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1.
J Microsc ; 294(3): 276-294, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38656474

RESUMO

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.


Assuntos
Disciplinas das Ciências Biológicas , Disciplinas das Ciências Biológicas/métodos
2.
Analyst ; 146(14): 4683-4699, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34195707

RESUMO

In this proof-of-principle study, we established and implemented a cross-modality imaging (CMI) pipeline to characterize and compare bisphosphonate (BIS)-treated jawbones of Sprague-Dawley rats after tooth extraction after physical therapies (photobiomodulation and extracorporeal shockwave therapy (PBMT and ESWT)). We showcase the feasibility of such a CMI approach and its compatibility across imaging modalities to probe the same region of interest (ROI) of the same jawbone. Jawbones were imaged in toto in 3D using micro-Computed Tomography to identify ROIs for subsequent sequential 2D analysis using well-established technologies such as Atomic Force Microscopy and Scanning Electron Microscopy, and recent imaging approaches in biomedical settings, such as micro-X-Ray Fluorescence Spectroscopy. By combining these four modalities, multiscale information on the morphology, topography, mechanical stiffness (Young's modulus), and calcium, zinc and phosphorus concentrations of the bone was collected. Based on the CMI pipeline, we characterized and compared the jawbones of a previously published clinically relevant rat model of BIS-related osteonecrosis of the jawbone (BRONJ) before and after treatment with BISs, PBMT and ESWT. While we did not find that physical therapies altered the mechanical and elemental jawbone parameters with significance (probably due to the small sample size of only up to 5 samples per group), both ESWT and PBMT reduced pore thicknesses and bone-to-enamel distances significantly compared to the controls. Although focused on BIS-treated jawbones, the established CMI platform can be beneficial in the study of bone-related diseases in general (such as osteoarthritis or -porosis) to acquire complementary hallmarks and better characterize disease status and alleviation potentials.


Assuntos
Tratamento por Ondas de Choque Extracorpóreas , Osteoartrite , Animais , Difosfonatos/toxicidade , Camundongos , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-X
3.
Int J Cosmet Sci ; 43(2): 263-268, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33236374

RESUMO

OBJECTIVE: The aim of the present study was to evaluate the effect of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in cosmetic submicron emulsions and submicron emulsion gels on physiological skin parameters during regular application in a clinical set-up. METHODS: Formulation morphology was investigated using cryo-transmission electron microscopy. Stability of the employed formulations was determined by photon correlation spectroscopy, measurement of pH and rheological properties. Effect on physiological skin parameters was evaluated during regular application over four weeks in a parallel group study (n = 15, healthy forearm skin) with a Corneometer, Sebumeter, skin-pH-Meter, Aquaflux and an Epsilon sensor. Confocal Raman spectroscopy was employed to monitor urea and NMF levels. RESULTS: Both submicron emulsions and gels showed satisfying storage stability irrespective of cyclodextrin incorporation. No statistically significant effects on skin barrier function and any of the observed parameters were obtained, indicating good skin tolerability of all tested formulations. CONCLUSION: Results suggest good skin tolerability of the developed cosmetic submicron emulsions and gels with HP-ß-CD.


OBJECTIF: Le but de la présente étude était d'évaluer l'effet de l'hydroxypropyl-ß-cyclodextrine (HP-ß-CD) dans les émulsions cosmétiques submicroniques et les gels d'émulsion submicronique sur les paramètres physiologiques de la peau lors d'une application régulière dans une configuration clinique. MÉTHODES: La morphologie de la formulation a été étudiée en utilisant la microscopie électronique à transmission cryo. La stabilité des formulations employées a été déterminée par spectroscopie de corrélation de photons, mesure du pH et des propriétés rhéologiques. L'effet sur les paramètres physiologiques de la peau a été évalué lors d'une application régulière pendant quatre semaines dans une étude de groupe parallèle (n = 15, peau d'avant-bras saine) avec un cornéomètre, un sébumètre, un pH-mètre cutané, un Aquaflux et un capteur Epsilon. La spectroscopie Raman confocale a été utilisée pour surveiller les niveaux d'urée et de NMF. RÉSULTATS: Les émulsions et les gels submicroniques ont montré une stabilité de stockage satisfaisante indépendamment de l'incorporation de cyclodextrine. Aucun effet statistiquement significatif sur la fonction de barrière cutanée et aucun des paramètres observés n'a été obtenu, indiquant une bonne tolérance cutanée de toutes les formulations testées. CONCLUSION: Les résultats suggèrent une bonne tolérance cutanée des émulsions et des gels cosmétiques submicroniques développés avec HP-ß-CD.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Absorção Cutânea/efeitos dos fármacos , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Humanos
4.
Proc Natl Acad Sci U S A ; 112(1): 262-7, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25535350

