641. 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, original scientific article 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: 349; Downloads: 301
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642. Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosusAlen Pšeničnik, Lucija Slemc, Martina Avbelj, Miha Tome, Martin Šala, Paul R. Herron, Maksym Shmatkov, Marko Petek, Špela Baebler, Peter Mrak, Daslav Hranueli, Antonio Starcevic, Iain S. Hunter, Hrvoje Petković, 2024, original scientific article Abstract: Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.
Keywords: genome reduction, antibiotic biosynthesis, oxytetracycline, cryptic metabolites
Published in DiRROS: 07.08.2024; Views: 286; Downloads: 233
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643. Hepatic alterations in a BTBR T + Itpr3tf/J mouse model of autism and improvement using melatonin via mitigation oxidative stress, inflammation and ferroptosisRita Rezzani, Marzia Gianò, Daniela Pinto, Fabio Rinaldi, Cornelis J. F. van Noorden, Gaia Favero, 2024, original scientific article Abstract: Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder, and its etiology is not well understood. It is known that genetic and nongenetic factors determine alterations in several organs, such as the liver, in individuals with this disorder. The aims of the present study were to analyze morphological and biological alterations in the liver of an autistic mouse model, BTBR T + Itpr3tf/J (BTBR) mice, and to identify therapeutic strategies for alleviating hepatic impairments using melatonin administration. We studied hepatic cytoarchitecture, oxidative stress, inflammation and ferroptosis in BTBR mice and used C57BL6/J mice as healthy control subjects. The mice were divided into four groups and then treated and not treated with melatonin, respectively. BTBR mice showed (a) a retarded development of livers and (b) iron accumulation and elevated oxidative stress and inflammation. We demonstrated that the expression of ferroptosis markers, the transcription factor nuclear factor erythroid-related factor 2 (NFR2), was upregulated, and the Kelch-like ECH-associated protein 1 (KEAP1) was downregulated in BTBR mice. Then, we evaluated the effects of melatonin on the hepatic alterations of BTBR mice; melatonin has a positive effect on liver cytoarchitecture and metabolic functions.
Keywords: autism spectrum disorder, liver, oxidative stress, inflammation, ferroptosis
Published in DiRROS: 07.08.2024; Views: 295; Downloads: 239
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644. Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assayŠpela Alič, Katarina Bačnik, Tanja Dreo, 2024, original scientific article Abstract: Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive.
Keywords: molecular testing, diagnostics, plant pathogen, real-time PCR, Dickeya, survey, water
Published in DiRROS: 07.08.2024; Views: 279; Downloads: 211
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645. The Superblock model : a review of an innovative urban model for sustainability, liveability, health and well-beingMark J. Nieuwenhuijsen, Audrey De Nazelle, Marta Cirach Pradas, Carolyn Daher, Angel M. Dzhambov, Cynthia Echave, Stefan Gössling, Tamara Iungman, Haneen Khreis, Nicolina Kirby, Jernej Tiran, 2024, original scientific article Abstract: Introduction
Current urban and transport planning practices have significant negative health, environmental, social and economic impacts in most cities. New urban development models and policies are needed to reduce these negative impacts. The Superblock model is one such innovative urban model that can significantly reduce these negative impacts through reshaping public spaces into more diverse uses such as increase in green space, infrastructure supporting social contacts and physical activity, and through prioritization of active mobility and public transport, thereby reducing air pollution, noise and urban heat island effects. This paper reviews key aspects of the Superblock model, its implementation and initial evaluations in Barcelona and the potential international uptake of the model in Europe and globally, focusing on environmental, climate, lifestyle, liveability and health aspects.
Methods
We used a narrative meta-review approach and PubMed and Google scholar databases were searched using specific terms.
Results
The implementation of the Super block model in Barcelona is slow, but with initial improvement in, for example, environmental, lifestyle, liveability and health indicators, although not so consistently. When applied on a large scale, the implementation of the Superblock model is not only likely to result in better environmental conditions, health and wellbeing, but can also contribute to the fight against the climate crisis. There is a need for further expansion of the program and further evaluation of its impacts and answers to related concerns, such as environmental equity and gentrification, traffic and related environmental exposure displacement. The implementation of the Superblock model gained a growing international reputation and variations of it are being planned or implemented in cities worldwide. Initial modelling exercises showed that it could be implemented in large parts of many cities.
Conclusion
The Superblock model is an innovative urban model that addresses environmental, climate, liveability and health concerns in cities. Adapted versions of the Barcelona Superblock model are being implemented in cities around Europe and further implementation, monitoring and evaluation are encouraged. The Superblock model can be considered an important public health intervention that will reduce mortality and morbidity and generate cost savings for health and other sectors.
