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Feasibility of droplet digital PCR analysis of plasma cell-free DNA from kidney transplant patients
Barbara 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: 10; Downloads: 4
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Is it time for ecotremology?
Rok Šturm, Juan José López Díez, Jernej Polajnar, Jérôme Sueur, Meta Virant-Doberlet, 2022, original scientific article

Abstract: Our awareness of air-borne sounds in natural and urban habitats has led to the recent recognition of soundscape ecology and ecoacoustics as interdisciplinary fields of research that can help us better understand ecological processes and ecosystem dynamics. Because the vibroscape (i.e., the substrate-borne vibrations occurring in a given environment) is hidden to the human senses, we have largely overlooked its ecological significance. Substrate vibrations provide information crucial to the reproduction and survival of most animals, especially arthropods, which are essential to ecosystem functioning. Thus, vibroscape is an important component of the environment perceived by the majority of animals. Nowadays, when the environment is rapidly changing due to human activities, climate change, and invasive species, this hidden vibratory world is also likely to change without our notice, with potentially crucial effects on arthropod communities. Here, we introduce ecotremology, a discipline that mainly aims at studying substrate-borne vibrations for unraveling ecological processes and biological conservation. As biotremology follows the main research concepts of bioacoustics, ecotremology is consistent with the paradigms of ecoacoustics. We argue that information extracted from substrate vibrations present in the environment can be used to comprehensively assess and reliably predict ecosystem changes. We identify key research questions and discuss the technical challenges associated with ecotremology studies.
Keywords: ecotremology, monitoring, hidden biodiversity, vibroscape, vibrational communication
Published in DiRROS: 16.07.2024; Views: 28; Downloads: 7
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Validated inventories of non-indigenous species (NIS) for the Mediterranean Sea as tools for regional policy and patterns of NIS spread
Marika Galanidi, Mehdi Aissi, Malek Ali, Ali Bakalem, Michel Bariche, Angela G. Bartolo, Borut Mavrič, Martina Orlando-Bonaca, 2023, original scientific article

Abstract: This work presents refined, updated subregional and regional non-indigenous species (NIS) inventories for the Mediterranean Sea, validated by national and taxonomic experts, with species records observed until December 2020. These datasets will be used as the baselines for the implementation of the Integrated Monitoring and Assessment Programme for the Mediterranean (IMAP) and the Mediterranean Quality Status Report 2023. In total, 1006 non-indigenous species have been found in Mediterranean marine and brackish waters. The highest numbers of NIS were observed in Israel, Türkiye, Lebanon and Italy. Approximately 45 species were categorized as data deficient, either due to lack of consensus on their alien status or the validity of their identification. Polychaeta, Foraminifera and macroalgae were the groups with the highest numbers of controversial species. There was a general increase in the yearly rate of new NIS introductions after the late 1990s, which appears to be slowing down in the last decade, but this may be confounded by reporting lags and differential research efforts. Between 1970 and 2020 there has been a steep increase in the proportion of shared species present throughout all four Mediterranean subregions, which are predominantly transported via shipping and recreational boating. While Lessepsian species are gradually spreading westwards and northwards, there is still a considerable invasion debt accumulating in the eastern and central Mediterranean.
Keywords: non-indigenous species, Mediterranean Sea, validation, non-indigenous species inventories, NIS, Integrated Monitoring and Assessment Programme for the Mediterranean, IMAP
Published in DiRROS: 12.07.2024; Views: 54; Downloads: 22
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Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera
Timotej Turk Dermastia, Ivano Vascotto, Janja Francé, David Stanković, Patricija Mozetič, 2023, original scientific article

Abstract: Diatoms are one of the most important phytoplankton groups in the world’s oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks.
Keywords: rbcL, metabarcoding, monitoring, diatoms, population genetics, Pseudo-nitzschia, Adriatic
Published in DiRROS: 12.07.2024; Views: 48; Downloads: 21
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Impact of deoxynivalenol and zearalenone as single and combined treatment on DNA, cell cycle and cell proliferation in HepG2 cells
Ana-Marija Domijan, Klara Hercog, Martina Štampar, Goran Gajski, Marko Gerić, Marijana Sokolović, Bojana Žegura, 2023, original scientific article

