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Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post‐transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas‐associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long‐lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

New psychoactive substances (NPS) constitute a group of psychotropic substances, designed to mimic the effects of traditional substances like cannabis, cocaine, MDMA, khat, which was not regulated by the 1961 United Nations Convention on Narcotics or the 1971 United Nations Convention on Psychotropic Substances. Illegal laboratories responsible for their production regularly developed new substances and placed them on the market to replace the ones that have been banned; for this reason, during the last decade this class of substances has represented a great challenge for the public health and forensic toxicologists. The spectrum of side effects caused by the intake of these drugs of abuse is very wide since they act on different systems with various mechanisms of action. To date most studies have focused on the neurotoxic effects, very few works focus on cardiotoxicity. Specifically, both synthetic cannabinoids and synthetic cathinones appear to be involved in different cardiac events, including myocardial infarction and sudden cardiac death due to fatal arrhythmias. Synthetic cannabinoids and cathinones cardiotoxicity are mainly mediated through activation of the CB1 receptor present on cardiomyocyte and involved with reactive oxygen species production, ATP depletion and cell death. Concerns with the adrenergic over-stimulation induced by this class of substances and increasing oxidative stress are mainly reported. In this systematic review we aim to summarize the data from all the works analyzing the possible mechanisms through which synthetic cannabinoids and synthetic cathinones damage the myocardial tissue.

Life cycle of the aquatic moth Cataclysta lemnata (Lepidoptera: Crambidae) was studied in laboratory conditions to obtain a basic biological knowledge useful for predicting the possible success of the herbivorous larvae of this insect as potential control agents in limiting the spread of the invasive American duckweed Lemna minuta (Alismatales: Araceae) in Italy. The multivoltinism of C. lemnata, as well as the high overall emergence from the pupal stage (85%), the high success in mating among the formed couples (>90%), and the high number of larvae born from each egg laying (on average 310 individuals), suggest that the insect can be successfully bred in the laboratory for the purposes of an augmentative biological control. Under experimental conditions, larvae developed in 23 days (through six larval instars, distinguishable by cephalic capsule dimensions) and pupae in 10, with no difference in duration between females and males. The larval phase resulted longer than the adult one (23 vs 10 days), therefore it can be considered the most suitable stage for releasing the insect in field for biocontrol purposes. Indeed, the larvae having a herbivorous diet might consume large amount of the invasive plant, contrarily to the adult phase which is focused exclusively on reproduction. The results emerged not only allow to contribute to the knowledge on aquatic lepidoptera that are scarcely known, but also support the effectiveness of a possible protocol for an augmentative biological control of the invasive alien duckweed L. minuta.

Several works have investigated the impact of urbanisation on carabid activity density using urban-rural gradients. Such works compared activity density recorded from green spaces located in different parts of a city and assigned to categories of increasing urban intensity, which poses two problems: (1) since the gradient is divided into categories, it is impossible to model continuous variations in biotic responses, and (2) sites representative of different urbanisation levels are not true segments of the same ecological continuum. To surpass these problems, we modelled variations in carabid activity density along an urban-rural transect within a single green space extending from the city centre of Rome to rural environments. Carabids were sampled by pitfall traps from sites distributed along the entire gradient. We used breakpoint regressions to model how (1) carabid activity density, (2) carabids/beetles ratio, (3) carabids/insects ratio and (3) carabids/arthropods ratio varied along the gradient. As already observed for various organisms in urban environments, we found that activity density of carabids and their contribution to the abundance of beetles, insects and arthropods, peaked in the middle of the gradient. This supports the intermediate disturbance hypothesis, according to which moderate urbanisation may favour diversity by increasing habitat heterogeneity.

