1511. Hay meadow vibroscape and interactions within insect vibrational communityRok Šturm, Behare Rexhepi, Juan José López Díez, Andrej Blejec, Jernej Polajnar, Jérôme Sueur, Meta Virant-Doberlet, 2021, original scientific article Abstract: Our experiences shape our knowledge and understanding of the world around us. The natural vibrational environment (vibroscape) is hidden to human senses but is nevertheless perceived and exploited by the majority of animals. Here, we show that the vibroscape recorded on plants in a temperate hay meadow is a dynamic low-frequency world, rich in species-specific vibrational signals. The overall vibroscape composition changed throughout the season and also depended on the plant species, as well as on the spatial position of individual plants within the meadow. Within the studied community, vibrationally signaling species sharing this communication channel avoided interference primarily by partitioning vibrational space on a fine temporal scale. The vibroscape is a reliable source of information in the environment and expands our understanding of ecological and evolutionary processes.
Published in DiRROS: 19.07.2024; Views: 380; Downloads: 259
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1512. Virome analysis of signal crayfish (Pacifastacus leniusculus) along its invasion range reveals diverse and divergent RNA virusesKatarina Bačnik, Denis Kutnjak, Silvija Černi, Ana Bielen, Sandra Hudina, 2021, original scientific article Abstract: Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.
Keywords: signal crayfish virome, RNA viruses, invasive alien species, invasion range, high-throughput sequencing
Published in DiRROS: 19.07.2024; Views: 473; Downloads: 226
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1513. A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter proteinMojca Juteršek, Marko Dolinar, 2021, original scientific article Abstract:
Background
Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803.
Methods
We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target.
Results
With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.
Keywords: bacteria, cyanobacteria, synthetic biology, proteolytic cleavage, cystatin
Published in DiRROS: 19.07.2024; Views: 341; Downloads: 409
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1514. Two new species of parastenocaris (Copepoda, Harpacticoida) from a hyporheic zone and overview of the present knowledge on Stygobiotic Copepoda in Vietnam : historical overview and new dataNgoc-Son Tran, Mau Trinh-Dang, Anton Brancelj, 2021, original scientific article Abstract: The number of freshwater species belonging to the genus Parastenocaris reported from ten countries of Southeast Asia is quite limited. Only two species have been reported so far from freshwater habitats there, compared to over 290 described species of the family Parastenocarididae worldwide. During the first study of the hyporheic zone of two small rivers in central Vietnam, two new species of the family Parastenocarididae were collected, Parastenocaris sontraensis sp. nov. and Parastencaris vugiaensis sp. nov. Both were collected from the gravel bar along the rivers (Suoi Da and Vu Gia river) using the Karaman–Chappuis method. Both the new species belong to the brevipes group of the genus Parastenocaris Kessler, 1913 sensu Lang (1948), and Reid (1995). Parastenocaris sontraensis sp. nov. is similar to P. hinumaensis Kikuchi, 1970 and Parastenocaris jane Karanovic, 2006 in the brevipes-group. Parastenocaris sontraensis sp. nov. differs from both Parastenocaris species by (i) Exp P3 with three segments in the male, (ii) caudal rami with seven setae, and (iii) caudal rami about 2.4 times as long as wide. Parastencaris vugiaensis sp. nov. can be distinguished from its congeners by the unique combination of the following characters: (i) the elliptical shape of caudal rami, (ii) apical seta (V) with bulbous base, and (iii) anal operculum extends beyond the end of anal somite. Until now, 14 stygobiotic species of Copepoda have been recorded in Vietnam (including two new species in this paper), which is relatively few compared with nearby Thailand with 25 species. Short comments on other stygobiotic Copepoda from Vietnam are added.
Keywords: Southeast Asia, groundwater fauna, hyporheic zone, Copepoda, new species, taxonomy
Published in DiRROS: 19.07.2024; Views: 314; Downloads: 191
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1515. 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: 442; Downloads: 193
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1516. 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: 379; Downloads: 215
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1517. 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: 427; Downloads: 252
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1518. 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: 368; Downloads: 199
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1519. 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: 311; Downloads: 222
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1520. 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: 355; Downloads: 215
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