11. Direct regulation of shikimate, early phenylpropanoid, and stilbenoid pathways by subgroup 2 R2R3-MYBs in grapevineLuis Orduña, Miaomiao Li, David Navarro-Payá, Chen Zhang, A. Santiago, Pablo Romero, Živa Ramšak, Gabriele Magon, Janine Höll, Patrick Merz, Kristina Gruden, Alessandro Vannozzi, Dario Cantu, Jochen Bogs, Darren C. J. Wong, Shao-shan Carol Huang, José Tomás Matus, 2022, original scientific article Abstract: The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we show how DNA affinity purification sequencing (DAP-Seq) allows for the genome-wide TF-binding site interrogation in grape. We obtained 5190 and 4443 binding events assigned to 4041 and 3626 genes for MYB14 and MYB15, respectively (approximately 40% of peaks located within −10 kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1400 transcriptomic datasets from leaves, fruits, and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15, and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H, and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates among which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes were induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism.
Keywords: secondary metabolism, regulatory networks, transcription factors, transcriptional regulation, DNA affinity purification sequencing
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 2
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12. Detection of the endangered stone crayfish Austropotamobius torrentium (Schrank, 1803) and its congeneric A. pallipes in its last italian biotope by eDNA analysisChiara Manfrin, Massimo Zanetti, David Stanković, Umberto Fattori, Victoria Bertucci-Maresca, Piero G. Giulianini, Alberto Pallavicini, 2022, original scientific article Abstract: The stone crayfish, Austropotamobius torrentium, is a European freshwater crayfish. Although this species is relatively widespread throughout the continent, it is undergoing significant declines throughout its range. However, as the decline rates have not been quantified in detail, this species is classified as data deficient by the IUCN Red List of Threatened Species. The present study describes the development and validation of two species-specific assays based on hydrolysis probe chemistry for the detection of A. torrentium and A. pallipes environmental DNA (eDNA) in water samples collected in the Julian Alps of Italy (Friuli Venezia Giulia). The eDNA-based method was applied to 14 sites within the Danubian Slizza basin, known to be inhabited by A. torrentium, but with insufficient information on their distribution. In addition, one station in the Tagliamento River basin was sampled to test the performance of the A. pallipes probe. The presence of A. torrentium is confirmed at 6 out of 15 sites. At four of these sites, A. torrentium is detected for the first time. In contrast, the presence of A. torrentium was not detected at two sites already known to harbour the species. Finally, the presence of A. pallipes was confirmed in the station belonging to the Tagliamento basin. The methodology described, which allows the distinction between the two species, paves the way for the parallel detection of the stone crayfish and the white-clawed crayfish (A. pallipes) through eDNA analysis.
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 0
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13. Fast assay to predict multipotent mesenchymal stromal cell replicative senescence dynamicsKatja Kološa, Aleš Leskovšek, Teja Rajar, Tamara Lah Turnšek, 2022, original scientific article Abstract: The major obstacle to the application of mesenchymal stromal cells (MSCs) in regenerative medicine is the expansion of the donor-derived cells in vitro to obtain high cell numbers in the shortest possible time. However, MSCs gradually undergo replicative senescence after a variable number of divisions that reduce their therapeutic efficacy, which needs to be determined before administration. The authors developed a fast and simple evaluation assay testing two senescence inducers, mitoxantrone (Mxt) and trichostatin A (TSA), to predict the onset of spontaneous replicative senescence of adipose-derived mesenchymal stromal cells (ASCs) and have confirmed the correlation between induced senescence and spontaneous replicative senescence in the assay using Mxt. This protocol facilitates the standardization of therapeutic ASCs and MSCs from other origins before application.
Keywords: adipose mesenchymal stromal (stem) cells (ASCs), cell longevity, induced senescence, mesenchymal stromal (stem) cells (MSCs), mitoxantrone, replicative senescence, trichostatin A
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 0
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14. 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, original scientific article Abstract: 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.
