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: 59; Prenosov: 45
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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: 103; Prenosov: 120
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3. Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCRAlexandra Bogožalec Košir, Carla Divieto, Jernej Pavšič, Stefano Pavarelli, David Dobnik, Tanja Dreo, Roberto Bellotti, Maria Paola Sassi, Jana Žel, 2017, izvirni znanstveni članek Povzetek: Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Ključne besede: droplet digital PCR, droplet volume, DNA quantification, optical microscopy imaging
Objavljeno v DiRROS: 25.07.2024; Ogledov: 131; Prenosov: 88
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4. 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: 148; Prenosov: 80
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5. Living inside a jellyfish : the symbiosis case study of host-specialized dinoflagellates, “Zooxanthellae”, and the Scyphozoan Cotylorhiza tuberculataAngélica Enrique- Navarro, Emma Huertas, Vesna Flander-Putrle, Ana Bartual, Gabriel Navarro, Javier Ruiz, Alenka Malej, Laura Prieto, 2022, izvirni znanstveni članek Povzetek: The photosymbiosis with host-specific dinoflagellates is a widespread relationship in marine organisms. Despite the evidenced biodiversity of this kind of mutualism, most research focuses on the study of scleractinian corals, and there is a lack of knowledge about other symbiotic cnidarians such as jellyfishes. The Mediterranean jellyfish Cotylorhiza tuberculata (Rhizostomae, Scyphozoa) harbors an endosymbiotic dinoflagellate of the family Symbiodiniaceae. In this study, we examine the algae distribution within the host body as well as, the pigment content and cell density of the symbiont. Furthermore, the size, morphology and fluorescence of cultured symbionts were studied under light microscopy, Imaging Flow Cytometry (IFC), and Scanning Electron Microscopy (SEM). The C:N composition and optical properties of the medusa tissue were measured to evaluate their role in the symbiosis. The medusae body was divided into two different sections to investigate the distribution of symbionts in hospite: oral arms (OA) and umbrella (UM). C:N composition of C. tuberculata was and symbiont density was significantly higher in the OA section. Mean chlorophyll a concentration of the algae was 1.33 (± 0.83) pg Chl a cell–1. The study of the pigment composition by HPLC (High Performance Liquid Chromatography), revealed the presence of 13 different pigments, being the most representative chlorophyll a, chlorophyll c2, and peridinin typical pigments of Symbiodiniaceae. Cell diameter of algae freshly isolated from the host was 8.71 ± 0.97 μm and cell growth rate was 0.52 (± 0.09) 106 cell ml–1 d–1. The presence of vegetative coccoid cells, doublet and motile mastigotes were revealed within the Symbiodiniaceae cultures. A calcifying matrix typical of Symbiodiniaceae and formed in partner with bacteria, was also observed most cultures. The umbrella tissue of the medusa absorbed at ultraviolet radiation (UVR) region, suggesting that medusae tissue protects photosymbionts from the negative effect of the high energetic UVR and attenuates the light intensity reaching algae inside the host. The presence of a dense Symbiodiniaceae population and the protection to UVR and elevated environmental irradiance provided by medusae tissue, maintain symbionts in optimal light conditions for photosynthesis and may be a reason added to explain the population success of Cotylorhiza tuberculata.
Ključne besede: HPLC, SEM microscopy, absorbance, photosymbiosis
Objavljeno v DiRROS: 16.07.2024; Ogledov: 116; Prenosov: 110
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6. Why are the Early Gothic murals in St. Jacob’s Church in Ormož, Slovenia, almost entirely black?Anabelle Križnar, Katja Kavkler, Sabina Dolenec, 2024, izvirni znanstveni članek Povzetek: In St. Jacob’s parish church in Ormož, Slovenia, mural paintings from around 1350–1370 are partially conserved in the northeastern corner of the main nave. They are almost completely black, indicating a large-scale pigment degradation. They were studied as a part of a larger research project aiming to identify materials applied and their possible degradation. First, they were studied in situ, and next, extracted samples of plaster, pigments, and colour layers were analysed by optical microscopy, Raman spectroscopy, FTIR spectroscopy, SEM-EDS, and XRD. Haematite, green earth, malachite, azurite, and tenorite were identified, showing that azurite and perhaps also malachite degraded to black tenorite, probably due to their fine grinding and their application directly on the fresh plaster. The plaster is made with small and large amounts of aggregate with mostly quartz with some impurities, which makes it fragile. The original appearance of these murals was of bright blue and green colours.
Ključne besede: mural painting, medieval painting, Gothic art, St. Jacob's Church, Ormož, Slovenia, pigments, chemical structure, colour degradation, optical microscopy, Raman microspectroscopy, FTIR, XRD
Objavljeno v DiRROS: 17.04.2024; Ogledov: 263; Prenosov: 258
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7. Understanding carbide evolution and surface chemistry during deep cryogenic treatment in high-alloyed ferrous alloyPatricia Jovičević Klug, Levi Tegg, Matic Klug Jovičević, Rahul Parmar, Matteo Amati, Luca Gregoratti, László Almásy, Julie M. Cairney, Bojan Podgornik, 2023, izvirni znanstveni članek Ključne besede: cryogenic temperature, surface chemistry, microstructure, atom probe tomography (APT), scanning photoelectron microscopy (SPEM), small angle neutron scattering (SANS)
Objavljeno v DiRROS: 08.03.2024; Ogledov: 408; Prenosov: 408
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8. 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: 254; Prenosov: 222
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9. Small cellular particles from European spruce needle homogenateMarko Jeran, Anna Romolo, Vesna Spasovski, Matej Hočevar, Urban Novak, Roman Štukelj, Vid Šuštar, Matic Kisovec, Apolonija Bedina Zavec, Ksenija Kogej, Aleš Iglič, Polonca Trebše, Veronika Kralj-Iglič, 2023, izvirni znanstveni članek Ključne besede: Picea abies, spruce needles, extracellular particles, extracellular vesicles, small cellular particles, interferometric light microscopy, exosomes, cell-to-cell communication, drug delivery
Objavljeno v DiRROS: 07.02.2024; Ogledov: 368; Prenosov: 205
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10. 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: 411; Prenosov: 194
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