Mountains are strongly seasonal habitats, which require special adaptations in wildlife species living on them. Population dynamics of mountain ungulates are largely determined by the availability of rich food resources to sustain lactation and weaning during summer. Increases of temperature affect plant phenology and nutritional quality. Cold-adapted plants occurring at lower elevations will shift to higher ones, if available. We predicted what could happen to populations of mountain ungulates based on how climate change could alter the distribution pattern and quality of high-elevation vegetation, using the “clover community-Apennine chamoisRupicapra pyrenaica ornata” system. From 1970 to 2014, increasing spring temperatures (2 °C) in our study area led to an earlier (25 days) onset of green-up in Alpine grasslands between 1700 and 2000 m, but not higher up. For 1970–2070, we have projected trends of juvenile winter survival of chamois, by simulating trajectories of spring temperatures and occurrence of clover, through models depicting four different scenarios. All scenarios have suggested a decline of Apennine chamois in its historical core range, during the next 50 years, from about 28% to near-extinction at about 95%. The negative consequences of climate changes presently occurring at lower elevations will shift to higher ones in the future. Their effects will vary with the species-specific ecological and behavioural flexibility of mountain ungulates, as well as with availability of climate refugia. However, global shifts in distributional ranges and local decreases or extinctions should be expected, calling for farsighted measures of adaptive management of mountain-dwelling herbivores.

1. Climate change is causing shifts in the distribution of many species and populations inhabiting mountain tops are particularly vulnerable to these threats because they are constrained in altitudinal shifts. Apennines are a relatively narrow and low mountain chain located in Southern Europe, which hosts many isolated populations of mountain species. The butterfly Erebia pandrose was recorded for the last time in the Apennines in 1977, on the top of a single massif (Monti della Laga). 2. We confirmed the presence of a small, isolated population of E. pandrose in the Apennines, at a distance of more than 400¿km to any other known populations. Then, we examined the cytochrome c oxidase subunit 1 mitochondrial DNA marker of this species across the Palaearctic area and estimated the potential decline over the Alps and the Apennines due to future climatic changes. 3. The Apennine population represents an endemic lineage characterised by eight mutations over the 658¿bp analysed (1.2%). In the Alps and Apennines, this species has shifted uphill more than 3 m per year since the end of the 19th century and more than 22 m per year since 1995. Species distribution models suggested that these mountain populations will experience a generalised loss of climatic suitability, which, according to our projections, could lead to the extinction of the Apennine population in a few decades. 4. Erebia pandrose has the potential to become a flagship species for advertising the risk of losing unique fractions of genetic diversity for mountain species., funded by the project: ‘Ricerca e conservazione sui lepidotteri diurni di sei Parchi Nazionali dell'Appennino centro-settentrionale’. Support for this research was also provided by ‘La Caixa’ Foundation (ID 100010434) to M.M. (grant LCF/BQ/DR20/11790020), by the Academy of Finland to V.D. (Academy Research Fellow, decision no. 328895) and by project PID2019-107078GB-I00/AEI/10.13039/501100011033 to R.V., 1 Introduction 2 Materials and methods 2.1 Data collection 2.2 COI analyses 2.3 Species distribution modelling 2.4 Elevational shifts of E. pandrose records through time 3 Results 3.1 COI structure 3.2 Climate change effects 4 Discussion 4.1 The population of Monte Gorzano 4.2 What is the future for E. pandrose on the Apennines? 4.3 Erebia pandrose in the context of preserving Apennine butterflies 5 Conclusions Acknowledgements Conflict of interest

Wildfires represent an important factor in the disturbance in Mediterranean ecosystems, although the effects of wildfires on the insect communities of mountain environments remain largely unknown. This research investigated the effect of fire on dung beetles in a Mediterranean high-altitude area, located in Central Italy (1500 m elevation). Sampling in each of the three main vegetation types of the study area (conifer plantation, beechwood forest and grassland) used pitfall traps during Spring to Autumn. For each habitat, three burnt and three unburnt sites were sampled. We considered the following community structure parameters: abundance, richness, Shannon diversity and Simpson diversity. Effects of fire, habitat and sampling period were investigated using generalised mixed-effects models. Abundance, species richness and diversity were higher in the grassland than in the two forest habitats and June was the month with the lowest values because of the late emergence of insects at high altitudes. Fire negatively affected dung beetle abundance, but not diversity. Forest habitats were less favourable to dung beetle recolonisation than the secondary grassland. Thus, dung beetles are sensitive to vegetation type, but not to fire, possibly because of their high recolonisation capabilities and preference for open habitats.