Keywords: urban geography, transport, public health, pollution, sustainable development, Barcelona (Spain)
Published in DiRROS: 07.08.2024; Views: 343; Downloads: 269
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646. Identification of triazolopyrimidinyl scaffold SARS-CoV-2 papain-like protease (PLpro) inhibitorSebastjan Kralj, Marko Jukič, Miha Bahun, Luka Kranjc, Anja Kolarič, Milan Hodošček, Nataša Poklar Ulrih, Urban Bren, 2024, original scientific article Abstract: The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic coronaviral research. In this study, a comprehensive approach using molecular docking, in vitro assays, and molecular dynamics simulations was applied to identify potential inhibitors for SARS-CoV-2 papain-like protease (PLpro), a key and underexplored viral enzyme target. A focused protease inhibitor library was initially created and molecular docking was performed using CmDock software (v0.2.0), resulting in the selection of hit compounds for in vitro testing on the isolated enzyme. Among them, compound 372 exhibited promising inhibitory properties against PLpro, with an IC50 value of 82 ± 34 μM. The compound also displayed a new triazolopyrimidinyl scaffold not yet represented within protease inhibitors. Molecular dynamics simulations demonstrated the favorable binding properties of compound 372. Structural analysis highlighted its key interactions with PLpro, and we stress its potential for further optimization. Moreover, besides compound 372 as a candidate for PLpro inhibitor development, this study elaborates on the PLpro binding site dynamics and provides a valuable contribution for further efforts in pan-coronaviral PLpro inhibitor development.
Keywords: drug design, protease inhibitor, SARS-CoV-2, papain-like protease, PLpro, antiviral design, in silico drug design, CADD, virtual screening, HTVS, structure-based design
Published in DiRROS: 07.08.2024; Views: 306; Downloads: 238
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647. On the occurrence of early life stage of the king of herrings, Regalectus glesne (Actinopterygii: Lampriformes: Regalectidae), in the Adriatic SeaBranko Dragičević, Armin Pallaoro, Robert Grgičević, Lovrenc Lipej, Jakov Dulčić, 2011, other scientific articles Abstract: The occurrence of an early life stage of the king of herrings, Regalecus glesne Ascanius, 1772, is reported for the first time from the Adriatic waters. A single larva (SL = 103.4 mm) was found alive in the waters of open Adriatic near a remote Island PalagruĹľa. Findings of early life stages of this species are very rare in the Mediterranean and have previously been reported only from the waters of the Strait of Sicily and Elba Island.
Keywords: fishes, ichthyology, Adriatic sea, Regalectidae, Regalectus glesne, oar fish, Adriatic Sea, larva, diversity, early life stage, record, king of herrings, Central Adriatic
Published in DiRROS: 07.08.2024; Views: 262; Downloads: 247
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648. First substantiated record of a lessepsian migrant - the dusky spinefood, Siganus luridus (Actinopterzgii: Perciformes: Siganidae), in the Adriatic SeaJakov Dulčić, Branko Dragičević, Robert Grgičević, Lovrenc Lipej, 2011, other scientific articles Abstract: One specimen of the dusky spinefoot, Siganus luridus (Rüppell, 1829) (+, total length = 17.3 cm, total weight = 87.61 g), a lessepsian migrant, was captured in the Mljet Channel (Southern Adriatic, Croatian coast) on 15 November 2010. This is the second record of this species from the Adriatic, but first well documented and based on the captured specimen. First record occurred earlier in the Northern Adriatic, but was based solely on underwater observations.
Keywords: fishes, ichthyology, Adriatic sea, Siganidae, lessepsian migrants, diversity, origins, Mediterranean, new record, Adriatic Sea
Published in DiRROS: 07.08.2024; Views: 332; Downloads: 142
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649. On the record of the moray eel (Muraena helena Linnaeus, 1758) in Slovenian coastal waters (Gulf of Trieste, northern Adriatic)Lovrenc Lipej, Sašo Moškon, 2011, other scientific articles Abstract: The authors report on the record of the moray eel (Muraena helena) in Slovenian coastal waters (Gulf of Trieste, northern Adriatic). The specimen was caught by a diver with a speargun in June 2011. The moray eel measured 1,127 mm in total length and weighed 4,120 g. The estimated age of the moray eel was about 12 years. This is the first recorded case of the occurrence of moray eel in Slovenian waters.
Keywords: moray eel, Murenae helena, zoogeography, Gulf of Trieste
Published in DiRROS: 07.08.2024; Views: 294; Downloads: 129
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650. Occurence of Egyptian Goose Alopochen aegyptiacus in SloveniaDejan Bordjan, Erik Šinigoj, 2011, other scientific articles Abstract: The article presents all known observations of the introduced Egyptian Goose Alopochen aegyptiacus made in Slovenia till the end of 2011. In Feb 2005, and eventually on 2 and 8 Mar 2008, a single individual was observed at Renče Clay Pits in the Vipava Valley while on 28 Oct 2010 two individuals were recorded only 2 km away at the confluence of the Lijak Stream and Vipava River (UTM UL98, SW Slovenia). The authors of the article presume that these individuals were escapees from the nearby pond situated along the Keramix factory at Volčja draga, where Egyptian Geese have been bred for a number of years as ornamental animals. On 8 Aug 2011, one individual was recorded on the bank of Ljubljanica River at Moste, Ljubljana (UTM VM60, Central Slovenia); in this case, too, an individual from captivity (E category) was presumably observed, judging by its unsuspicious behaviour. Apart from these records, a single individual was registered on 29 Mar 2008 at Rački Ponds (UTM WM54, NE Slovenia). Considering the circumstances of the above observations, as well as increase and expansion of the naturalised population north of the Alps, the authors infer that this individual belonged to the above mentioned population and that it should thus be most appropriately included, in the list of birds registered in Slovenia, in the category of species occurring as vagrants from naturalized populations abroad (C5).
Keywords: gosi, Alopochen aegyptiacus, zoologija, Slovenija, zoogeografija
Published in DiRROS: 07.08.2024; Views: 281; Downloads: 145
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