1181. Feasibility of droplet digital PCR analysis of plasma cell-free DNA from kidney transplant patientsBarbara Jerič Kokelj, Maja Štalekar, Sebastian Vencken, David Dobnik, Polona Kogovšek, Matjaž Stanonik, Miha Arnol, Maja Ravnikar, 2021, original scientific article Abstract: Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patientsʼ plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA.
Keywords: kidney transplantation, droplet digital PCR, plasma cell-free DNA, minor allele quantification, assay evaluation, graft health monitoring
Published in DiRROS: 19.07.2024; Views: 359; Downloads: 162
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1182. Interlaboratory comparison study on ribodepleted total RNA high-throughput sequencing for plant virus diagnostics and bioinformatic competenceYahya Gaafar, Marcel Westenberg, Marleen Botermans, László Krizbai, Kris De Jonghe, Yoika Foucart, Luca Ferretti, Denis Kutnjak, Anja Pecman, Nataša Mehle, Jan F. Kreuze, Giovanna Müller, Nikolaos Vakirlis, Despoina Beris, Christina Varveri, Heiko Ziebell, 2021, original scientific article Abstract: High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.
Keywords: high-throughput sequencing, ribodepletion, interlaboratory comparison, test performance study, proficiency test, Virtool
Published in DiRROS: 19.07.2024; Views: 303; Downloads: 178
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1183. The European stag beetle (Lucanus cervus) Monitoring network: international citizen science cooperation reveals regional differences in phenology and temperature responseArno Thomaes, Sylvie Barbalat, Marco Bardiani, Laura Bower, Alessandro Campanaro, Natalia Fanega Sleziak, João Gonçalo Soutinho, Sanne Govaert, Deborah J. Harvey, Colin Hawes, Marcin Kadej, Marcos Méndez, Bruno Meriguet, Markus Rink, Sarah Rossi De Gasperis, Sanne Ruyts, Lucija Šerić-Jelaska, Adrian Smolis, Eduard Snegin, Arianna Tagliani, Al Vrezec, 2021, original scientific article Abstract: To address the decline in biodiversity, international cooperation in monitoring of threatened species is needed. Citizen science can play a crucial role in achieving this challenging goal, but most citizen science projects have been established at national or regional scales. Here we report on the establishment and initial findings of the European Stag Beetle Monitoring Network (ESBMN), an international network of stag beetle (Lucanus cervus) monitoring schemes using the same protocol. The network, started in 2016, currently includes 14 countries (see results) but with a strong variation in output regarding the number of transects (148 successful transects in total) and transect walks (1735). We found differences across European regions in the number of stag beetles recorded, related to phenology and temperature, but not for time of transect start. Furthermore, the initial experiences of the ESBMN regarding international cooperation, citizen science approach, and drop-out of volunteers is discussed. An international standardised protocol that allows some local variation is essential for international collaboration and data management, and analysis is best performed at the international level, whereas recruiting, training, and maintaining volunteers is best organised locally. In conclusion, we appeal for more joint international citizen science-based monitoring initiatives assisting international red-listing and conservation actions.
Published in DiRROS: 19.07.2024; Views: 307; Downloads: 197
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1184. Microplastic contamination in protected areas of the Gulf of VeniceMichele Mistri, Marco Scoponi, Andrea Augusto Sfriso, Marta Curiotto, Adriano Sfriso, Martina Orlando-Bonaca, Lovrenc Lipej, 2021, original scientific article Abstract: We investigated the concentration and composition of plastics in 7 Special Areas of Conservation (SACs) from the Gulf of Venice (northern Adriatic Sea). A total of 42 sediment samples were analyzed from 21 sites from 2017 to 2018. All sites except one were found to be polluted by plastics, with density ranging between 2250 and 28.4 items kg−1. Microplastics ranged from 100 to 61.6% of the collected plastics. Fragments were more represented than filaments. The greatest plastic concentrations were generally recorded in western SACs. Identification through FT-IR spectroscopy evidenced the presence of 8 polymer types: in western SACS, the majority were low-density polymers (PE, PP, PS, and TPU), while in eastern SACs they were high-density polymers (PET, nylon, and PVC). In addition to the role of large rivers (all on the western side of the Gulf) in conveying plastics into the sea, a possible role of the cyclonic water circulation of the northern Adriatic Sea on distribution and composition of plastics along the Gulf coasts is likely.