The peculiar morphology of the discotelic larvae of three species of Pachyteles (Coleoptera, Carabidae, Paussinae, ozaenini) collected in an Andean cloud forest in Ecuador (otonga nature Reserve, Cotopaxi) is here analyzed. All instars of Pachyteles vignai Deuve, 1999, 2nd and 3rd instars of P. digiulioi Deuve, 1999, and the 3rd instar of another undetermined Pachyteles species are described. A particular emphasis on the illustration of the aberrant larval morphology is given by using both traditional drawing techniques and macro multifocal image acquisition, combined with scanning electron microscopy (SEM) for analyzing the details. The diagnostic characters of these larvae are compared to those of the other known larvae of ozaenini.

There is a growing interest on the potential interplay between weather, habitat, and interspecific competition on population dynamics of wild herbivores. Favorable environmental conditions may buffer the negative effects of competition; conversely, competition may be expected to be stronger under harsh environmental conditions. We investigated relationships between competitor abundance, weather, and habitat cover on density and local distribution of a medium‐sized herbivore, the roe deer Capreolus capreolus, as well as its spatial overlap with fallow deer Dama dama in a Mediterranean protected area. Over 11 years (2007–2017), roe deer density was not affected by spring–summer rainfall in the previous year and decreased with increasing density of fallow deer in the previous year. Hence, over the considered temporal scale, results supported a major role of competition over weather in influencing population trends of roe deer. At a finer spatial scale, roe deer occupancy was negatively affected by local abundance of fallow deer, especially in “poorer” habitats. We found a slight support for a positive effect of fallow deer density on interspecific spatial overlap. Moreover, fine‐scale spatial overlap between deer species increased with decreasing rainfall in spring–summer. Fallow deer were introduced to our study area in historical times and their role as superior competitors over roe deer has been found also in other study areas. We suggest a potential role of harsh weather conditions during the growing season of vegetation (i.e. scarce rainfall) in triggering the potential for ecological overlap, emphasizing the negative effects of interspecific competition., We investigated relationships between competitor abundance, weather and habitat on density and distribution of the roe deer Capreolus capreolus, as well as its spatial overlap with fallow deer Dama dama in a Mediterranean protected area. Over 11‐years, roe deer density was not affected by spring‐summer rainfall in the previous year and decreased with increasing density of fallow deer in the previous year. At a finer spatial scale, roe deer occupancy was negatively affected by local abundance of fallow deer, especially in “poorer” habitats; interspecific spatial overlap was influenced both by habitat and rainfall, being negatively related to water precipitation in spring‐summer.

The mitigation of ecological/economic impacts of wild boar Sus scrofa is one of the most challenging issues in wildlife management worldwide. Monitoring population density and impact of wild boar is crucial to plan appropriate management actions to reduce its density, environmental impact and epidemiological risk, as well as to evaluate control effectiveness. In 2018–2019, we used plot-based faeces counts, coupled with specific estimates of daily defecation rates, to estimate wild boar density and rooting activity in natural/semi-natural habitats, in a Mediterranean protected area. Daily defecation rate was 6.7 faeces/individual, much lower than that of ruminants. We obtained estimates of 70.0–70.5 faeces/km2, corresponding to wild boar densities of c. 10.5 individuals/km2 (relative standard error: 18%) in both years. Low daily defecation rates and skewed distribution frequencies of wild boar faeces should be considered to plan surveys with an adequate sampling design and intensity. Faeces abundance and rooting activity peaked in ecotones, i.e. open areas at the interface of wood patches, whereas they did not differ between other habitat types, suggesting a fine-scale concordance between the two indicators. Long-term monitoring is needed to assess relationships between indicators of impact and abundance at a broad scale. Our work shows how to undertake simultaneously wild boar density estimates and impact assessment in natural environments, which is particularly important for protected areas and/or habitats of conservation concern across the globally distributed Mediterranean biome.

The ecological drivers underlying breeding performance are expected to differ across the geographical range of seabird species, but few studies have compared trade-offs between colonies with different local conditions. During chick-rearing (2000–01), we compared the foraging trips, diet and breeding parameters of two Adélie penguin colonies in the Ross Sea, at Edmonson Point (EdPo; ~2000 breeding pairs) and Inexpressible Island (InIs; ~24 000 breeding pairs). Penguins from InIs travelled farther and performed longer feeding trips. The quantity of food brought to the nest was the same for the two colonies, but penguins from InIs brought more fish and less krill. Eggs hatched earlier at EdPo. Breeding success did not differ, but chick weight during hatching–fledging was greater at InIs. Despite worse weather conditions at InIs, the larger proportion of high-energy food brought by penguins from InIs (i.e. fish) may explain their offspring‘s better performance. In addition, the persistence of fast ice at EdPo may have led to greater energy expenditure of breeding individuals, possibly reducing chick growth. The greater intraspecific competition expected at InIs may have been reduced by longer foraging trips and/or counteracted by the more nutritious diet. Our findings reveal complex trade-offs between foraging effort and environmental constraints in determining the breeding performance of Adélie penguins.

