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1.
Dalton Trans ; 49(7): 2323-2330, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32022053

RESUMO

A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or Fx-r-Fx-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn2+, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 µM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.

2.
Chem Commun (Camb) ; 55(90): 13530-13533, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31647088

RESUMO

Biocompatible polymersomes are prepared from amphiphilic block copolypeptoids with aggregation-induced emission, where the hydrophobic block P(TPE-NAG) is a tetraphenylethylene (TPE)-modified poly(N-allylglycine) and the hydrophilic block is polysarcosine. These nanoparticles are non-cytotoxic and show strong fluorescence emission in aqueous solution.

3.
Materials (Basel) ; 12(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315199

RESUMO

The reduction of the electron dose in electron tomography of biological samples is of high significance to diminish radiation damages. Simulations have shown that sparse data collection can perform efficient electron dose reduction. Frameworks based on compressive-sensing or inpainting algorithms have been proposed to accurately reconstruct missing information in sparse data. The present work proposes a practical implementation to perform tomographic collection of block-based sparse images in scanning transmission electron microscopy. The method has been applied on sections of chemically-fixed and resin-embedded Trypanosoma brucei cells. There are 3D reconstructions obtained from various amounts of downsampling, which are compared and eventually the limits of electron dose reduction using this method are explored.

4.
Biomacromolecules ; 20(9): 3435-3444, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31361468

RESUMO

Stimuli-responsive polymersomes formed by amphiphilic block copolymers have attracted substantial attention as smart and robust containers for drug delivery and nano/microreactors. Biosourced amphiphilic diblock copolypeptoids were developed that can self-assemble into oxidation-responsive unilamellar vesicles. These vesicles can burst under the action of reactive oxygen species which can be the hydrogen peroxide or the singlet oxygen produced by light-activation of a photosensitizer with spatiotemporal control. Polysarcosine (PSar, also called poly(N-methyl glycine)) was selected as the hydrophilic block because of its resistance to protein adsorption and low toxicity, similar to poly(ethylene glycol) (PEG). We designed and synthesized poly(N-3-(methylthio)propyl glycine) as the hydrophobic block. Its polyglycine backbone is the same as that of PSar, and especially, its hydrophobic N-substituents, thioether side chains, can be oxidized to hydrophilic sulfoxides. These oxidation-responsive polymersomes entirely based on N-substituted poly(amino acid)s were biocompatible as confirmed by cell viability tests and may find applications in drug delivery, biosensing, biodetection, and nano/microreactors.

5.
Angew Chem Int Ed Engl ; 58(30): 10260-10265, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31145525

RESUMO

Fluorescent polymersomes with both aggregation-induced emission (AIE) and CO2 -responsive properties were developed from amphiphilic block copolymer PEG-b-P(DEAEMA-co-TPEMA) in which the hydrophobic block was a copolymer made of tetraphenylethene functionalized methacrylate (TPEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) with unspecified sequence arrangement. Four block copolymers with different DEAEMA/TPEMA and hydrophilic/hydrophobic ratios were synthesized, and bright AIE polymersomes were prepared by nanoprecipitation in THF/water and dioxane/water systems. Polymersomes of PEG45 -b-P(DEAEMA36 -co-TPEMA6 ) were chosen to study the CO2 -responsive property. Upon CO2 bubbling vesicles transformed to small spherical micelles, and upon Ar bubbling micelles returned to vesicles with the presence of a few intermediate morphologies. These polymersomes might have promising applications as sensors, nanoreactors, or controlled release systems.

6.
J Microsc ; 274(1): 23-31, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30649833

RESUMO

Propagation of structural information through conformational changes in host-encoded amyloid proteins is at the root of many neurodegenerative disorders. Although important breakthroughs have been made in the field, fundamental issues like the 3D-structures of the fibrils involved in some of those disorders are still to be elucidated. To better characterise those nanometric fibrils, a broad range of techniques is currently available. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of the C-terminal region of a bacterial protein called Hfq as a model amyloidogenic protein, using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM-IR. We introduce and discuss the strategy that we have implemented as well as the protocol, challenges and difficulties encountered during this study to characterise amyloid assemblies at the nearly single-molecule level. LAY DESCRIPTION: Propagation of structural information through conformational changes in amyloid proteins is at the root of many neurodegenerative disorders. Amyloids are nanostructures originating from the aggregation of multiple copies of peptide or protein monomers that eventually form fibrils. Often described as being the cause for the development of various diseases, amyloid fibrils are of major significance in the public health domain. While important breakthroughs have been made in the field, fundamental issues like the 3D-structures of the fibrils implied in some of those disorders are still to be elucidated. To better characterise these fibrils, a broad range of techniques is currently available for the detection and visualisation of amyloid nanostructures. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of model amyloidogenic fibrils using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM-IR that allows chemical characterisation at the nanometric scale. The strategy, protocol, challenges and difficulties encountered in this approach are introduced and discussed herein.