Keywords: microplastics, special areas of conservation, polymer composition, Gulf of Venice, FT-IR spectroscopy
Published in DiRROS: 19.07.2024; Views: 271; Downloads: 159
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1185. TRIM28 selective nanobody reduces glioblastoma stem cell invasionAndrej Porčnik, Metka Novak, Barbara Breznik, Bernarda Majc, Barbara Hrastar, Neja Šamec, Alja Zottel, Ivana Jovchevska, Miloš Vittori, Ana Rotter, Radovan Komel, Tamara Lah Turnšek, 2021, original scientific article Abstract: Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.
Published in DiRROS: 19.07.2024; Views: 227; Downloads: 192
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1186. DNA damage in circulating leukocytes measured with the comet assay may predict the risk of deathStefano Bonassi, Marcello Ceppi, Peter Møller, Amaya Azqueta, Mirta Milić, Gunnar Brunborg, Gudrun Koppen, Marco Bruzzone, Juliana Da Silva, Danieli Benedetti, Silvia Moretti, Patrizia Riso, Patrizia Russo, Ricardo Marcos, Goran Gajski, Biljana Spremo-Potparević, Lada Živković, Elisa Boutet-Robinet, Maria Dusinska, Andrew Collins, Bojana Žegura, 2021, original scientific article Abstract: The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan–Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06–1.90) for overall mortality, and 1.94 (1.04–3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.
Published in DiRROS: 19.07.2024; Views: 286; Downloads: 183
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1187. Polysaccharide-based bilayer coatings for biofilm-inhibiting surfaces of medical devicesUrban Ajdnik, Thomas Luxbacher, Alenka Vesel, Alja Štern, Bojana Žegura, Janja Trček, Lidija Fras Zemljič, 2021, original scientific article Abstract: Chitosan (Chi) and 77KS, a lysine-derived surfactant, form polyelectrolyte complexes that reverse their charge from positive to negative at higher 77KS concentrations, forming aggregates that have been embedded with amoxicillin (AMOX). Dispersion of this complex was used to coat polydimethylsiloxane (PDMS) films, with an additional layer of anionic and hydrophilic hyaluronic acid (HA) as an outer adsorbate layer to enhance protein repulsion in addition to antimicrobial activity by forming a highly hydrated layer in combination with steric hindrance. The formed polysaccharide-based bilayer on PDMS was analyzed by water contact angle measurements, X-ray photoelectron spectroscopy (XPS), and surface zeta (ζ)-potential. All measurements show the existence and adhesion of the two layers on the PDMS surface. Part of this study was devoted to understanding the underlying protein adsorption phenomena and identifying the mechanisms associated with biofouling. Thus, the adsorption of a mixed-protein solution (bovine serum albumin, fibrinogen, γ-globulin) on PDMS surfaces was studied to test the antifouling properties. The adsorption experiments were performed using a quartz crystal microbalance with dissipation monitoring (QCM-D) and showed improved antifouling properties by these polysaccharide-based bilayer coatings compared to a reference or for only one layer, i.e., the complex. This proves the benefit of a second hyaluronic acid layer. Microbiological and biocompatibility tests were also performed on real samples, i.e., silicone discs, showing the perspective of the prepared bilayer coating for medical devices such as prostheses, catheters (balloon angioplasty, intravascular), delivery systems (sheaths, implants), and stents.
Keywords: polysaccharides, chitosan, hyaluronic acid, anti-biofilm, silicone
Published in DiRROS: 19.07.2024; Views: 376; Downloads: 189
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1188. Succinylation of polyallylamine: influence on biological efficacy and the formation of electrospun fibersLucija Jurko, Matej Bračič, Silvo Hribernik, Damjan Makuc, Janez Plavec, Filip Jerenec, Sonja Žabkar, Nenad Gubeljak, Alja Štern, Rupert Kargl, 2021, original scientific article Abstract: Succinylation of proteins is a commonly encountered reaction in biology and introduces negatively charged carboxylates on previously basic primary amine groups of amino acid residues. In analogy, this work investigates the succinylation of primary amines of the synthetic polyelectrolyte polyallylamine (PAA). It investigates the influence of the degree of succinylation on the cytotoxicity and antibacterial activity of the resulting polymers. Succinylation was performed in water with varying amounts of succinic anhydride and at different pH values. The PAA derivatives were analyzed in detail with respect to molecular structure using nuclear magnetic resonance and infrared absorbance spectroscopy. Polyelectrolyte and potentiometric charge titrations were used to elucidate charge ratios between primary amines and carboxylates in the polymers. The obtained materials were then evaluated with respect to their minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa. The biocompatibility was assessed using mouse L929 fibroblasts. The degree of succinylation decreased cytotoxicity but more significantly reduced antibacterial efficacy, demonstrating the sensitivity of the fibroblast cells against this type of ampholytic polyelectrolytes. The obtained polymers were finally electrospun into microfiber webs in combination with neutral water-soluble polyvinyl alcohol. The resulting non-woven could have the potential to be used as wound dressing materials or coatings.