Movements, habitat use, and activity of herbivorous species are mainly influenced by trade-offs between food availability and daily/seasonal variation in predation risk. Nevertheless, studies conducted across several seasons and evaluating both spatial and temporal responses of meso-small herbivores are still scanty. Additionally, information on spatiotemporal behaviour is often lacking for cryptic, localised species, which may limit conservation actions. We evaluated how sex, time of day, seasonality, and habitat type—reflecting trade-offs in predation risk and access to food resources—influenced the spatial behaviour and locomotor activity of an herbivorous mammal, the Apennine hare, a threatened species endemic to central-southern Italy. In a 4-year study, we intensively radio-tracked 12 individuals, providing the first insights on the spatiotemporal ecology of this lagomorph. Sex affected neither home range size nor habitat selection. Home range size was larger during the night and in warm months, when hares moved to the most energetically rewarding habitat type for feeding activities, i.e., cultivations. At both study area and home range spatial scales, habitat selection did not vary between the cold and the warm months. At the study area scale, hares avoided deciduous woodland and human settlements, whereas selected the Mediterranean scrubwood and cultivations. Within home ranges, at night, Apennine hares selected risky patches, i.e., cultivations as feeding grounds, whereas in daylight, the safer Mediterranean scrubwood was used for cover and resting. Throughout the year, Apennine hares were mostly nocturnal and were more active in open than in concealed habitats, with no differences between sexes. Bright nights, i.e., with full moon and clear sky, inhibited activity in open areas but not in cover, likely to reduce predation risk. Our findings emphasised the role of feeding/antipredatory requirements in shaping spatiotemporal behaviour of meso-small mammals, with potential consequences for the conservation of threatened species.

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of -1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

11 páginas, 2 figuras, 1 tabla, Inappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance., I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)

All methods currently employed in cladistic biogeography usually give contrasting results and are theoretically disputed. In two overlooked papers, Hovenkamp (1997, 2001) strongly criticized methods currently used by biogeographers and proposed two other methods. However, his criticisms have remained unanswered and his methods rarely applied. I used three different data sets to show the superiority of Hovenkamp's methods. Both methods proposed by Hovenkamp do not suffer from the unrealistic assumptions that underlie other methods commonly used in cladistic biogeography. The method proposed in 2001 is more powerful than the previous method published in 1997, because it does not use a priori assumptions about the areas involved. However, the method proposed in 1997 may be a valid alternative for large data sets. © The Willi Hennig Society 2007.

Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.

The genetic identification of skeletal remains found in Second World War mass graves is complicated because of the poor quality of the samples. The aim of this study was to set up a workflow for STR typing of such samples combining PCR/CE and PCR/NGS technologies. To this end, 57 DNA samples from an equal number of 75-year-old femurs were studied. After a first round of PCR typing using GlobalFiler CE, 42 samples yielded a full profile and were therefore submitted to our standard workflow. The 15 samples that yielded no or a limited number (2–17/21) of autosomal STR markers as well four bone control samples that provided a full profile with the conventional PCR/CE test were typed in duplicate by the GlobalFiler NGS kit. Despite the degradation of the samples, which resulted in lower coverage and a lower % of on-target reads, reliable sequencing data were obtained from 16/19 samples. The use of a threshold of 30× for the locus call led to a consensus profile (cp) of 20–31/31 STR autosomal loci in 10 samples and to a cp of 8–10/31 loci in two samples, whereas the four control samples yielded a cp of 26–31/31 loci. Finally, the data of the NGS typing were combined with those of the CE typing. This last task allowed us to recover (on average) three alleles per sample and to increase the number of the heterozygous patterns in 37 cases. In total, the combined approach proposed here made possible the genetic typing of 65–100% of the autosomal STR markers in 10/15 (66.6 %) skeletal remains that yielded no or very poor results with the conventional PCR/CE approach. However, because several artefacts (such as allelic drop-out and allelic drop-in) were scored, the risk of mistyping cannot be neglected.