Abstract: The study aimed to investigate toxicity and the mechanism of toxicity of two Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA). DON and ZEA were applied to HepG2 cells as single compounds and in combination at low environmentally relevant concentrations. HepG2 cells were exposed to DON (0.5, 1, and 2 µM), ZEA (5, 10, and 20 µM) or their combinations (1 µM DON + 5 µM ZEA, 1 µM DON + 10 µM ZEA and 1 µM DON + 20 µM ZEA) for 24 h and cell viability, DNA damage, cell cycle and proliferation were assessed. Both mycotoxins reduced cell viability, however, combined treatment with DON and ZEA resulted in higher reduction of cell viability. DON (1 µM) induced primary DNA damage, while DON (1 µM) in combination with higher ZEA concentrations showed antagonistic effects compared to DON alone at 1 µM. DON arrested HepG2 cells in G2 phase and significantly inhibited cell proliferation, while ZEA had no significant effect on cell cycle. The combined treatment with DON and ZEA arrested cells in G2 phase to a higher extend compared to treatment with single mycotoxins. Potentiating effect observed after DON and ZEA co-exposure at environmentally relevant concentrations indicates that in risk assessment and setting governments’ regulations, mixtures of mycotoxins should be considered.
Keywords: mycotoxins, comet assay, flow cytometry, co-exposure, food monitoring
Published in DiRROS: 12.07.2024; Views: 69; Downloads: 21
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Vsebnosti potencialno strupenih elementov v sedimentih in vodah reke Meže in njenih pritokov, ki odvodnjavajo odlagališča rudarskih odpadkov
Mateja Gosar, Špela Bavec, Miloš Miler, Martin Gaberšek, 2024, original scientific article

Abstract: Predstavljeni so rezultati spremljanja vsebnosti potencialno strupenih elementov (PSE) v sedimentih (v letih 2013, 2017, 2020) in vodah (v letih 2017, 2020) reke Meže ter njenih pritokov, ki odvodnjavajo odlagališča rudarskih odpadkov. Skupno 13 vzorčnih mest je vzpostavljenih v vzorčni shemi, ki omogoča dolgoročno opazovanje vpliva odlagališč rudarskih odpadkov. V sedimentih so zaradi vplivov več kot 300-letnega delovanja rudarsko-predelovalne industrije močno povečane vsebnosti PSE, predvsem Pb, Zn, Cd, Mo in As, ki s časom precej nihajo. Razlike v vsebnostih na istih lokacijah v različnih letih so najbolj izrazite v pritokih reke Meže, ki drenirajo odlagališča rudarskih odpadkov. Na vsebnosti imajo pomemben vpliv hidrološki pogoji, saj so ob višjem vodostaju in višjem pretoku vsebnosti PSE večje. Vodna erozija odlagališč ima pomemben vpliv na dotok onesnaženega materiala v vodotoke. V nasprotju s pritoki, v zgornjem toku reke Meže nismo opazili večjega vpliva višjega vodostaja in pretoka na vsebnosti PSE v sedimentih. Dolvodno od Žerjava so nihanja vsebnosti med posameznimi leti oz. različnimi hidrološkimi pogoji tudi v Meži večja. Predstavljeni rezultati kažejo, da so v sedimentih reke Meže in njenih pritokov vsebnosti Pb, Zn, Cd, Mo in As zelo velike ter krepko presegajo zakonsko določeno kritično vrednost za tla. V površinski vodi so vsebnosti PSE lokalno povečane in se s časom bistveno ne spreminjajo. Glede na primerjavo z zakonodajnimi smernicami, so v obravnavanih vodah lokalno presežene koncentracije Pb, Cd in Zn. Ocenjujemo, da je dinamika obremenjenosti sedimentov reke Meže s PSE vzdolž krajev Črna na Koroškem, Žerjav in Mežica zelo kompleksna. Poleg odlagališč rudarskih odpadkov na vsebnosti PSE v sedimentih in vodah vplivajo tudi razpršeni viri v okolju, kot so onesnažena tla in poplavne ravnice ter njihova različna stopnja onesnaženosti, saj je okolje obremenjeno zaradi dolgoletnih rudarskih in talilniških dejavnosti. Dodaten okoljski vpliv ima morda tudi sedanja industrijska dejavnost v dolini reke Meže.
Keywords: rudarjenje, rudarski odpadki, odlagališča odpadkov, potencialno strupeni elementi, rečni sedimenti, monitoring, onesnaženje okolja
Published in DiRROS: 09.07.2024; Views: 65; Downloads: 14
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Distribution of toxigenic cyanobacteria in Alpine lakes and rivers as revealed by molecular screening
Maša Jablonska, Leonardo Cerasino, Adriano Boscaini, Camilla Capelli, Claudia Greco, Aleksandra Krivograd-Klemenčič, Ute Mischke, Nico Salmaso, Rainer Kurmayer, 2024, original scientific article

Abstract: The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel study.
Keywords: cyanotoxins, planktic and benthic habitats, water quality monitoring, early warning, toxigenic cyanobacteria, European Alps
Published in DiRROS: 03.06.2024; Views: 199; Downloads: 119
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