Keywords: polyallylamine hydrochloride, succinylation, aqueous chemistry, cytotoxicity, antimicrobial effect, electrospinning, nanofibers, mouse L929 fibroblasts, Staphylococcus aureus, Pseudomonas aeruginosa
Published in DiRROS: 19.07.2024; Views: 212; Downloads: 234
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1189. Single-use plastic bans pollution : exploring stakeholder perspectives on best practices for reducing plasticEmily Cowan, Andy M. Booth, Andreas Misund, Katja Klun, Ana Rotter, Rachel Tiller, 2021, original scientific article Abstract: Abstract
In this study, we conducted and documented workshops and interviews in Norway and Slovenia to identify stakeholder and future generation opinions and mitigation strategies for solving one of the most prominent environmental issues: plastic pollution. As part of the EU H2020 project GoJelly, stakeholders were brought together to explore their perceptions on considering jellyfish mucus as a new resource to contribute to reducing plastic pollution from entering the marine environment. The study was conducted in the spring of 2019, in a context directly after the European Union (EU) announced its Directive to ban the most commonly used single-use plastic (SUP) items. The study applied the snowball method as a methodological choice to identify relevant stakeholders. Systems thinking was utilized as a participatory modelling approach, which allowed for the creation of conceptual mind maps from the various workshops and interviews, to understand consumers’ consciousness, and to map out ideas on plastic pollution reduction. Plastic pollution takes place on a global scale and stakeholders discussed their individual perceptions of national and international solutions that could be put in place to solve it, including the opportunities around utilizing jellyfish mucus to filter and capture micro- and nanoplastic. We found that industry stakeholders in both case areas were generally more accepting of policy and increased innovation moving forward, but placed weight on the scientific community to conduct more research on the pollution issue and propose solutions. Future generation stakeholders (youth aged 14–18), however, put emphasis on consumer behavior and buying patterns of single-use products fueling the plastic crisis.
Keywords: ecology, plastics, single-use plastic, stakeholder integration, plastic pollution, systems thinking
Published in DiRROS: 19.07.2024; Views: 320; Downloads: 176
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1190. Grass growth and ▫$N_2O$▫ emissions from soil after application of jellyfish in coastal areasFlorian Borchert, Iraj Emadodin, Christof Kluß, Ana Rotter, Thorsten Reinsch, 2021, original scientific article Abstract: The supply of nutrients for agricultural production faces enormous challenges as food security and sustainability goals have to be ensured. Processing of marine biomass has high potential to provide nutrients for agricultural purposes in coastal areas. One underexplored resource are jellyfish, which occur as blooms and by-catch of the fishing industry. In this context, a pot experiment investigated the effects of jellyfish as a fertilizer on biomass accumulation of annual ryegrass (Lolium multiflorum), and its effect on the important greenhouse gas N2O as a sustainability indicator of novel fertilizers. Dried and ground jellyfish was applied [3 species: Aurelia aurita (AA), Cyanea capillata (CC), Periphylla periphylla (PP)] and compared with an unfertilized and a mineral fertilized (calcium-ammonium-nitrate, CAN) treatment. Dried jellyfish and CAN were applied at equal N rates of 5 g N per m2. The N2O-fluxes from soil were measured over 56 days after fertilizer application. Grass dry matter yields, when using CC and PP treatments, were not significantly different to the CAN treatment (p > 0.05). After reducing its salinity, AA also showed no differences to CAN on plant growth and the lowest coefficient of variation for dry matter yield as an indicator for yield stability. Accumulated N2O-emissions were lowest in the control and were 3-times higher in AA and CC compared to CAN (p < 0.05). If salinity levels are moderate, jellyfish application to soil can compete with artificial mineral fertilizers in terms of N-supply for above- and belowground yield response, regardless of jellyfish species used. However, elevated N2O-emissions are likely to affect its suitability for large-scale application. Nevertheless, if energy-efficient methods of drying and desalination of jellyfish can be developed, in coastal areas dried jellyfish is a valuable fertilizer in coastal areas, particularly in situations where nutrient supplies for agriculture are limited.
Published in DiRROS: 19.07.2024; Views: 262; Downloads: 187
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