1. In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separationMojca Janc, Kaja Zevnik, Ana Dolinar, Tjaša Jakomin, Maja Štalekar, Katarina Bačnik, Denis Kutnjak, Magda Tušek-Žnidarič, Lorena Zentilin, Dmitri G. Fedorov, David Dobnik, 2024, izvirni znanstveni članek Povzetek: Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.
Ključne besede: recombinant adeno-associated viruses (rAAVs), CsCl ultracentrifugation gradient, analytical methods, digital droplet PCR (ddPCR), transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), Illumina sequencing, virology
Objavljeno v DiRROS: 07.08.2024; Ogledov: 92; Prenosov: 73
 Celotno besedilo (8,47 MB)
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2. Microscopy of crustacean cuticle : formation of a flexible extracellular matrix in moulting sea slaters Ligia pallasiiJasna Štrus, Magda Tušek-Žnidarič, Urška Repnik, Andrej Blejec, Adam P. Summers, 2019, izvirni znanstveni članek Povzetek: Structural and functional properties of exoskeleton in moulting sea slaters Ligia pallasii from the Eastern Pacific coast were investigated with CT scanning and electron microscopy. Ultrastructure of preecdysial and postecdysial cuticular layers was described in premoult, intramoult and postmoult animals. Cuticle is a flexible extracellular matrix connected to the epidermal cells through pore channels. During premoult epicuticle and exocuticle are formed and during intramoult and postmoult endocuticular lamellae are deposited and the cuticle is progressively constructed by thickening and mineralization. Cuticle permeability, flexibility and waterproofing capacity change accordingly. Elaboration of epicuticular scales connected to an extensive network of nanotubules, establish its anti-adhesive and hydrophobic properties. Labelling with gold conjugated WGA lectins on Tokuyashu thawed cryosections exposes differences in chitin content between exocuticle and endocuticle. Histochemical staining of cuticle shows presence of acidic carbohydrates/glycoconjugates and lipoproteins in epicuticular layer. Chitin microfibrils are formed at the microvillar border of epidermal cells with abundant Golgi apparatus and secretory vesicles. Numerous spherules associated with nanotubules were observed in the ecdysial space in intramoult animals. The mineral component of the cuticle as visualized with CT scanning indicates progressive mineral resorption from the posterior to the anterior half of the body in premoult animals, its translocation from the anterior to posterior part during intramoult and its progressive deposition in the posterior and anterior exoskeleton during postmoult. Cuticle of sea slaters is a unique biocomposite and biodynamic material constantly reconstructed during frequent moults, and adapted to specific physical and biotic conditions of the high intertidal rocky zone.
Ključne besede: cuticle ultrastructure, micro CT scanning, moult cycle, SEM, TEM, terrestrial isopods
Objavljeno v DiRROS: 23.07.2024; Ogledov: 234; Prenosov: 222
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3. Nanoscale transformations of amphiboles within human alveolar epithelial cellsRuggero Vigliaturo, Maja Jamnik, Goran Dražić, Marjetka Podobnik, Magda Tušek-Žnidarič, Giancarlo Della Ventura, Günther Redhammer, Nada Žnidaršič, Simon Caserman, Reto Gieré, 2022, izvirni znanstveni članek Povzetek: Amphibole asbestos is related to lung fibrosis and several types of lung tumors. The disease-triggering mechanisms still challenge our diagnostic capabilities and are still far from being fully understood. The literature focuses primarily on the role and formation of asbestos bodies in lung tissues, but there is a distinct lack of studies on amphibole particles that have been internalized by alveolar epithelial cells (AECs). These internalized particles may directly interact with the cell nucleus and the organelles, exerting a synergistic action with asbestos bodies (AB) from a different location. Here we document the near-atomic- to nano-scale transformations induced by, and taking place within, AECs of three distinct amphiboles (anthophyllite, grunerite, “amosite”) with different Fe-content and morphologic features. We show that: (i) an Fe-rich layer is formed on the internalized particles, (ii) particle grain boundaries are transformed abiotically by the internal chemical environment of AECs and/or by a biologically induced mineralization mechanism, (iii) the Fe-rich material produced on the particle surface does not contain large amounts of P, in stark contrast to extracellular ABs, and (iv) the iron in the Fe-rich layer is derived from the particle itself. Internalized particles and ABs follow two distinct formation mechanisms reaching different physicochemical end-states.
Ključne besede: amphiboles, human alveolar epithelial cells, asbestos, lung cancer, nanoscale investigation, acS/TEM-EDXS, dual-EELS, asbestos-related tumors
Objavljeno v DiRROS: 16.07.2024; Ogledov: 241; Prenosov: 106
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5. Synthesis and advanced electrochemical characterization of multifunctional electrocatalytic composite for unitized regenerative fuel cellLeonard Moriau, Marjan Bele, Alen Vižintin, Francisco Ruiz-Zepeda, Urša Petek, Primož Jovanovič, Martin Šala, Miran Gaberšček, Nejc Hodnik, 2019, izvirni znanstveni članek Ključne besede: electrocatalysis, bifunctional electrocatalysts, oxygen evolution reaction, oxygen reduction reaction, IL-TEM, IL-SEM, EFC-ICP-MS
Objavljeno v DiRROS: 24.11.2020; Ogledov: 1677; Prenosov: 1082
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6. Atomically resolved anisotropic electrochemical shaping of nano-electrocatalystFrancisco Ruiz-Zepeda, Matija Gatalo, Andraž Pavlišič, Goran Dražić, Primož Jovanovič, Marjan Bele, Miran Gaberšček, Nejc Hodnik, 2019, izvirni znanstveni članek Ključne besede: electrocatalysis, dealloying, fuel cells, identical location TEM, kinetic Monte Carlo
Objavljeno v DiRROS: 23.11.2020; Ogledov: 1655; Prenosov: 1139
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