7.
Pathogens ; 7(4)2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30513780

RESUMO

Hfq is a pleiotropic regulator that has key roles in the control of genetic expression. The protein noticeably regulates translation efficiency and RNA decay in Gram-negative bacteria, due to the Hfq-mediated interaction between small regulatory noncoding RNA and mRNA. This property is of primary importance for bacterial adaptation and virulence. We have previously shown that the Hfq E. coli protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure. In the present work, we demonstrate that epigallocatechin gallate (EGCG), a major green tea polyphenol compound, targets the Hfq amyloid region and can be used as a potential antibacterial agent. We analysed the effect of this compound on Hfq amyloid fibril stability and show that EGCG both disrupts Hfq-CTR fibrils and inhibits their formation. We show that, even if EGCG affects other bacterial amyloids, it also specifically targets Hfq-CTR in vivo. Our results provide an alternative approach for the utilisation of EGCG that may be used synergistically with conventional antibiotics to block bacterial adaptation and treat infections.

8.
J Cell Biol ; 217(12): 4284-4297, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30275108

RESUMO

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3-4 and 7-8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3-4 s while remaining on the same side of the axoneme.


Assuntos
Flagelos/metabolismo , Microtúbulos/metabolismo , Trypanosoma brucei brucei/metabolismo , Transporte Biológico Ativo/fisiologia , Flagelos/genética , Flagelos/ultraestrutura , Microtúbulos/genética , Microtúbulos/ultraestrutura , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestrutura
9.
ACS Nano ; 12(4): 4025-4035, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29617555

RESUMO

Fluorescent polymersomes are interesting systems for cell/tissue imaging and in vivo study of drug distribution and delivery. We report on bright fluorescent polymersomes with aggregation-induced emission self-assembled by a series of tetraphenylethylene (TPE)-containing amphiphilic biodegradable block copolymers, where the hydrophilic block is a polyethylene glycol and hydrophobic block is a TPE-substituted trimethylenecarbonate polymer P(TPE-TMC). Their self-assemblies in water were prepared by nanoprecipitation using dioxane or tetrahydrofuran as co-solvent, and the self-assembling processes were studied in detail by cryo-electron microscopy, dynamic light scattering, and spectrofluorometer. The polymersomes are formed via the closure of bilayer lamellae self-assembled first by amphiphilic block copolymers. The polymersome membrane affords a nanosize bright fluorescent system with self-assembly induced emission in the thickness scale of 10-15 nm. The control of the whole size of polymersome is achieved by the choice of co-solvent for self-assembling and by the design of a suitable hydrophilic/hydrophobic ratio of block copolymers. These polymersomes can be potentially used as a stable fluorescent tool to monitor the transportation and distribution of drugs and bioconjugates in living cells.

10.
Methods Mol Biol ; 1737: 321-340, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29484601

RESUMO

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-assembly, with a special focus on the detection of amyloidogenic polymers.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Multimerização Proteica , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/genética , Técnicas In Vitro , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética
11.
J Struct Biol ; 202(1): 51-60, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29248600

RESUMO

The protist Trypanosoma brucei is an emerging model for the study of cilia and flagella. Here, we used scanning transmission electron microscopy (STEM) tomography to describe the structure of the trypanosome transition zone (TZ). At the base of the TZ, nine transition fibres irradiate from the B microtubule of each doublet towards the membrane. The TZ adopts a 9 + 0 structure throughout its length of ∼300 nm and its lumen contains an electron-dense structure. The proximal portion of the TZ has an invariant length of 150 nm and is characterised by a collarette surrounding the membrane and the presence of electron-dense material between the membrane and the doublets. The distal portion exhibits more length variation (from 55 to 235 nm) and contains typical Y-links. STEM analysis revealed a more complex organisation of the Y-links compared to what was reported by conventional transmission electron microscopy. Observation of the very early phase of flagellum assembly demonstrated that the proximal portion and the collarette are assembled early during construction. The presence of the flagella connector that maintains the tip of the new flagellum to the side of the old was confirmed and additional filamentous structures making contact with the membrane of the flagellar pocket were also detected. The structure and potential functions of the TZ in trypanosomes are discussed, as well as its mode of assembly.


Assuntos
Cílios/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Flagelos/ultraestrutura , Trypanosoma brucei brucei/ultraestrutura , Axonema/metabolismo , Axonema/ultraestrutura , Cílios/metabolismo , Flagelos/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Trypanosoma brucei brucei/metabolismo
12.
Sci Rep ; 7(1): 15651, 2017 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-29127300

RESUMO

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

13.
Trends Microbiol ; 25(10): 782-784, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28869086

RESUMO

Ever since their discovery, bacterial tubulins, found in several Prosthecobacter species, have raised curiosity as they are closely related to eukaryotic tubulin. Deng and colleagues now present new evidence for the functional homology of the two cytoskeletal systems where in vitro reconstituted Btub-microtubules display eukaryote-like biochemical and dynamic properties.