RESUMO

The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022-12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8-106 fAm(2)) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation.


Assuntos
Óxido Ferroso-Férrico/metabolismo , Espaço Intracelular/metabolismo , Receptores de Superfície Celular/metabolismo , Vertebrados/metabolismo , Animais , Forma Celular , Cóclea/citologia , Cóclea/ultraestrutura , Columbidae , Fenômenos Magnéticos , Frações Subcelulares/metabolismo , Truta
5.
Nano Lett ; 17(8): 4989-4995, 2017 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-28656771

RESUMO

Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, nonlinear pH changes. The time scales are tunable by variation of the enzyme concentrations and additional buffer substances. The resulting material system operates in full autonomy after injection of the chemical fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.


Assuntos
DNA/química , Esterases/química , Urease/química , Biocatálise , Soluções Tampão , Retroalimentação , Hidrogéis , Concentração de Íons de Hidrogênio , Cinética , Conformação de Ácido Nucleico , Transição de Fase
6.
Nano Lett ; 16(12): 7870-7874, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27802042

RESUMO

The rational design of anisotropic interaction patterns is a key step for programming colloid and nanoparticle self-assembly and emergent functions. Herein, we demonstrate a concept for harnessing the capabilities of 3D DNA origami for extensive supracolloidal self-assembly and showcase its use for making truly monodisperse, patchy, divalent nanocuboids that can self-assemble into supracolloidal fibrils via programmable DNA hybridization. A change in the number of connector duplexes at the patches reveals that multivalency and cooperativity play crucial roles to enhance superstructure formation. We further show thermal and chemical switching of the superstructures as the first steps toward reconfigurable self-assemblies. This concept lays the groundwork for merging monodisperse 3D DNA origami, featuring programmable patchiness and interactions, with nanoparticle self-assembly.


Assuntos
Coloides/química , DNA/química , Nanopartículas , Anisotropia , Hibridização de Ácido Nucleico
7.
Macromol Rapid Commun ; 37(16): 1323-30, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27322217

RESUMO

This study reports a simple and versatile synthesis route for the preparation of highly uniform and densely functionalized aqueous microgels by modification of latex particles composed of an active ester monomer (pentafluorophenyl acrylate; PFPA). The hydrophobic nature of the PFPA allows synthesizing very uniform latex particles via emulsion polymerization, whose size can be controlled by the surfactant concentration, while the degree of crosslinking is a function of the added crosslinker. The high reactivity of the PFPA groups toward nucleophilic substitution delivers a platform method to synthesize functional microgels by reaction with functional amines. This study demonstrates this process for the dense functionalization of the entire particle with an amine carrying a pH-responsive unit. This study further describes the influence of the crosslinking degree on the ability for swelling of the resulting microgels in aqueous dispersion.


