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Article ; Online: Length-Controlled Nanofiber Micelleplexes as Efficient Nucleic Acid Delivery Vehicles.

Street, Steven T G / Chrenek, Josie / Harniman, Robert L / Letwin, Keiran / Mantell, Judith M / Borucu, Ufuk / Willerth, Stephanie M / Manners, Ian

Journal of the American Chemical Society

2022 Volume 144, Issue 43, Page(s) 19799–19812

Abstract: Micelleplexes show great promise as effective polymeric delivery systems for nucleic acids. Although studies have shown that spherical micelleplexes can exhibit superior cellular transfection to polyplexes, to date there has been no report on the effects ...

Abstract	Micelleplexes show great promise as effective polymeric delivery systems for nucleic acids. Although studies have shown that spherical micelleplexes can exhibit superior cellular transfection to polyplexes, to date there has been no report on the effects of micelleplex morphology on cellular transfection. In this work, we prepared precision, length-tunable poly(fluorenetrimethylenecarbonate)-
MeSH term(s)	Micelles ; Nanofibers ; Nucleic Acids ; Transfection ; Polymers ; DNA
Chemical Substances	Micelles ; Nucleic Acids ; Polymers ; DNA (9007-49-2)
Language	English
Publishing date	2022-10-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.2c06695
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Article ; Online: A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function.

McCaughey, Janine / Stevenson, Nicola L / Mantell, Judith M / Neal, Chris R / Paterson, Alex / Heesom, Kate / Stephens, David J

Journal of cell science

2021 Volume 134, Issue 17

Abstract: Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 ( ... ...

Abstract	Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER-Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.
MeSH term(s)	Animals ; Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism ; COP-Coated Vesicles/genetics ; COP-Coated Vesicles/metabolism ; Endoplasmic Reticulum/genetics ; Endoplasmic Reticulum/metabolism ; Golgi Apparatus/genetics ; Golgi Apparatus/metabolism ; Humans ; Membrane Proteins/metabolism ; Protein Transport ; Secretory Pathway
Chemical Substances	Membrane Proteins ; SURF4 protein, human ; Aryl Hydrocarbon Receptor Nuclear Translocator (138391-32-9)
Language	English
Publishing date	2021-09-07
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2993-2
ISSN	1477-9137 ; 0021-9533
ISSN (online)	1477-9137
ISSN	0021-9533
DOI	10.1242/jcs.259075
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Article ; Online: Quantitative silencing of EGFP reporter gene by self-assembled siRNA lipoplexes of LinOS and cholesterol.

Metwally, Abdelkader A / Blagbrough, Ian S / Mantell, Judith M

Molecular pharmaceutics

2012 Volume 9, Issue 11, Page(s) 3384–3395

Abstract: Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N(4),N(9)-diacyl spermine conjugate, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios ... ...

Abstract	Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N(4),N(9)-diacyl spermine conjugate, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N(4),N(9)-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106-118 nm, compared to 194-356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.
MeSH term(s)	Cell Survival ; Cholesterol/chemistry ; Cholesterol/metabolism ; Cryoelectron Microscopy ; Fatty Acids/chemistry ; Fatty Acids/metabolism ; Flow Cytometry ; Gene Silencing ; Genetic Therapy ; Genetic Vectors ; Green Fluorescent Proteins/antagonists & inhibitors ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; HeLa Cells ; Humans ; Lipids/chemistry ; Liposomes ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Particle Size ; RNA, Small Interfering/genetics ; Spermine/analogs & derivatives ; Spermine/metabolism ; Transfection
Chemical Substances	Fatty Acids ; Lipids ; Liposomes ; RNA, Small Interfering ; enhanced green fluorescent protein ; Green Fluorescent Proteins (147336-22-9) ; Spermine (2FZ7Y3VOQX) ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2012-10-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2138405-8
ISSN	1543-8392 ; 1543-8384
ISSN (online)	1543-8392
ISSN	1543-8384
DOI	10.1021/mp300435x
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Article ; Online: De Novo Designed Peptide and Protein Hairpins Self-Assemble into Sheets and Nanoparticles.

Galloway, Johanna M / Bray, Harriet E V / Shoemark, Deborah K / Hodgson, Lorna R / Coombs, Jennifer / Mantell, Judith M / Rose, Ruth S / Ross, James F / Morris, Caroline / Harniman, Robert L / Wood, Christopher W / Arthur, Christopher / Verkade, Paul / Woolfson, Derek N

Small (Weinheim an der Bergstrasse, Germany)

2021 Volume 17, Issue 10, Page(s) e2100472

Abstract: The design and assembly of peptide-based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible ... ...

Abstract	The design and assembly of peptide-based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a "disulfide pin" between two elements of secondary structure that drive self-assembly. Specifically, homodimerizing and homotrimerizing de novo coiled-coil α-helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.
MeSH term(s)	Biophysical Phenomena ; Nanoparticles ; Peptides ; Protein Structure, Secondary ; Proteins
Chemical Substances	Peptides ; Proteins
Language	English
Publishing date	2021-02-15
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2168935-0
ISSN	1613-6829 ; 1613-6810
ISSN (online)	1613-6829
ISSN	1613-6810
DOI	10.1002/smll.202100472
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Article ; Online: Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network.