The HIrisPlex-S system, targeting a total of 41 SNPs, allows the simultaneous eye, hair and skin color prediction from DNA. In the present study, we developed a massive parallel sequencing (MPS) multiplex assay in order to genotype all the HIrisPlex-S markers in degraded casework samples. PCR amplicons sizes of target regions were kept below 180 bp, in order to allow analysis of degraded DNA samples. Individuals with known phenotype, artificially degraded DNA samples and a set of 2800M control DNA dilutions were sequenced on a Ion PGM System, in order to evaluate the concordance testing results and the forensic suitability of this 41-plex MPS assay. Full and reliable profiles could be obtained with 0.1 ng of input DNA. The increment of the number of PCR cycles results in improvement of sensitivity or in typing results but an increase of artifacts were also observed.

Relationships between meteorological factors and airborne fungal spore concentrations at high altitudes are virtually unknown. We used cross-correlation analyses to test the relationships between daily variation in meteorological factors (i.e., temperature, humidity and wind speed) and airborne spore concentration, diversity (Shannon and Simpson indices) and evenness (Pielou index) in an Apennine high altitude site (Gran Sasso Massif, 2117 m elevation). Daily average concentration of spores in this high altitude site was much lower than that observed at a low altitude site in the same area, which can be explained by the environmental conditions at high altitudes. We found that diversity and evenness tended to be negatively correlated with temperature and positively with humidity and wind speed with some delay, whereas abundance tended to be positively correlated with temperature and wind, but negatively with humidity. These relationships can be explained by the fact that rain increases turnover by removing dry spores of Cladosporium (the most abundant taxon) and aerosolizing wet ones. The high dominance of the most abundant taxon is a reflection of the extreme climatic conditions at high altitudes.

Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.

Auxins induce adventitious roots (ARs) in numerous culture-systems, and indole-3-butyric acid (IBA) is frequently the best AR-inducer. Vitamin requirements vary according to species, explant, and culture-conditions. Arabidopsis thaliana thin cell layers (AtTCLs) are uncapable of AR-formation on hormone-free medium containing thiamine and myo-inositol, whereas ARs are induced when IBA (10 μM), with/without kinetin (Kin, 0.1 μM), is added. The research first aim was to determine whether a synergism between IBA and myo-inositol and thiamine was necessary for AR-formation. Results showed that IBA induced AR-formation without myo-inositol and thiamine, but better when both vitamins were also present. Deciphering hormonal action on AR formation under optimal vitamin content would be essential for improving the AR process. Ethylene (ET)/jasmonic acid (JA) signaling cross-talk has been demonstrated as being involved in AR-formation in IBA + Kin-cultured AtTCLs, by using ein3eil1 and coi1-16 mutants. ETHYLENE INSENSITIVE3 (EIN3)/EIN3-LIKE1 (EIL1) are positive regulators of ethylene (ET)-signaling, whereas CORONATINE INSENSITIVE1 (COI1) is involved in JA-signaling. The ETHYLENE INSENSITIVE2 (EIN2) protein activates EIN3/EIL1 in ET-presence. To understand whether EIN2 was also involved, the AR-response of ein2-1 and coi1-16 TCLs was evaluated adding the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC, 0.1 μM) and/or the JA-donor methyl jasmonate (JAMe, 0.01 μM) to IBA + vitamins-containing medium. AR-formation was enhanced by JAMe, reduced by ACC, but unchanged by JAMe + ACC in the wild type TCLs, whereas remained similarly low in ein2-1 and coi1-16 under all treatments. Collectively, these results demonstrate that the antagonism between JA and ET in AR-formation from AtTCLs involves a cross-talk by EIN2 and COI1. The reciprocal regulation of ethylene and jasmonate perception through EIN2 and COI1 cross-talk is involved in root formation and branching in Arabidopsis stem explants, cultured with IBA, myo-inositol and thiamine.

