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: 91; Prenosov: 66
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2. "Only introduced" or "invasive" : spread of the alga Aulacoseira ambigua f. japonica from Asia to Africa and EuropeTina Eleršek, 2018, izvirni znanstveni članek Povzetek: Many non-native algal species are found in waters all over the world. Many terms are used to describe such organisms that have expanded their distribution. However, a unified model or concept remains to be defined, as how biological ‘invasions’ are seen depends on the perspective. Understanding invasive organisms is important for biodiversity, science policy and water management. During monitoring sampling at Slivniško Lake (Slovenia) in 2016, the curved diatom Aulacoseira ambigua f. japonica Tuji & D.M. Williams was identified. This species originates from Japan, from where it was described more than 100 years ago. The chronology of the published distributions of A. ambigua f. japonica defines its spread from Japan to Asia, then to western Russia and South Africa, and now to central Europe. This study provides further evidence that A. ambigua f. japonica has become established in Europe (Slovenia), as supported by light microscopy and scanning electron microscopy. To the best of our knowledge, this is the first qualitative and quantitative description of A. ambigua f. japonica in Europe. It has been suggested that the shape of these algal colonies is significant for the interpretation of ecological information, and indeed, also in the present case, spiral colonies were found in this eutrophic water body. Although curved cells and colonies can also be interpreted as (sub)populations or morphological variants, only detailed molecular studies can reveal if these also have taxonomic significance. Ključne besede: algae, introduced species, invasive species, scanning electron microscopy Objavljeno v DiRROS: 01.08.2024; Ogledov: 115; Prenosov: 128
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3. Accurate quantification and characterization of adeno-associated viral vectorsDavid Dobnik, Polona Kogovšek, Tjaša Jakomin, Nejc Košir, Magda Tušek-Žnidarič, Maja Leskovec, Stephen M. Kaminsky, Janet Mostrom, Hyunmi Lee, Maja Ravnikar, 2019, izvirni znanstveni članek 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
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4. In vitro and in vivo degradation behavior of Mg-0.45Zn-0.45Ca (ZX00) screws for orthopedic applicationsDiana C. Martinez, Anna Dobkowska, Romy Marek, Hanna Ćwieka, Jakub Jaroszewicz, T. Plociński, Črtomir Donik, Heike Helmholz, Bérengère Luthringer-Feyerabend, Berit Zeller-Plumhoff, Regine Willumeit-Römer, Wojciech Święszkowski, 2023, izvirni znanstveni članek Ključne besede: magnesium alloys, biodegradable implants, microstructure, electron microscopy, corrosion layers Objavljeno v DiRROS: 08.03.2024; Ogledov: 259; Prenosov: 228
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5. Morphological characteristics of young and old murine hematopoietic stem cell niches, as modeled in vitroMojca Justin, Ema Rogac Randl, Veno Kononenko, Matej Hočevar, Damjana Drobne, Primož Rožman, 2023, izvirni znanstveni članek 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
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6. Bringing into play automated electron microscopy data processing for understanding nanoparticulate electrocatalysts’ structure–property relationshipsAna Rebeka Kamšek, Francisco Ruiz-Zepeda, Andraž Pavlišič, Armin Hrnjić, Nejc Hodnik, 2022, pregledni znanstveni članek Ključne besede: identical location electron microscopy, image analysis, machine learning, electrocatalysis, metallic nanoparticles Objavljeno v DiRROS: 01.07.2022; Ogledov: 772; Prenosov: 519
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7. Attic dust: an archive of historical air contamination of the urban environment and potential hazard to health?Martin Gaberšek, Michael J. Watts, Mateja Gosar, 2022, izvirni znanstveni članek 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
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8. The effect of Eutypella parasitica on the wood decay of three maple speciesAna Brglez, Barbara Piškur, Miha Humar, Jožica Gričar, Nikica Ogris, 2020, izvirni znanstveni članek Povzetek: 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. Ključne besede: wood decay, mass loss, moisture content, mini-block test, decay test, Acer spp., Eutypella parasitica, fungi, light microscopy, scanning electron microscopy Objavljeno v DiRROS: 13.07.2020; Ogledov: 2000; Prenosov: 1376
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9. 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: 3348; Prenosov: 1518
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