Roybal, Kole T / Mace, Emily M / Mantell, Judith M / Verkade, Paul / Orange, Jordan S / Wülfing, Christoph

PloS one

2015 Volume 10, Issue 8, Page(s) e0133299

Abstract: Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of ... ...

Abstract	Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches.
MeSH term(s)	Actins/metabolism ; Animals ; Antigen-Presenting Cells/immunology ; Antigen-Presenting Cells/metabolism ; Lymphocyte Activation/immunology ; Mice ; Receptors, Antigen, T-Cell/metabolism ; Signal Transduction/immunology ; T-Lymphocytes/immunology ; T-Lymphocytes/metabolism
Chemical Substances	Actins ; Receptors, Antigen, T-Cell
Language	English
Publishing date	2015-08-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0133299
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Article ; Online: Using size-selected gold clusters on graphene oxide films to aid cryo-transmission electron tomography alignment.

Arkill, Kenton P / Mantell, Judith M / Plant, Simon R / Verkade, Paul / Palmer, Richard E

Scientific reports

2015 Volume 5, Page(s) 9234

Abstract: A three-dimensional reconstruction of a nano-scale aqueous object can be achieved by taking a series of transmission electron micrographs tilted at different angles in vitreous ice: cryo-Transmission Electron Tomography. Presented here is a novel method ... ...

Abstract	A three-dimensional reconstruction of a nano-scale aqueous object can be achieved by taking a series of transmission electron micrographs tilted at different angles in vitreous ice: cryo-Transmission Electron Tomography. Presented here is a novel method of fine alignment for the tilt series. Size-selected gold clusters of ~2.7 nm (Au₅₆₁±₁₄ ), ~3.2 nm (Au₉₂₃± ₂₂ ), and ~4.3 nm (Au₂₀₅₇±₄₅) in diameter were deposited onto separate graphene oxide films overlaying holes on amorphous carbon grids. After plunge freezing and subsequent transfer to cryo-Transmission Electron Tomography, the resulting tomograms have excellent (de-)focus and alignment properties during automatic acquisition. Fine alignment is accurate when the evenly distributed 3.2 nm gold particles are used as fiducial markers, demonstrated with a reconstruction of a tobacco mosaic virus. Using a graphene oxide film means the fiducial markers are not interfering with the ice bound sample and that automated collection is consistent. The use of pre-deposited size-selected clusters means there is no aggregation and a user defined concentration. The size-selected clusters are mono-dispersed and can be produced in a wide size range including 2-5 nm in diameter. The use of size-selected clusters on a graphene oxide films represents a significant technical advance for 3D cryo-electron microscopy.
MeSH term(s)	Cryoelectron Microscopy ; Electron Microscope Tomography ; Gold/chemistry ; Graphite/chemistry ; Metal Nanoparticles/chemistry ; Oxides/chemistry ; Particle Size
Chemical Substances	Oxides ; Gold (7440-57-5) ; Graphite (7782-42-5)
Language	English
Publishing date	2015-03-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep09234
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Article ; Online: Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

Stinchcombe, Jane C / Randzavola, Lyra O / Angus, Karen L / Mantell, Judith M / Verkade, Paul / Griffiths, Gillian M

Current biology : CB

2015 Volume 25, Issue 24, Page(s) 3239–3244

Abstract: Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological ... ...

Abstract	Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome.
MeSH term(s)	Animals ; Cells, Cultured ; Centrioles/physiology ; Cilia/physiology ; Immunological Synapses/physiology ; Mice, Inbred C57BL ; Microscopy, Electron, Transmission ; T-Lymphocytes, Cytotoxic/physiology ; T-Lymphocytes, Cytotoxic/ultrastructure
Language	English
Publishing date	2015-12-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1071731-6
ISSN	1879-0445 ; 0960-9822
ISSN (online)	1879-0445
ISSN	0960-9822
DOI	10.1016/j.cub.2015.10.028
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Article ; Online: Bioinspired Silicification Reveals Structural Detail in Self-Assembled Peptide Cages.

Galloway, Johanna M / Senior, Laura / Fletcher, Jordan M / Beesley, Joseph L / Hodgson, Lorna R / Harniman, Robert L / Mantell, Judith M / Coombs, Jennifer / Rhys, Guto G / Xue, Wei-Feng / Mosayebi, Majid / Linden, Noah / Liverpool, Tanniemola B / Curnow, Paul / Verkade, Paul / Woolfson, Derek N

ACS nano

2018 Volume 12, Issue 2, Page(s) 1420–1432

Abstract: Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or ... ...

