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Single molecule microscopy

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https://www.readbyqxmd.com/read/28102201/direct-pip2-binding-mediates-stable-oligomer-formation-of-the-serotonin-transporter
#1
Andreas Anderluh, Tina Hofmaier, Enrico Klotzsch, Oliver Kudlacek, Thomas Stockner, Harald H Sitte, Gerhard J Schütz
The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization...
January 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/28099797/epitaxy-induced-assembly-and-enantiomeric-switching-of-an-on-surface-formed-dinuclear-organocobalt-complex
#2
Raphael Hellwig, Tobias Paintner, Zhi Chen, Mario Ruben, Ari P Seitsonen, Florian Klappenberger, Harald Brune, Johannes V Barth
We report on the surface-guided synthesis of an unprecedented dinuclear organocobalt complex, its self-assembly into a complex nanoarchitecture, and a single-molecule level investigation of its switching behavior. Initially, an organic layer is prepared by depositing hexakis((trimethylsilyl)ethynyl)-benzene under ultra-high vacuum conditions onto Ag(111). After Co dosage at 200 Kelvin, low-temperature scanning tunneling microscopy (STM) reveals an epitaxy-mediated organization mechanism of molecules and on-surface formed host-guest complexes...
January 18, 2017: ACS Nano
https://www.readbyqxmd.com/read/28098202/superresolution-imaging-of-clinical-formalin-fixed-paraffin-embedded-breast-cancer-with-single-molecule-localization-microscopy
#3
Matthew K Creech, Jing Wang, Xiaolin Nan, Summer L Gibbs
Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries...
January 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28097000/biased-small-molecule-ligands-for-selective-inhibition-of-hiv-1-cell-entry-via-ccr5
#4
Christian Berg, Katja Spiess, Hans R Lüttichau, Mette M Rosenkilde
Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 agonists as HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIV Env and luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout...
December 2016: Pharmacology Research & Perspectives
https://www.readbyqxmd.com/read/28092434/3d-probed-lipid-dynamics-in-small-unilamellar-vesicles
#5
Meng-Hsuan Chao, Yen-Ting Lin, Namasivayam Dhenadhayalan, Hsin-Lung Lee, Hsin-Yen Lee, King-Chuen Lin
Single-molecule fluorescence correlation spectroscopy overcomes the resolution barrier of optical microscopy (10≈-20 nm) and is utilized to look into lipid dynamics in small unilamellar vesicles (SUVs; diameter < 100 nm). The fluorescence trajectories of lipid-like tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in the membrane bilayers are acquired at a single-molecule level. The autocorrelation analysis yields the kinetic information on lipid organization, oxygen transport, and lateral diffusion in SUVs' membrane...
January 16, 2017: Small
https://www.readbyqxmd.com/read/28091541/apelin-receptor-homodimer-oligomers-revealed-by-single-molecule-imaging-and-novel-g-protein-dependent-signaling
#6
Xin Cai, Bo Bai, Rumin Zhang, Chunmei Wang, Jing Chen
The apelin receptor (APJ) belongs to family A of the G protein-coupled receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation...
January 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28080023/microbial-nanoscopy-breakthroughs-challenges-and-opportunities
#7
Yves F Dufrêne
Studying the structure, properties, and interactions of microbial cells is key to understanding the functions of the microbiome. Recent advances in nanotechnology have offered new tools to probe microbes at the single-molecule and single-cell levels. In this issue of ACS Nano, Kumar et al. present an atomic force microscopy method that is capable of imaging the nanoscale organization of bacterial proteins in native, curved membranes. This study represents an important step forward in the development of nanoscopy techniques for analyzing biological systems with large curvature and vertical dimensions, such as membrane vesicles and bacterial cells...