Keywords: HPLC, SEM microscopy, absorbance, photosymbiosis
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 0
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15. An analysis of Adriatic ichthyofauna : ecology, zoogeography, and conservation statusLovrenc Lipej, Marcelo Kovačić, Jakov Dulčić, 2022, original scientific article Abstract: The paper presents an analysis of biogeographic and habitat distribution patterns, and the conservation status data of Adriatic fishes, based on the last published checklist and evidence-based critical analyses of species presence. The total number of species recorded in the Adriatic is 449. The Adriatic has 58.8% of Mediterranean species richness, 76.1% of its families, and 87.8% of its orders. Among species discovered in the Adriatic after 2010, twelve species were attributed to biological invasion, mostly Atlantic immigrants or alien species, and ten species were attributed to improved research on the native ichthyofauna of the Adriatic area. About 58% of species are native species of Atlanto-Mediterranean origin, 21% are native species of wider global occurrence, 15% are Mediterranean or Mediterranean and Black Sea endemics and 5% originated outside Mediterranean Sea. The majority of species inhabit the benthic environment (71.9%), while others occur in the pelagic environment (20.7%) or are euryhaline (7.3%). The benthic littoral species are the most numerous Adriatic fishes, representing 40% of all species richness, whereas pelagic fishes are mainly eurybathic or epipelagic; only 3.6% of species are deep pelagic species. A Red Book of marine fishes of the Adriatic Sea is urgently needed to assess their conservation status, covering the entire Adriatic Sea and reviewing all fish species to assess their conservation status.
Keywords: Adriatic ichthyofauna, evidence-based approach, zoogeography, habitat distribution
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 2
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16. From causal networks to adverse outcome pathways : a developmental neurotoxicity case studyŽiva Ramšak, Vid Modic, Roman A. Li, Colette vom Berg, Anže Županič, 2022, original scientific article Abstract: The last decade has seen the adverse outcome pathways (AOP) framework become one of the most powerful tools in chemical risk assessment, but the development of new AOPs remains a slow and manually intensive process. Here, we present a faster approach for AOP generation, based on manually curated causal toxicological networks. As a case study, we took a recently published zebrafish developmental neurotoxicity network, which contains causally connected molecular events leading to neuropathologies, and developed two new adverse outcome pathways: Inhibition of Fyna (Src family tyrosine kinase A) leading to increased mortality via decreased eye size (AOP 399 on AOP-Wiki) and GSK3beta (Glycogen synthase kinase 3 beta) inactivation leading to increased mortality via defects in developing inner ear (AOP 410). The approach consists of an automatic separation of the toxicological network into candidate AOPs, filtering the AOPs according to available evidence and length as well as manual development of new AOPs and weight-of-evidence evaluation. The semiautomatic approach described here provides a new opportunity for fast and straightforward AOP development based on large network resources.
Keywords: systems toxicology, adverse outcome pathway, casual network, toxicological network, neurotoxicity
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 2
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17. 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: 2; Downloads: 2
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18. Development and validation of a one-step reverse transcription real-time PCR assay for simultaneous detection and identification of tomato mottle mosaic virus and tomato brown rugose fruit virusAntonio Tiberini, Ariana Manglli, Anna Taglienti, Ana Vučurović, Jakob Brodarič, Luca Ferretti, Marta Luigi, Andrea Gentili, Nataša Mehle, 2022, original scientific article Abstract: Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10−5–10−6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.
Keywords: real-time PCR, tomato mottle mosaic virus, tomato brown rugose fruit virus, leaves detection, seeds detections, performance criteria
Published in DiRROS: 16.07.2024; Views: 0; Downloads: 4
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20. Is it function or fashion? : an integrative analysis of morphology, performance, and metabolism in a colour polymorphic lizardVeronica Gomes, Anamarija Žagar, Guillem Pérez i de Lanuza, Tatjana Simčič, Miguel A. Carretero, 2022, original scientific article Abstract: Colour polymorphism may be accompanied by alternative expressions of phenotypic traits that are directly or indirectly related to fitness, and selection forces may act on the traits separately or concurrently. In polymorphic species, natural selection may act through frequency- or density-dependent mechanisms and maintain polymorphism through interactions among morphs. We used an integrative approach to examine functionally relevant and interrelated life history traits in the context of colour polymorphism in the European wall lizard (Podarcis muralis). Body, head and limb morphology, bite and locomotor performance, and physiology, particularly metabolic and antioxidant capacity, were analysed in the three pure colour morphs (yellow, orange, white) and the two mosaic morphs (yellow-orange and white-orange). Morphological differences between morphs were present but subtle and consisted mainly of variations in head length. Head size and bite force were strongly associated between and within morphs. Limb and boot morphology and locomotor performance (sprinting and climbing) were variably associated among morphs. Finally, variation in biochemical indicators of cellular metabolism and antioxidant capacity appeared to be largely independent of morphology and performance. The results provide evidence for existing and non-existing relationships between colour and morphology, performance, and physiology that could have short- and long-term effects on selection.
Keywords: colour morphs, sprint speed, climbing, bite force, physiology
Published in DiRROS: 16.07.2024; Views: 8; Downloads: 4
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