Assuntos
Bactérias/genética , Citoesqueleto/genética , Eucariotos/genética , Microtúbulos/genética , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Células Eucarióticas/fisiologia , Tubulina (Proteína)/genética
14.
Sci Rep ; 7(1): 10724, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28878270

RESUMO

Hfq is a bacterial RNA binding protein that carries out several roles in genetic expression regulation, mainly at the post-transcriptional level. Previous studies have shown its importance in growth and virulence of bacteria. Here, we provide the direct observation of its ability to interact with membranes. This was established by co-sedimentation assay, cryo-transmission electron (cryo-TEM) and atomic force (AFM) microscopies. Furthermore, our results suggest a role for its C-terminus amyloidogenic domain in membrane disruption. Precisely, AFM images of lipid bilayers in contact with Hfq C-terminus fibrils show the emergence of holes with a size dependent on the time of interaction. Cryo-TEM observations also show that liposomes are in contact with clusters of fibrils, with occasional deformation of the vesicles and afterward the apparition of a multitude of tiny vesicles in the proximity of the fibrils, suggesting peptide-induced breakage of the liposomes. Finally, circular dichroism spectroscopy demonstrated a change in the secondary structure of Hfq C-terminus upon interaction with liposomes. Altogether, these results show an unexpected property of Hfq and suggest a possible new role for the protein, exporting sRNA outside of the bacterial cell.

15.
Nat Plants ; 3: 17082, 2017 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-28604682

RESUMO

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.


Assuntos
Citocinese , Plasmodesmos/metabolismo , Actinas/metabolismo , Comunicação Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Permeabilidade , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plasmodesmos/ultraestrutura
16.
J Vis Exp ; (121)2017 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-28362414

RESUMO

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.


Assuntos
Imagem Tridimensional/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Manejo de Espécimes/métodos , Tomografia Computadorizada por Raios X/métodos , Trypanosoma brucei brucei/ultraestrutura
17.
Ultramicroscopy ; 179: 47-56, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28411510

RESUMO

A central challenge in scanning transmission electron microscopy (STEM) is to reduce the electron radiation dosage required for accurate imaging of 3D biological nano-structures. Methods that permit tomographic reconstruction from a reduced number of STEM acquisitions without introducing significant degradation in the final volume are thus of particular importance. In random-beam STEM (RB-STEM), the projection measurements are acquired by randomly scanning a subset of pixels at every tilt view. In this work, we present a tailored RB-STEM acquisition-reconstruction framework that fully exploits the compressed sensing principles. We first demonstrate that RB-STEM acquisition fulfills the "incoherence" condition when the image is expressed in terms of wavelets. We then propose a regularized tomographic reconstruction framework to recover volumes from RB-STEM measurements. We demonstrate through simulations on synthetic and real projection measurements that the proposed framework reconstructs high-quality volumes from strongly downsampled RB-STEM data and outperforms existing techniques at doing so. This application of compressed sensing principles to STEM paves the way for a practical implementation of RB-STEM and opens new perspectives for high-quality reconstructions in STEM tomography.

18.
Sci Rep ; 7: 45668, 2017 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-28358387

RESUMO

Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today's eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.


Assuntos
Proteínas de Bactérias/metabolismo , Citoesqueleto/metabolismo , Lipídeos de Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas de Bactérias/química , Proteínas do Citoesqueleto/metabolismo , Verrucomicrobia/metabolismo , Verrucomicrobia/ultraestrutura
19.
Micron ; 84: 23-36, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26922256

RESUMO

Chemical imaging offers extensive possibilities for better understanding of biological systems by allowing the identification of chemical components at the tissue, cellular, and subcellular levels. In this review, we introduce modern methods for chemical imaging that can be applied to biological samples. This work is mainly addressed to the biological sciences community and includes the bases of different technologies, some examples of its application, as well as an introduction to approaches on combining multimodal data.


Assuntos
Elementos , Microscopia Eletrônica/métodos , Imagem Molecular/métodos , Imagem Tridimensional/métodos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica de Transmissão e Varredura/métodos , Nanopartículas , Neurônios/química , Neurônios/ultraestrutura
20.
PLoS One ; 10(12): e0144829, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26714308

RESUMO

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.


Assuntos
NADPH Oxidases/metabolismo , Nanopartículas/toxicidade , Superóxidos/metabolismo , Titânio/química , Titânio/toxicidade , Animais , Bovinos , Humanos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Fatores de Tempo
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