Assuntos
Acrilatos/química , Ésteres/química , Fluorbenzenos/química , Reagentes de Ligações Cruzadas/química , Emulsões , Géis , Interações Hidrofóbicas e Hidrofílicas , Microesferas , Tamanho da Partícula , Polimerização
8.
Nano Lett ; 15(4): 2213-9, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25393204

RESUMO

Nature regulates complex structures in space and time via feedback loops, kinetically controlled transformations, and under energy dissipation to allow non-equilibrium processes. Although man-made static self-assemblies realize excellent control over hierarchical structures via molecular programming, managing their temporal destiny by self-regulation is a largely unsolved challenge. Herein, we introduce a generic concept to control the time domain by programming the lifetimes of switchable self-assemblies in closed systems. We conceive dormant deactivators that, in combination with fast promoters, enable a unique kinetic balance to establish an autonomously self-regulating, transient pH-state, whose duration can be programmed over orders of magnitude-from minutes to days. Coupling this non-equilibrium state to pH-switchable self-assemblies allows predicting their assembly/disassembly fate in time, similar to a precise self-destruction mechanism. We demonstrate a platform approach by programming self-assembly lifetimes of block copolymers, nanoparticles, and peptides, enabling dynamic materials with a self-regulation functionality.

9.
Soft Matter ; 11(42): 8342-53, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26350118

RESUMO

Microgels with internal and reconfigurable complex nanostructure are emerging as possible adaptive particles, yet they remain challenging to design synthetically. Here, we report the synthesis of highly charged poly(methacrylic acid) (PMAA) microgels incorporating permanent (poly(methyl methacrylate) (PMMA)) and switchable hydrophobic pockets (poly(N,N'-diethylaminoethyl methacrylate) (PDEAEMA)) via emulsion polymerization. We demonstrate detailed tuning of the size, crosslinking density and tailored incorporation of functional comonomers into the polyacid microgels. Analysis via cryo-TEM and pyrene probe measurements reveal switchable hydrophobic pockets inside the microgels as a function of pH. The particles show a rich diversity of internal phase-segregation, that adapts to the surrounding conditions. Large amounts of hydrophobic pockets even lead to hydrophobic bridging between particles. The study shows ways towards tailored polyelectrolyte microgels with narrow dispersity, high charge density, as well as tailored and reconfigurable hydrophobic compartments and interactions.

10.
Proc Natl Acad Sci U S A ; 109(30): E2067-76, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22733763

RESUMO

Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.


Assuntos
Axonema/química , Chlamydomonas/química , Microscopia Crioeletrônica/métodos , Dineínas/química , Microtúbulos/química , Dineínas/genética , Processamento de Imagem Assistida por Computador , Immunoblotting , Mutação/genética , Proteômica , Transdução de Sinais/genética
11.
Angew Chem Int Ed Engl ; 54(45): 13258-62, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26249239

RESUMO

Switchable self-assemblies respond to external stimuli with a transition between near-equilibrium states. Although being a key to present-day advanced materials, these systems respond rather passively, and do not display autonomous dynamics. For autonomous behavior, approaches must be found to orchestrate the time domain of self-assemblies, which would lead to new generations of dynamic and self-regulating materials. Herein, we demonstrate catalytic control of the time domain of pH-responsive peptide hydrogelators in a closed system. We program transient acidic pH states by combining a fast acidic activator with the slow, enzymatic, feedback-driven generation of a base (dormant deactivator). This transient state can be programmed over orders of magnitude in time. It is coupled to dipeptides to create autonomously self-regulating, dynamic gels with programmed lifetimes, which are used for fluidic guidance, burst release, and self-erasing rapid prototyping.


Assuntos
Biocatálise , Dipeptídeos/metabolismo , Retroalimentação Fisiológica , Hidrogéis/metabolismo , Urease/metabolismo , Dipeptídeos/química , Hidrogéis/química , Concentração de Íons de Hidrogênio , Cinética , Fatores de Tempo , Urease/química
12.
Bioscience ; 64(12): 1073-1083, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26955066

RESUMO

The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke-central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation.

13.
Proc Natl Acad Sci U S A ; 108(42): E845-53, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-21930914

RESUMO

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.


