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Article: Chlamydia Uses K+ Electrical Signalling to Orchestrate Host Sensing, Inter-Bacterial Communication and Differentiation

Andrew, Susan C / Dumoux, Maud / Hayward, Richard D

Microorganisms. 2021 Jan. 15, v. 9, no. 1

2021

Abstract: Prokaryotic communities coordinate quorum behaviour in response to external stimuli to control fundamental processes including inter-bacterial communication. The obligate intracellular bacterial pathogen Chlamydia adopts two developmental forms, invasive ...

Abstract	Prokaryotic communities coordinate quorum behaviour in response to external stimuli to control fundamental processes including inter-bacterial communication. The obligate intracellular bacterial pathogen Chlamydia adopts two developmental forms, invasive elementary bodies (EBs) and replicative reticulate bodies (RBs), which reside within a specialised membrane-bound compartment within the host cell termed an inclusion. The mechanisms by which this bacterial community orchestrates different stages of development from within the inclusion in coordination with the host remain elusive. Both prokaryotic and eukaryotic kingdoms exploit ion-based electrical signalling for fast intercellular communication. Here we demonstrate that RBs specifically accumulate potassium (K⁺) ions, generating a gradient. Disruption of this gradient using ionophores or an ion-channel inhibitor stalls the Chlamydia lifecycle, inducing persistence. Using photobleaching approaches, we establish that the RB is the master regulator of this [K⁺] differential and observe a fast K⁺ exchange between RBs revealing a role for this ion in inter-bacterial communication. Finally, we demonstrate spatio-temporal regulation of bacterial membrane potential during RB to EB differentiation within the inclusion. Together, our data reveal that Chlamydia harnesses K⁺ to orchestrate host sensing, inter-bacteria communication and pathogen differentiation.
Keywords	Chlamydia ; bacterial communities ; behavior ; cell communication ; developmental stages ; ionophores ; ions ; membrane potential ; microorganisms ; pathogens ; photobleaching ; potassium
Language	English
Dates of publication	2021-0115
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
Note	NAL-light
ZDB-ID	2720891-6
ISSN	2076-2607
ISSN	2076-2607
DOI	10.3390/microorganisms9010173
Database	NAL-Catalogue (AGRICOLA)

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Article: [No title information]

Andrew, Susan C / Dumoux, Maud / Hayward, Richard D

Microorganisms

2021 Volume 9, Issue 1

Abstract	Prokaryotic communities coordinate quorum behaviour in response to external stimuli to control fundamental processes including inter-bacterial communication. The obligate intracellular bacterial pathogen
Language	English
Publishing date	2021-01-15
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720891-6
ISSN	2076-2607
ISSN	2076-2607
DOI	10.3390/microorganisms9010173
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: A protocol for cryogenic volumetric imaging using serial plasma FIB/SEM.

Dumoux, Maud / Smith, Jake L R / Glen, Thomas / Grange, Michael / Darrow, Michele C / Naismith, James H

Methods in cell biology

2023 Volume 177, Page(s) 327–358

Abstract: Cryogenic volumetric imaging using serial plasma focused ion beam scanning electron microscopy (serial pFIB/SEM) is a new and exciting correlative volume electron microscopy (vEM) technique. It enables visualization of un-stained, cryogenically ... ...

Abstract	Cryogenic volumetric imaging using serial plasma focused ion beam scanning electron microscopy (serial pFIB/SEM) is a new and exciting correlative volume electron microscopy (vEM) technique. It enables visualization of un-stained, cryogenically immobilized cells and tissues with ∼20-50nm resolution and a field of view of ∼10-30μm resulting in near-native state imaging and the possibility of microscale, mesoscale and nanoscale correlative imaging. We have written a detailed protocol for optimization of FIB and SEM parameters to reduce imaging artefacts and enable downstream computational processing and analysis. While our experience is based on use of a single system, the protocol has been written to be as hardware and software agnostic as possible, with a focus on the purpose of each step rather than a fully procedural description to provide a useful resource regardless of the system/software in use.
MeSH term(s)	Microscopy, Electron, Scanning ; Imaging, Three-Dimensional/methods ; Volume Electron Microscopy ; Software
Language	English
Publishing date	2023-03-09
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	0091-679X
ISSN	0091-679X
DOI	10.1016/bs.mcb.2023.01.015
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Ot2Rec: A semi-automatic, extensible, multi-software tomographic reconstruction workflow.

Yee, Neville B-Y / Ho, Elaine M L / Tun, Win / Smith, Jake L R / Dumoux, Maud / Grange, Michael / Darrow, Michele C / Basham, Mark

Biological imaging

2023 Volume 3, Page(s) e10

Abstract: Electron cryo-tomography is an imaging technique for probing 3D structures with at the nanometer scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the ... ...

