained obscure. Here, we describe how to employ Xenopus laevis egg extract for the study of NHEJ. The egg extract is easy to prepare in large quantities, and it performs efficient end joining that requires the core end joining proteins Ku, DNA-PKcs, XLF, XRCC4, and DNA ligase IV. These factors, along with the rest of the soluble proteome, are present at endogenous concentrations, allowing mechanistic analysis in a system that begins to approximate the complexity of cellular end joining...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28644628/stereoelectronics-of-the-hydrogen-bond-induced-fluorescence-quenching-of-3-aminofluorenones-with-alcohols
#10
Isaac G Alty, Christopher John Abelt
Two derivatives of 3-amino-9-fluorenone (1) bearing one (2) and two methyl (3) groups flanking carbonyl group are prepared. Comparison of their photophysical properties show that all suffer efficient radiationless deactivation in the presence of alcohols. Preferential solvation studies with mono alcohols reveal that a single H-bonding interaction quenches the excited states of 1 and 2, but not that of 3. In contrast, a single molecule of ethylene glycol quenches all three. These results are interpreted in a quenching mechanism similar to one proposed by Inoue and coworkers, but where an out-of-plane H-bond with the carbonyl group gives rise to an emissive species, while an in-plane H-bond results in quenching...
June 23, 2017: Journal of Physical Chemistry. A
https://www.readbyqxmd.com/read/28643869/mechanotransduction-properties-of-the-cytoplasmic-tail-of-pecam-1
#11
Jessica L Snyder, Elena McBeath, Tamlyn N Thomas, Yi Jen Chiu, Robert L Clark, Keigi Fujiwara
BACKGROUND: Vascular endothelial cells (ECs) are a well-known cell system used in the study of mechanobiology. Using cultured ECs, we found that platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31), a cell adhesion protein localized to regions of EC-EC contact, was rapidly tyrosine phosphorylated in ECs exposed to shear or cyclic stretch. Src-homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) binds phosphorylated PECAM-1 and activates the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade, a known flow-activated signaling pathway...
June 23, 2017: Biology of the Cell
https://www.readbyqxmd.com/read/28642595/3d-bayesian-cluster-analysis-of-super-resolution-data-reveals-lat-recruitment-to-the-t-cell-synapse
#12
Juliette Griffié, Leigh Shlomovich, David J Williamson, Michael Shannon, Jesse Aaron, Satya Khuon, Garth L Burn, Lies Boelen, Ruby Peters, Andrew P Cope, Edward A K Cohen, Patrick Rubin-Delanchy, Dylan M Owen
Single-molecule localisation microscopy (SMLM) allows the localisation of fluorophores with a precision of 10-30 nm, revealing the cell's nanoscale architecture at the molecular level. Recently, SMLM has been extended to 3D, providing a unique insight into cellular machinery. Although cluster analysis techniques have been developed for 2D SMLM data sets, few have been applied to 3D. This lack of quantification tools can be explained by the relative novelty of imaging techniques such as interferometric photo-activated localisation microscopy (iPALM)...
June 22, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28642368/the-influence-of-disulfide-bonds-on-the-mechanical-stability-of-proteins-is-context-dependent
#13
Aitor Manteca, Álvaro Alonso-Caballero, Marie Fertin, Simon Poly, David De Sancho, Raul Perez-Jimenez
Disulfide bonds play a crucial role in proteins, modulating their stability and constraining their conformational dynamics. A particularly important case is that of proteins that need to withstand forces arising from their normal biological function and that are often disulfide bonded. However, the influence of disulfides on the overall mechanical stability of proteins is poorly understood. Here, we used single-molecule force spectroscopy (smFS) to study the role of disulfide bonds in different mechanical proteins in terms of their unfolding forces...
June 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28640936/preparation-single-molecule-manipulation-and-energy-transfer-investigation-of-a-polyfluorene-graft-dna-polymer
#14
Mikael Madsen, Rasmus S Christensen, Abhichart Krissanaprasit, Mette R Bakke, Camilla F Riber, Karina S Nielsen, Alexander Zelikin, Kurt Vesterager Gothelf
Conjugated polymers have been intensively studied due to their unique optical and electronic properties combined with their physical flexibility and scalable bottom up synthesis. While the bulk qualities of conjugated polymers have been extensively utilized in research and industry, the ability to handle and manipulate conjugated polymers at the nanoscale lacks significantly behind. Here we extend the toolbox for controlled manipulation of conjugated polymers through the synthesis of a polyfluorene-DNA graft type polymer (poly(F-DNA))...