Assuntos
Axonema/ultraestrutura , Flagelos/ultraestrutura , Animais , Axonema/química , Chlamydomonas/química , Chlamydomonas/genética , Chlamydomonas/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Flagelos/química , Imageamento Tridimensional , Masculino , Modelos Moleculares , Proteínas de Plantas/química , Subunidades Proteicas , Cauda do Espermatozoide/química , Cauda do Espermatozoide/ultraestrutura , Strongylocentrotus purpuratus/química , Strongylocentrotus purpuratus/ultraestrutura , Tubulina (Proteína)/química
14.
J Extracell Biol ; 3(1): e134, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38938681

RESUMO

Extracellular vesicles (EVs) are crucial mediators of cell-to-cell communication in physiological and pathological conditions. Specifically, EVs released from the vasculature into blood were found to be quantitatively and qualitatively different in diseases compared to healthy states. However, our understanding of EVs derived from the lymphatic system is still scarce. In this study, we compared the mRNA and microRNA (miRNA) expression in blood vascular (BEC) and lymphatic (LEC) endothelial cells. After characterization of the EVs by fluorescence-triggered flow cytometry, nanoparticle tracking analysis and cryo-transmission electron microscopy (cryo-TEM) we utilized small RNA-sequencing to characterize miRNA signatures in the EVs and identify cell-type specific miRNAs in BEC and LEC. We found miRNAs specifically enriched in BEC and LEC on the cellular as well as the extracellular vesicle level. Our data provide a solid basis for further functional in vitro and in vivo studies addressing the role of EVs in the blood and lymphatic vasculature.

15.
Viruses ; 15(6)2023 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-37376661

RESUMO

Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte's pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle's shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution.


Assuntos
Dependovirus , Dependovirus/genética , Eletroforese/métodos , Microscopia de Força Atômica , Concentração de Íons de Hidrogênio
16.
Biomacromolecules ; 13(12): 4205-12, 2012 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-23102411

RESUMO

Sustainable alternatives for high-performance and functional materials based on renewable resources are intensely needed as future alternatives for present-day, fossil-based materials. Nanochitin represents an emerging class of highly crystalline bionanoparticles with high intrinsic mechanical properties and the ability for conjugation into functional materials owing to reactive amine and hydroxyl groups. Herein we demonstrate that hydrogels containing surface-deacetylated chitin nanofibrils of micrometer length and average diameters of 9 nm, as imaged by cryogenic transmission electron microscopy, can be wet-spun into macrofibers via extrusion in a coagulation bath, a simple low energy and large-scale processing route. The resulting biofibers display attractive mechanical properties with a large plastic region of about 12% in strain, in which frictional sliding of nanofibrils allows dissipation of fracture energy and enables a high work-of-fracture of near 10 MJ/m3. We further show how to add functionality to these macrofibers by exploiting the amine functions of the surface chitosan groups to host catalytically active noble metal nanoparticles, furnishing biobased, renewable catalytic hybrids. These inorganic/organic macrofibers can be used repeatedly for fast catalytic reductions of model compounds without loss of activity, rendering the concept of hybridized chitin materials interesting as novel bioderived supports for nanoparticle catalysts.


Assuntos
Materiais Biocompatíveis/química , Quitina/química , Hidrogéis/química , Nanopartículas Metálicas/química , Platina/química , Catálise , Quitosana/química , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Propriedades de Superfície , Difração de Raios X
17.
J Extracell Vesicles ; 11(12): e12282, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36437554

RESUMO

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.


Assuntos
Vesículas Extracelulares , Dióxido de Silício , Dióxido de Silício/metabolismo , Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Diagnóstico por Imagem , Biomarcadores/metabolismo
18.
J Extracell Vesicles ; 11(4): e12207, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35398993

RESUMO

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super-resolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nano-therapy design.


Assuntos
Vesículas Extracelulares , Coroa de Proteína , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Imunomodulação , Placenta , Gravidez , Coroa de Proteína/metabolismo , Proteômica
19.
Cells ; 10(12)2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34943829

RESUMO

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.


Assuntos
Vesículas Extracelulares/metabolismo , Imunomodulação , Leucemia Mieloide Aguda/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Vesículas Extracelulares/ultraestrutura , Humanos , Terapia de Imunossupressão , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia
20.
Mol Metab ; 54: 101329, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34454092

RESUMO

OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.


Assuntos
Proteína Forkhead Box O1/metabolismo , Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adulto , Animais , Desdiferenciação Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade
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