Abstract	Electron cryo-tomography is an imaging technique for probing 3D structures with at the nanometer scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the advancement in technology, microscopes are currently capable of producing images amounting to terabytes of data per day, posing great challenges for scientists as the speed of processing of the images cannot keep up with the ever-higher throughput of the microscopes. Therefore, automation is an essential and natural pathway on which image processing-from individual micrographs to full tomograms-is developing. In this paper, we present Ot2Rec, an open-source pipelining tool which aims to enable scientists to build their own processing workflows in a flexible and automatic manner. The basic building blocks of Ot2Rec are plugins which follow a unified application programming interface structure, making it simple for scientists to contribute to Ot2Rec by adding features which are not already available. In this paper, we also present three case studies of image processing using Ot2Rec, through which we demonstrate the speedup of using a semi-automatic workflow over a manual one, the possibility of writing and using custom (prototype) plugins, and the flexibility of Ot2Rec which enables the mix-and-match of plugins. We also demonstrate, in the Supplementary Material, a built-in reporting feature in Ot2Rec which aggregates the metadata from all process being run, and output them in the Jupyter Notebook and/or HTML formats for quick review of image processing quality. Ot2Rec can be found at https://github.com/rosalindfranklininstitute/ot2rec.
Language	English
Publishing date	2023-03-29
Publishing country	England
Document type	Journal Article
ISSN	2633-903X
ISSN (online)	2633-903X
DOI	10.1017/S2633903X23000107
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Article ; Online: Optimization of Small-Scale Production of Zika Virus Envelope Glycoprotein by Transient Expression in HEK293 Cells for ELISA.

Kim, Young Chan / Dumoux, Maud / Owens, Raymond J / Reyes-Sandoval, Arturo

Methods in molecular biology (Clifton, N.J.)

2020 Volume 2142, Page(s) 103–112

Abstract: Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which has recently caused global epidemics with its association with congenital Zika syndrome such as severe microcephaly. The recombinant ZIKV envelope (Env) glycoprotein is useful for ... ...

Abstract	Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which has recently caused global epidemics with its association with congenital Zika syndrome such as severe microcephaly. The recombinant ZIKV envelope (Env) glycoprotein is useful for immunological applications such as serodiagnosis of ZIKV infection and for monitoring immune responses in preclinical and clinical ZIKV vaccine developments. In this chapter, we describe the optimization of production of Zika virus envelope glycoprotein in Human Embryonic Kidney (HEK 293T) cells by small-scale expression followed by large-scale protein production. Small-scale expression of HEK 293T cells allows screening of a large number of vectors simultaneously to select the vectors with best secretory profiles for scale-up in Expi293 mammalian system to maximize the protein yield followed by purification for research and clinical applications.
MeSH term(s)	CD4 Antigens/chemistry ; CD4 Antigens/genetics ; CD4 Antigens/metabolism ; Calibration ; Enzyme-Linked Immunosorbent Assay/methods ; Enzyme-Linked Immunosorbent Assay/standards ; Gene Expression ; Gene Products, env/genetics ; Gene Products, env/isolation & purification ; Gene Products, env/metabolism ; HEK293 Cells ; Humans ; Proteomics/methods ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/isolation & purification ; Recombinant Fusion Proteins/metabolism ; Secretory Pathway ; Serologic Tests/methods ; Transfection/methods ; Viral Envelope/chemistry ; Viral Envelope/metabolism ; Viral Envelope Proteins/genetics ; Viral Envelope Proteins/isolation & purification ; Viral Envelope Proteins/metabolism ; Zika Virus/chemistry ; Zika Virus/genetics ; Zika Virus/metabolism
Chemical Substances	CD4 Antigens ; Gene Products, env ; Recombinant Fusion Proteins ; Viral Envelope Proteins
Language	English
Publishing date	2020-05-04
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-0581-3_9
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Membrane contact sites between pathogen-containing compartments and host organelles.

Dumoux, Maud / Hayward, Richard D

Biochimica et biophysica acta

2016 Volume 1861, Issue 8 Pt B, Page(s) 895–899

Abstract: Intracellular pathogens survive and replicate within specialised membrane-bound compartments that can be considered as pseudo-organelles. Using the obligate intracellular bacterium Chlamydia as an illustrative example, we consider the modes of lipid ... ...