January 12, 2017: ACS Nano
https://www.readbyqxmd.com/read/28078590/quantifying-cd95-cl-cd95l-implications-in-cell-mechanics-and-membrane-tension-by-atomic-force-microscopy-based-force-measurements
#8
Anaïs Sadoun, Pierre-Henri Puech
Atomic force microscopy (AFM) is an invaluable tool to investigate the structure of biological material surfaces by imaging them at nanometer scale in physiological conditions. It can also be used to measure the forces and mechanics from single molecule interaction to cell-cell adhesion. Here, we present a methodology that allows to quantify cell elastic properties (using the Young modulus) and cell membrane tension modulated by CD95/cl-CD95L interactions by coupling nanoindentation and membrane tube pulling using suitably decorated AFM levers...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28078539/unfolding-of-the-myosin-head-by-purealin-in-glycerol
#9
Jiro Takito, Jun'ichi Kobayashi, Masanori Nakamura, Yasushi Ohizumi, Yoshiaki Nonomura
Purealin is a small bioactive compound obtained from the marine sponge. The compound modulates various types of ATPase activity of myosin from skeletal muscle, cardiac muscle, and smooth muscle. To elucidate the structural basis of these effects of purealin on myosin ATPases, we examined the effect of purealin on the conformation of skeletal muscle myosin in aqueous solution and in glycerol. Analysis of the circular dichroism spectrum of subfragment 1, a single-headed fragment of myosin, revealed that in 10% glycerol purealin decreased the β-sheet content of S1, but in aqueous solution it had little effect on the secondary structure of S1...
January 11, 2017: Anatomical Science International
https://www.readbyqxmd.com/read/28076805/regulation-of-active-icam-4-on-normal-and-sickle-cell-disease-rbcs-via-akaps-is-revealed-by-afm
#10
Jing Zhang, Krithika Abiraman, Sasia-Marie Jones, George Lykotrafitis, Biree Andemariam
Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD...
January 10, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28075577/reversible-unfolding-and-folding-of-the-metalloprotein-ferredoxin-revealed-by-single-molecule-atomic-force-microscopy
#11
Hai Lei, Yabin Guo, Xiaodong Hu, Chunguang Hu, Xiaotang Hu, Hongbin Li
Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding-folding of ferredoxins in vitro is challenging, since the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron-sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here we employed atomic force microscopy-based single molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena...
January 11, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28073817/paranodal-dissection-in-chronic-inflammatory-demyelinating-polyneuropathy-with-antineurofascin-155-and-anticontactin-1-antibodies
#12
Haruki Koike, Masato Kadoya, Ken-Ichi Kaida, Shohei Ikeda, Yuichi Kawagashira, Masahiro Iijima, Daisuke Kato, Hidenori Ogata, Ryo Yamasaki, Noriyuki Matsukawa, Jun-Ichi Kira, Masahisa Katsuno, Gen Sobue
OBJECTIVE: To investigate the morphological features of chronic inflammatory demyelinating polyneuropathy (CIDP) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. METHODS: We assessed sural nerve biopsy specimens obtained from 9 patients with CIDP with antineurofascin-155 antibodies and 1 patient with anticontactin-1 antibodies. 13 patients with CIDP without these antibodies were also examined to compare pathological findings...
January 10, 2017: Journal of Neurology, Neurosurgery, and Psychiatry
https://www.readbyqxmd.com/read/28071667/photogate-microscopy-to-track-single-molecules-in-crowded-environments
#13
Vladislav Belyy, Sheng-Min Shih, Jigar Bandaria, Yongjian Huang, Rosalie E Lawrence, Roberto Zoncu, Ahmet Yildiz
Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signalling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density. Here we develop the PhotoGate technique that controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. PhotoGate bypasses the requirement of photoactivation to track single particles at surface densities two orders of magnitude greater than the single-molecule detection limit...
January 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/28070848/intravaginal-delivery-of-polyphenon-60-and-curcumin-nanoemulsion-gel
#14
Atinderpal Kaur, Yashaswee Saxena, Rakhi Bansal, Sonal Gupta, Amit Tyagi, Rakesh Kumar Sharma, Javed Ali, Amulya Kumar Panda, Reema Gabrani, Shweta Dang
Polyphenon 60 (P60) and curcumin (CUR) were loaded in a single nanoemulsion system and their combined antibacterial action was studied against uropathogenic Escherichia coli. To enhance availability at target organs and to inhibit enzymatic degradation in gastro intestinal tract, vaginal route of administration was explored. P60 + CUR nanoemulsion (NE) was formulated by ultra-sonication and optimized using Box-Behnken design. Optimized NE showed Z-average of 211.2 nm, polydispersity index of 0.343, and zeta potential of -32...
January 9, 2017: AAPS PharmSciTech
https://www.readbyqxmd.com/read/28069377/development-of-an-aptamer-ampicillin-conjugate-for-treating-biofilms
#15
Cheng Lijuan, Yan Xing, Wu Minxi, Li Wenkai, Deng Le
Biofilm formation involves the development of extracellular matrix and initially depends on adherence and tropism by flagellar movement. With the widespread development of antibiotic resistance and tolerance of biofilms, there is a growing need for novel anti-infective strategies. No currently approved medications specifically target biofilms. Aptamers are single-stranded nucleic acid molecules that may bind to their targets with high affinity and affect the target functions. We developed a bifunctional conjugate by linking an aptamer targeting bacterial flagella with ampicillin...
