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
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2. Annual cambial rhythm in Pinus halepensis and Pinus sylvestris as indicator for climate adaptationPeter Prislan, Jožica Gričar, Martin De Luis, Klemen Novak, Edurne Martinez Del Castillo, Uwe Schmitt, Gerald Koch, Jasna Štrus, Polona Mrak, Magda Tušek-Žnidarič, Katarina Čufar, 2016, izvirni znanstveni članek 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: 3355; Prenosov: 1519
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