Abstract	Intracellular pathogens survive and replicate within specialised membrane-bound compartments that can be considered as pseudo-organelles. Using the obligate intracellular bacterium Chlamydia as an illustrative example, we consider the modes of lipid transport between pathogen-containing compartments and host organelles, including the formation of static membrane contact sites. We discuss how lipid scavenging can be mediated via the reprogramming of cellular transporters at these interfaces and describe recent data suggesting that pathogen effectors modulate the formation of specific membrane contacts. Further study of these emerging mechanisms is likely to yield new insights into the cell biology of lipid transport and organelle communication, which highlights potential new targets and strategies for future therapeutics. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.
MeSH term(s)	Animals ; Biological Transport ; Cell Membrane/metabolism ; Chlamydia/metabolism ; Golgi Apparatus/metabolism ; Host-Pathogen Interactions/physiology ; Humans ; Inclusion Bodies/metabolism ; Inclusion Bodies/microbiology ; Organelles/metabolism ; Sphingomyelins/metabolism
Chemical Substances	Sphingomyelins
Language	English
Publishing date	2016-01-26
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	60-7
ISSN	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
ISSN (online)	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
ISSN	0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
DOI	10.1016/j.bbalip.2016.01.018
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Article: Membrane contact sites between pathogen-containing compartments and host organelles

Dumoux, Maud / Richard D. Hayward

Biochimica et biophysica acta. 2016 Aug., v. 1861, no. 8

2016

Abstract	Intracellular pathogens survive and replicate within specialised membrane-bound compartments that can be considered as pseudo-organelles. Using the obligate intracellular bacterium Chlamydia as an illustrative example, we consider the modes of lipid transport between pathogen-containing compartments and host organelles, including the formation of static membrane contact sites. We discuss how lipid scavenging can be mediated via the reprogramming of cellular transporters at these interfaces and describe recent data suggesting that pathogen effectors modulate the formation of specific membrane contacts. Further study of these emerging mechanisms is likely to yield new insights into the cell biology of lipid transport and organelle communication, which highlights potential new targets and strategies for future therapeutics. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.
Keywords	bacteria ; Chlamydia ; landscapes ; lipids ; organelles ; pathogens ; therapeutics ; transporters
Language	English
Dates of publication	2016-08
Size	p. 895-899.
Publishing place	Elsevier B.V.
Document type	Article
ISSN	1388-1981
DOI	10.1016/j.bbalip.2016.01.018
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae.

Parkhurst, James M / Crawshaw, Adam D / Siebert, C Alistair / Dumoux, Maud / Owen, C David / Nunes, Pedro / Waterman, David / Glen, Thomas / Stuart, David I / Naismith, James H / Evans, Gwyndaf

IUCrJ

2023 Volume 10, Issue Pt 3, Page(s) 270–287

Abstract: Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between ... ...

Abstract	Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between electrons and matter. In recent years, focused-ion-beam (FIB) milling has been used in the preparation of thin samples for 3DED. These instruments typically use a gallium liquid metal ion source. Inductively coupled plasma (ICP) sources in principle offer faster milling rates. Little work has been done to quantify the damage these sources cause to delicate biological samples at cryogenic temperatures. Here, an analysis of the effect that milling with plasma FIB (pFIB) instrumentation has on lysozyme crystals is presented. This work evaluates both argon and xenon plasmas and compares them with crystals milled with a gallium source. A milling protocol was employed that utilizes an overtilt to produce wedge-shaped lamellae with a shallow thickness gradient which yielded very thin crystalline samples. 3DED data were then acquired and standard data-processing statistics were employed to assess the quality of the diffraction data. An upper bound to the depth of the pFIB-milling damage layer of between 42.5 and 50 nm is reported, corresponding to half the thickness of the thinnest lamellae that resulted in usable diffraction data. A lower bound of between 32.5 and 40 nm is also reported, based on a literature survey of the minimum amount of diffracting material required for 3DED.
Language	English
Publishing date	2023-05-01
Publishing country	England
Document type	Journal Article
ZDB-ID	2754953-7
ISSN	2052-2525 ; 2052-2525
ISSN (online)	2052-2525
ISSN	2052-2525
DOI	10.1107/S2052252523001902
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Plasma FIB milling for the determination of structures in situ.

Berger, Casper / Dumoux, Maud / Glen, Thomas / Yee, Neville B-Y / Mitchels, John M / Patáková, Zuzana / Darrow, Michele C / Naismith, James H / Grange, Michael

Nature communications

2023 Volume 14, Issue 1, Page(s) 629

Abstract: Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an ... ...

Abstract	Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.
MeSH term(s)	Humans ; Microscopy ; Electrons ; Workflow ; Carmustine
Chemical Substances	Carmustine (U68WG3173Y)
Language	English
Publishing date	2023-02-06
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-36372-9
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Okapi-EM: A napari plugin for processing and analyzing cryogenic serial focused ion beam/scanning electron microscopy images.

Perdigão, Luís M A / Ho, Elaine M L / Cheng, Zhiyuan C / Yee, Neville B-Y / Glen, Thomas / Wu, Liang / Grange, Michael / Dumoux, Maud / Basham, Mark / Darrow, Michele C

Biological imaging

2023 Volume 3, Page(s) e9

Abstract: An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at ... ...

Abstract	An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualization and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This article presents Okapi-EM, an open-source napari plugin developed to process and analyze cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming.
Language	English
Publishing date	2023-03-27
Publishing country	England
Document type	Journal Article
ISSN	2633-903X
ISSN (online)	2633-903X
DOI	10.1017/S2633903X23000119
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