January 6, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28068246/intercellular-adhesion-molecule-1-and-vascular-cell-adhesion-molecule-are-induced-by-ionizing-radiation-on-lymphatic-endothelium
#16
María E Rodriguez-Ruiz, Saray Garasa, Inmaculada Rodriguez, Jose Luis Solorzano, Benigno Barbes, Alba Yanguas, Alvaro Teijeira, Iñaki Etxeberria, José Javier Aristu, Cornelia Halin, Ignacio Melero, Ana Rouzaut
PURPOSE/OBJECTIVES: The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. MATERIALS/METHODS: Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays...
February 1, 2017: International Journal of Radiation Oncology, Biology, Physics
https://www.readbyqxmd.com/read/28066821/evaluation-of-receptor-ligand-mechanisms-of-dual-targeted-particles-to-an-inflamed-endothelium
#17
Catherine A Fromen, Margaret B Fish, Anthony Zimmerman, Reheman Adili, Michael Holinstat, Omolola Eniola-Adefeso
Vascular-targeted carriers (VTCs) are designed as leukocyte mimics, decorated with ligands that target leukocyte adhesion molecules (LAMs) and facilitate adhesion to diseased endothelium. VTCs require different design considerations than other targeted particle therapies; adhesion of VTCs in regions with dynamic blood flow requires multiple ligand-receptor (LR) pairs that provide particle adhesion and disease specificity. Despite the ultimate goal of leukocyte mimicry, the specificity of multiple LAM-targeted VTCs remains poorly understood, especially in physiological environments...
March 2016: Bioeng Transl Med
https://www.readbyqxmd.com/read/28065316/single-molecule-measurements-to-study-polymerization-dynamics-of-ftsz-ftsa-copolymers
#18
N Baranova, M Loose
Bacterial cytokinesis is commonly initiated by the Z-ring, a dynamic cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin-like GTPase, that like its eukaryotic relative forms protein filaments in the presence of GTP. Since the discovery of the Z-ring 25years ago, various models for the role of FtsZ have been suggested. However, important information about the architecture and dynamics of FtsZ filaments during cytokinesis is still missing. One reason for this lack of knowledge has been the small size of bacteria, which has made it difficult to resolve the orientation and dynamics of individual FtsZ filaments in the Z-ring...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28063442/a-multi-technique-study-of-co2-adsorption-on-fe3o4-magnetite
#19
Jiri Pavelec, Jan Hulva, Daniel Halwidl, Roland Bliem, Oscar Gamba, Zdenek Jakub, Florian Brunbauer, Michael Schmid, Ulrike Diebold, Gareth S Parkinson
The adsorption of CO2 on the Fe3O4(001)-(2 × 2)R45° surface was studied experimentally using temperature programmed desorption (TPD), photoelectron spectroscopies (UPS and XPS), and scanning tunneling microscopy. CO2 binds most strongly at defects related to Fe(2+), including antiphase domain boundaries in the surface reconstruction and above incorporated Fe interstitials. At higher coverages,CO2 adsorbs at fivefold-coordinated Fe(3+) sites with a binding energy of 0.4 eV. Above a coverage of 4 molecules per (2 × 2)R45° unit cell, further adsorption results in a compression of the first monolayer up to a density approaching that of a CO2 ice layer...
January 7, 2017: Journal of Chemical Physics
https://www.readbyqxmd.com/read/28063045/induction-and-analysis-of-bronchus-associated-lymphoid-tissue
#20
Henrike Fleige, Reinhold Förster
Bronchus-associated lymphoid tissue (BALT) forms spontaneously in the lung after pulmonary infection and has been identified as a highly organized lymphoid structure supporting the efficient priming of T cells in the lung. To explore the mechanisms and instructive signals controlling BALT neogenesis we used both, a single dose of vaccinia virus MVA and repeated inhalations of heat-inactivated Pseudomonas aeruginosa (P. aeruginosa). Intranasal administration of both pathogens induces highly organized BALT but distinct pathways and molecules are used to promote the development of BALT...
2017: Methods in Molecular Biology
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