| 1. |
- Kay, E., et al.
(författare)
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NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:2
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Tidskriftsartikel (refereegranskat)abstract
- The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted en-docytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
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| 2. |
- Becquart, Cécile, et al.
(författare)
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Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:29, s. 10549-10556
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Tidskriftsartikel (refereegranskat)abstract
- Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity. Here, we used a combination of electron microscopy and NanoSIMS to assess the subcellular concentrations of a 34S-labeled GalNAc-ASO and a naked ASO in the organelles of primary human hepatocytes. We first cross-validated the method by including a 127I-labeled ASO, finding that the absolute concentration of the lysosomal ASO using two independent labeling strategies gave matching results, demonstrating the strength of our approach. This work also describes the preparation of external standards for absolute quantification by NanoSIMS. For both the 34S and 127I approaches used for our quantification methodology, we established the limit of detection (5 and 2 μM, respectively) and the lower limit of quantification (14 and 5 μM, respectively).
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| 3. |
- Gupta, G., et al.
(författare)
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Exploiting Mass Spectrometry to Unlock the Mechanism of Nanoparticle-Induced Inflammasome Activation
- 2023
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Ingår i: Acs Nano. - : AMER CHEMICAL SOC. - 1936-0851 .- 1936-086X. ; 17:17, s. 17451-17467
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Tidskriftsartikel (refereegranskat)abstract
- Nanoparticles (NPs) elicit sterile inflammation, but the underlying signaling pathways are poorly understood. Here, we report that human monocytes are particularly vulnerable to amorphous silica NPs, as evidenced by single-cell-based analysis of peripheral blood mononuclear cells using cytometry by time-of-flight (CyToF), while silane modification of the NPs mitigated their toxicity. Using human THP-1 cells as a model, we observed cellular internalization of silica NPs by nanoscale secondary ion mass spectrometry (nanoSIMS) and this was confirmed by transmission electron microscopy. Lipid droplet accumulation was also noted in the exposed cells. Furthermore, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed specific changes in plasma membrane lipids, including phosphatidylcholine (PC) in silica NP-exposed cells, and subsequent studies suggested that lysophosphatidylcholine (LPC) acts as a cell autonomous signal for inflammasome activation in the absence of priming with a microbial ligand. Moreover, we found that silica NPs elicited NLRP3 inflammasome activation in monocytes, whereas cell death transpired through a non-apoptotic, lipid peroxidation-dependent mechanism. Together, these data further our understanding of the mechanism of sterile inflammation.
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| 4. |
- Konrad-Schmolke, Matthias, 1970, et al.
(författare)
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Mineral dissolution and reprecipitation mediated by an amorphous phase
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Fluid-mediated mineral dissolution and reprecipitation processes are the most common mineral reaction mechanism in the solid Earth and are fundamental for the Earth's internal dynamics. Element exchange during such mineral reactions is commonly thought to occur via aqueous solutions with the mineral solubility in the coexisting fluid being a rate limiting factor. Here we show in high-pressure/low temperature rocks that element transfer during mineral dissolution and reprecipitation can occur in an alkali-Al-Si-rich amorphous material that forms directly by depolymerization of the crystal lattice and is thermodynamically decoupled from aqueous solutions. Depolymerization starts along grain boundaries and crystal lattice defects that serve as element exchange pathways and are sites of porosity formation. The resulting amorphous material occupies large volumes in an interconnected porosity network. Precipitation of product minerals occurs directly by repolymerization of the amorphous material at the product surface. This mechanism allows for significantly higher element transport and mineral reaction rates than aqueous solutions with major implications for the role of mineral reactions in the dynamic Earth.
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| 5. |
- Lindgren, Johan, et al.
(författare)
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Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur
- 2018
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 564:7736
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Tidskriftsartikel (refereegranskat)abstract
- Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.
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| 6. |
- Nguyen, Tho D. K., et al.
(författare)
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Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells
- 2022
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Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:3, s. 4831-4842
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Tidskriftsartikel (refereegranskat)abstract
- For decades, "all-or-none"and "kiss-and-run"were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically. © 2022 The Authors. Published by American Chemical Society.
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| 7. |
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| 8. |
- Nieves-Morión, Mercedes, et al.
(författare)
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Single-Cell Measurements of Fixation and Intercellular Exchange of C and N in the Filaments of the HeterocystForming Cyanobacterium Anabaena sp. Strain PCC 7120
- 2021
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Ingår i: Mbio. - 2150-7511 .- 2161-2129. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- Under diazotrophic conditions, the model filamentous, heterocystforming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N-2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [C-13]bicarbonate and N-15(2) and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG- SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The C-13/C-12 and N-15/N-14 ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-L-arginyl-poly(L-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, C-13/C-12 and N-15/N-14 ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N-2, and the C-13/C-12 ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions. IMPORTANCE Filamentous, heterocyst- forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are crucial to understand metabolic activities and qualify specific aspects related to multicellularity. Here, we used stable isotopes (C-13 and N-15) coupled to LG-SIMS and NanoSIMS imaging to follow single-cell C and N-2 fixation and metabolic dynamics along the filaments in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. Our results show a close relationship between C and N fixation and distribution in the filaments and indicate that wild-type filaments in a culture can exhibit a substantial variability of metabolic states. This illustrates how some novel properties can be appreciated by studying microbial cultures at the single-cell level.
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| 9. |
- Thomen, Aurélien, et al.
(författare)
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Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles
- 2020
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 14:4, s. 4316-4325
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.
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| 10. |
- Walbrühl, Martin, et al.
(författare)
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Effective diffusion in cemented carbide systems : Geometrical effect of the labyrinth factor
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In cemented carbides the effective diffusivities are associated with the carbides acting as obstacles that increase the diffusion distance, thus altering the overall diffusion in the composite. From an industrial point of view, the prediction of the surface gradient formation is important to develop state-of-the-art cemented carbide cutting tools and require an understanding of the liquid binder diffusivities and the effective diffusion reduction at typical sintering temperatures where the binder is molten. Recently, a full description of the diffusivities in the liquid binder has become available and the focus of the present work is thus the effective diffusion reduction. Isotope diffusion couple experiments have been successfully performed to investigate the effective diffusion in a WC-Ni liquid binder-carbide composite material, i.e. a cemented carbide. The 58Ni isotope diffusion profiles have been measured with Secondary Ion Mass Spectroscopy (SIMS) and the results have been compared to DICTRA simulations using an updated kinetic database. The agreement between the experimental and simulated diffusion profiles is excellent showing that the theoretical geometrical limit, simulated with the upper Hashin-Shtrikman bound, is obeyed in simple cemented carbide systems. For complex cemented carbide systems, where gradient sintering is relevant, the effective diffusion is insufficiently explained by the geometrical reduction.
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