Search the repository

A+ | A- | | SLO | ENG

Abstract: 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.
Keywords: 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
Published in DiRROS: 07.08.2024; Views: 464; Downloads: 357
Full text (8,47 MB)
This document has many files! More...

Abstract: Eutypella parasitica R.W. Davidson & R.C. Lorenz is the causative agent of Eutypella canker of maple, a destructive disease of maples in Europe and North America. The fungus E. parasitica is known to cause wood stain and decay. However, it is not known how effectively it decomposes the wood of the most widespread maple species in Europe. Wood samples of Acer pseudoplatanus L., A. platanoides L., and A. campestre L. were exposed to four isolates of E. parasitica and nine other fungal species for comparison, according to the modified EN 113 standard. After 15 weeks of incubation, mass loss and microscopical analysis of samples showed evidence of colonization and different wood decay potentials among fungal species. A highly significant positive correlation was found between mass loss and moisture content for all fungal species. Similarly, the measured cell wall thickness correlated well with the calculated mass loss of the samples. On average, the fungal species caused the lowest mass loss in A. pseudoplatanus (10.0%) and the highest in A. campestre (12.6%) samples. Among the samples exposed to E. parasitica isolates, the highest mass loss was recorded in A. pseudoplatanus (6.6%). Statistical analysis showed significant differences in mass loss and moisture content between different E. parasitica isolates. Based on the results of staining, we discuss the type of decay caused by E. parasitica. Although E. parasitica isolates caused smaller mass loss of samples compared to other more effective decay species, we should not disregard its capability of degrading maple wood. Because E. parasitica usually infects the lower portion of the trunk, which is the largest and most valuable part of the tree, any damage can cause significant economic and resource loss.
Keywords: wood decay, mass loss, moisture content, mini-block test, decay test, Acer spp., Eutypella parasitica, fungi, light microscopy, scanning electron microscopy
Published in DiRROS: 13.07.2020; Views: 2239; Downloads: 1563
Full text (8,28 MB)
This document has many files! More...

Abstract: 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.
Keywords: Aleppo pine, Pinus halepensis, Scots pine, Pinus sylvestris, cambium, light microscopy, Mediterranean environment, temporate environment, transmission electron microscopy, xylem
Published in DiRROS: 12.07.2017; Views: 3632; Downloads: 1638
Full text (4,63 MB)
This document has many files! More...