June 22, 2017: Chemistry: a European Journal
https://www.readbyqxmd.com/read/28640691/the-nesprin-cytoskeleton-interface-probed-directly-on-single-nuclei-is-a-mechanically-rich-system
#15
Daniel A Balikov, Sonia K Brady, Ung Hyun Ko, Jennifer H Shin, Jose M de Pereda, Arnoud Sonnenberg, Hak-Joon Sung, Matthew J Lang
The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex...
June 22, 2017: Nucleus
https://www.readbyqxmd.com/read/28639669/plasmon-enhanced-fluorescence-spectroscopy
#16
REVIEW
Jian-Feng Li, Chao-Yu Li, Ricardo F Aroca
Fluorescence spectroscopy with strong emitters is a remarkable tool with ultra-high sensitivity for detection and imaging down to the single-molecule level. Plasmon-enhanced fluorescence (PEF) not only offers enhanced emissions and decreased lifetimes, but also allows an expansion of the field of fluorescence by incorporating weak quantum emitters, avoiding photobleaching and providing the opportunity of imaging with resolutions significantly better than the diffraction limit. It also opens the window to a new class of photostable probes by combining metal nanostructures and quantum emitters...
June 22, 2017: Chemical Society Reviews
https://www.readbyqxmd.com/read/28639652/probing-single-molecule-electron-hole-transfer-dynamics-at-a-molecule-nio-semiconductor-nanocrystalline-interface
#17
Bharat Dhital, Vishal Govind Rao, H Peter Lu
Interfacial charge transfer dynamics in dye-sensitized NiO nanoparticles are being investigated for photocathodes in p-type dye-sensitized solar cells. In the photoreaction, after fast electron transfer from NiO to a molecule, the recombination of the hole in the nanoparticles with the electron in a reduced molecule plays an important role in the charge separation process and solar energy harvesting. Nevertheless, knowledge of the interfacial charge recombination (CR) rate and its mechanism is still limited due to the complex photoinduced electron and hole dynamics and lack of characterization of the inhomogeneity of the dynamics...
June 22, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28638910/dark-plasmonic-mode-based-perfect-absorption-and-refractive-index-sensing
#18
W H Yang, C Zhang, S Sun, J Jing, Q Song, S Xiao
Dark plasmonic resonances in metallic nanostructures are essential for many potential applications such as refractive index sensing, single molecule detection, nanolasers etc. However, it is difficult to excite the dark modes in optical experiments and thus the practical applications are severely limited. Herein, we demonstrate a simple method to experimentally excite the quadrupolar and higher-order plasmonic modes with normal incident light. By directionally depositing silver films onto the sidewalls of metal-covered one-dimensional grating, we have experimentally observed a series of asymmetrical resonances at the plasmonic ranges of silver gratings...
June 22, 2017: Nanoscale
https://www.readbyqxmd.com/read/28638076/single-molecule-analysis-reveals-that-dna-replication-dynamics-vary-across-the-course-of-schizogony-in-the-malaria-parasite-plasmodium-falciparum
#19
Slavica Stanojcic, Nada Kuk, Imran Ullah, Yvon Sterkers, Catherine J Merrick
The mechanics of DNA replication and cell cycling are well-characterized in model organisms, but less is known about these basic aspects of cell biology in early-diverging Apicomplexan parasites, which do not divide by canonical binary fission but undergo unconventional cycles. Schizogony in the malaria parasite, Plasmodium, generates ~16-24 new nuclei via independent, asynchronous rounds of genome replication prior to cytokinesis and little is known about the control of DNA replication that facilitates this...
June 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28637867/full-length-cellular-beta-secretase-has-a-trimeric-subunit-stoichiometry-and-its-sulfur-rich-transmembrane-interaction-site-modulates-cytosolic-copper-compartmentalization
#20
Filip Liebsch, Mark R P Aurousseau, Tobias Bethge, Hugo McGuire, Silvia Scolari, Andreas Herrmann, Rikard Blunck, Derek Bowie, Gerd Multhaup
The beta-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells...
June 21, 2017: Journal of Biological Chemistry
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