Abstract	Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or embedding, which can damage or obscure features. An alternative is to use bioinspired mineralization, mimicking how certain organisms use biomolecules to template mineral formation. Previously, we have reported the design and characterization of Self-Assembled peptide caGEs (SAGEs) formed from de novo peptide building blocks. In SAGEs, two complementary, 3-fold symmetric, peptide hubs combine to form a hexagonal lattice, which curves and closes to form SAGE nanoparticles. As hexagons alone cannot tile onto spheres, the network must also incorporate nonhexagonal shapes. While the hexagonal ultrastructure of the SAGEs has been imaged, these defects have not been observed. Here, we show that positively charged SAGEs biotemplate a thin, protective silica coating. Electron microscopy shows that these SiO
MeSH term(s)	Particle Size ; Peptides/chemical synthesis ; Peptides/chemistry ; Silicon Dioxide/chemistry ; Surface Properties
Chemical Substances	Peptides ; Silicon Dioxide (7631-86-9)
Language	English
Publishing date	2018-01-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1936-086X
ISSN (online)	1936-086X
DOI	10.1021/acsnano.7b07785
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Article ; Online: Resolution of the three dimensional structure of components of the glomerular filtration barrier.

Arkill, Kenton P / Qvortrup, Klaus / Starborg, Tobias / Mantell, Judith M / Knupp, Carlo / Michel, C Charles / Harper, Steve J / Salmon, Andy H J / Squire, John M / Bates, Dave O / Neal, Chris R

BMC nephrology

2014 Volume 15, Page(s) 24

Abstract: Background: The human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers - the endothelium, the basement membrane and the podocyte foot processes. ... ...

Abstract	Background: The human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers - the endothelium, the basement membrane and the podocyte foot processes. However, recent studies have suggested that at least two additional layers contribute to the function of the GFB, the endothelial glycocalyx on the vascular side, and the sub-podocyte space on the urinary side. To investigate the structure of these additional layers is difficult as it requires three-dimensional reconstruction of delicate sub-microscopic (<1 μm) cellular and extracellular elements. Methods: Here we have combined three different advanced electron microscopic techniques that cover multiple orders of magnitude of volume sampled, with a novel staining methodology (Lanthanum Dysprosium Glycosaminoglycan adhesion, or LaDy GAGa), to determine the structural basis of these two additional layers. Serial Block Face Scanning Electron Microscopy (SBF-SEM) was used to generate a 3-D image stack with a volume of a 5.3 x 105 μm3 volume of a whole kidney glomerulus (13% of glomerular volume). Secondly, Focused Ion Beam milling Scanning Electron Microscopy (FIB-SEM) was used to image a filtration region (48 μm3 volume). Lastly Transmission Electron Tomography (Tom-TEM) was performed on a 0.3 μm3 volume to identify the fine structure of the glycocalyx. Results: Tom-TEM clearly showed 20 nm fibre spacing in the glycocalyx, within a limited field of view. FIB-SEM demonstrated, in a far greater field of view, how the glycocalyx structure related to fenestrations and the filtration slits, though without the resolution of TomTEM. SBF-SEM was able to determine the extent of the sub-podocyte space and glycocalyx coverage, without additional heavy metal staining. Neither SBF- nor FIB-SEM suffered the anisotropic shrinkage under the electron beam that is seen with Tom-TEM. Conclusions: These images demonstrate that the three dimensional structure of the GFB can be imaged, and investigated from the whole glomerulus to the fine structure of the glycocalyx using three dimensional electron microscopy techniques. This should allow the identification of structural features regulating physiology, and their disruption in pathological states, aiding the understanding of kidney disease.
MeSH term(s)	Animals ; Glomerular Filtration Barrier/ultrastructure ; Glycocalyx/ultrastructure ; Imaging, Three-Dimensional/methods ; Male ; Microscopy, Electron/methods ; Rats ; Rats, Sprague-Dawley ; Reproducibility of Results ; Sensitivity and Specificity
Language	English
Publishing date	2014-02-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2041348-8
ISSN	1471-2369 ; 1471-2369
ISSN (online)	1471-2369
ISSN	1471-2369
DOI	10.1186/1471-2369-15-24
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Article ; Online: 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography.

Arkill, Kenton P / Neal, Chris R / Mantell, Judith M / Michel, Charles C / Qvortrup, Klaus / Rostgaard, Jørgen / Bates, Dave O / Knupp, Carlo / Squire, John M

Microcirculation (New York, N.Y. : 1994)

2012 Volume 19, Issue 4, Page(s) 343–351

Abstract: Objective: Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways ...

Abstract	Objective: Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. Methods: Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). Results: 3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. Conclusion: The tomographic reconstructions show a potentially significant improvement in determining glycocalyx structure over standard transmission electron microscopy.
MeSH term(s)	Animals ; Capillaries/ultrastructure ; Electron Microscope Tomography ; Endothelium, Vascular/ultrastructure ; Glycocalyx/ultrastructure ; Imaging, Three-Dimensional ; Microscopy, Electron, Transmission ; Rats ; Rats, Wistar
Language	English
Publishing date	2012-02-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1217758-1
ISSN	1549-8719 ; 1073-9688
ISSN (online)	1549-8719
ISSN	1073-9688
DOI	10.1111/j.1549-8719.2012.00168.x
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