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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: 91; Prenosov: 66
Celotno besedilo (8,47 MB)
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Povzetek: One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.
Ključne besede: absolute quantification, AAV, gene therapy, electron microscopy, digital PCR, real-time PCR
Objavljeno v DiRROS: 23.07.2024; Ogledov: 157; Prenosov: 87
Celotno besedilo (3,23 MB)
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Povzetek: The hematopoietic stem cell (HSC) niche undergoes detrimental changes with age. The molecular differences between young and old niches are well studied and understood; however, young and old niches have not yet been extensively characterized in terms of morphology. In the present work, a 2D stromal model of young and old HSC niches isolated from bone marrow was investigated using light and scanning electron microscopy (SEM) to characterize cell density after one, two, or three weeks of culturing, cell shape, and cell surface morphological features. Our work is aimed at identifying morphological differences between young and old niche cells that could be used to discriminate between their respective murine HSC niches. The results show several age- specific morphological characteristics. The old niches differ from the young ones in terms of lower cell proliferating capacity, increased cell size with a flattened appearance, increased number of adipocytes, and the presence of tunneling nanotubes. In addition, proliferating cell clusters are present in the young niches but not in the old niches. Together, these characteristics could be used as a relatively simple and reliable tool to discriminate between young and old murine HSC niches and as a complementary approach to imaging with specific cellular markers.
Ključne besede: bone marrow, hematoopetic stem cell niche, aging, adipocytes, scanning electron microscopy
Objavljeno v DiRROS: 26.01.2024; Ogledov: 416; Prenosov: 198
Celotno besedilo (2,47 MB)
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Povzetek: A comprehensive study of attic dust in an urban area is presented. Its entire life cycle, from determining historical emission sources to recognising the processes that take place in attic dust and its potential to impact human health is discussed. Its chemical composition and morphological characteristics of individual solid particles reflect past anthropogenic activities. High levels of Be-Cd-Cu-Sb-Sn-Pb-Te-Zn and occurrence of Cu-Zn shavings are typical for an industrial zone characterised by a foundry and a battery factory. High levels of Co-Fe-Mo-Ni-W-Ba-Cr-Mg-Mn-Nb-Ti and occurrence of various solid Fe-oxides, particularly spherical particles, were identified in another industrial zone, which was dominated by the automotive and metal-processing industries. Emissions from coal combustion affected the distribution of S-Se-Hg-Tl-As-Ag-U. The predominant mineral in attic dust is gypsum, which was presumably formed in situ by the reaction of carbonate dust particles and atmospheric SO2 gas. The high oral bioaccessibility of As-Cd-Cu-Pb-Zn in the gastric phase and high bioaccessibility of As-Cu-Cd-Ni in the gastrointestinal phase were identified. Determined characteristics of attic dust and identified possibilities of prolonged human exposure to it indicate that attic dust should be treated as an excellent proxy for historical air contamination as well as a potentially hazardous material for human health.
Ključne besede: multi-element composition, scanning electron microscopy, oral bioaccessibility, unified BARGE method, urban geochemistry
Objavljeno v DiRROS: 23.06.2022; Ogledov: 755; Prenosov: 282
Celotno besedilo (1,39 MB)

Povzetek: To understand better the adaptation strategies of intra-annual radial growth in Pinus halepensis and Pinus sylvestris to local environmental conditions, we examined the seasonal rhythm of cambial activity and cell differentiation at tissue and cellular levels. Two contrasting sites differing in temperature and amount of precipitation were selected for each species, one typical for their growth and the other represented border climatic conditions, where the two species coexisted. Mature P. halepensis trees from Mediterranean (Spain) and sub-Mediterranean (Slovenia) sites, and P. sylvestris from sub-Mediterranean (Slovenia) and temperate (Slovenia) sites were selected. Repeated sampling was performed throughout the year and samples were prepared for examination with light and transmission electron microscopes. We hypothesized that cambial rhythm in trees growing at the sub-Mediterranean site where the two species co-exist will be similar as at typical sites for their growth. Cambium in P. halepensis at the Mediterranean site was active throughout the year and was never truly dormant, whereas at the sub-Mediterranean site it appeared to be dormant during the winter months. In contrast, cambium in P. sylvestris was clearly dormant at both sub-Mediterranean and temperate sites, although the dormant period seemed to be significantly longer at the temperate site. Thus, the hypothesis was only partly confirmed. Different cambial and cell differentiation rhythms of the two species at the site where both species co-exist and typical sites for their growth indicate their high but different adaptation strategies in terms of adjustment of radial growth to environmental heterogeneity, crucial for long-term tree performance and survival.
Ključne besede: Aleppo pine, Pinus halepensis, Scots pine, Pinus sylvestris, cambium, light microscopy, Mediterranean environment, temporate environment, transmission electron microscopy, xylem
Objavljeno v DiRROS: 12.07.2017; Ogledov: 3348; Prenosov: 1518
Celotno besedilo (4,63 MB)
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