A worldwide effort is ongoing to discover drugs against the Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2), which has so far caused >3.5 million fatalities (https://covid19.who.int/). The virus essential RNA-dependent RNA polymerase complex is targeted by several nucleoside/tide analogues whose mechanisms of action and clinical potential are currently evaluated. The guanosine analogue AT-527, a double prodrug of its 5'-triphosphate AT-9010, is currently in phase III clinical trials as a COVID19 treatment. Here we report the cryo-EM structure at 2.98 Å resolution of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 complex with RNA showing AT-9010 bound at three sites of nsp12. At the RdRp active-site, one AT-9010 is incorporated into the RNA product. Its 2'-methyl group prevents correct alignment of a second AT-9010 occupying the incoming NTP pocket. The 2'-F, 2'-methyl 3'-OH ribose scaffold explains the non-obligate RNA chain-termination potency of this NA series for both HCV NS5 and SARS-CoV RTCs. A third AT-9010 molecule 5'-diphosphate binds to a coronavirus-specific pocket in the nsp12 N-terminus NiRAN domain, a SelO pseudo-kinase structural and functional homologue. This unique binding mode impedes NiRAN-mediated UMPylation of SARS-CoV-2 nsp8 and nsp9 proteins. Our results suggest a mechanism of action for AT-527 in line with a therapeutic use for COVID19.

SummaryHow viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and −2 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5’-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 Å resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and −2, representing a promising drug for COVID-19 treatment.

Diatoms are unicellular phototrophic organisms with huge ecological impact. Characteristic for these organisms is their peculiar cell wall, which is composed of inorganic and organic components. Cell wall formation is a highly complex and orchestrated process, and in the last years has been studied intensively, also on the molecular level. Here, we review on the cell wall proteins of diatoms, with a focus on the species Thalassiosira pseudonana. We report on the expression patterns of these proteins in synchronized cultures, as well as their modifications and intracellular targeting.

According to the ISFG guidelines on the use of X-chromosome, the biostatistical evaluation in kinship analysis is based on a likelihood ratio approach, but since in calculation linkage and recombination events should be accounted for, an accurate estimate of mutation and recombination rates of X-markers analyzed is required. Due to the mode of genetic transmission and physical location, sometimes X-chromosome markers can be more informative than autosomal STRs and their analysis could be considered a supplementary tool in DNA testing. The increased demand to forensic laboratories for kinship investigations in complex cases explains the need not only to enlarge the Italian population database for 12 X-STRs routinely used for forensic application, but also to investigate the recombination fractions among markers. A collaborative exercise of the Italian Speaking Working Group Ge.F.I. was organized to evaluate mutation and recombination events in 12 X-STRs included in the Investigator Argus X12 kit. In order to explore the segregation stability, three-generation families (grandpa-mother-son) and two-generation families (mother-sons, father-daughters) for a total of 269 pedigrees were analyzed and calculations to estimate the recombination fractions between pairs of markers and mutation rates were performed. The statistical analysis showed evidence of inter- and intra-cluster recombination events, with almost free recombination for junction markers of linkage groups I and II and reduced recombination for linkage groups III and VI. We observed one- and two-step mutations, with an average value of 2.9 × 10−3.

The social parasitic beetle Paussus favieri (Coleoptera, Carabidae, Paussini) performs different types of stridulations, which selectively mimic those emitted by different ant castes of its host Pheidole pallidula (Hymenoptera, Formicidae, Myrmicinae). However, the significance of this acoustical mimicry for the success of the parasitic strategy and the behaviors elicited in the host ants by stridulations was unknown. We reared Paussus favieri in Pheidole pallidula colonies and filmed their interacting behaviors. We analyzed in slow motion the behavior of ants near a stridulating beetle. We analyzed separately trains of pulse (Pa + Pb, produced by repeated rubbings) and single pulse (Pc, produced by a single rubbing) of stridulations, clearly recognizable from the shaking up and down of the beetle hind legs, and associated them with different ant responses. The full repertoire of sounds produced by P. favieri elicited benevolent responses both in workers and soldiers. We found that different signals elicit different (sometimes multiple) behaviors in ants, with different frequency in the two ant castes. However, Pc (alone or in conjunction with other types of pulses) appears to be the type of acoustic signal mostly responsible for all recorded behaviors. These results indicate that the acoustic channel plays a pivotal role in the host-parasite interaction. Finding that a parasite uses the acoustical channel so intensively, and in such a complicated way to trigger ant behaviors, indicates that acoustic signals may be more important in ant societies than commonly recognized.

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

In clinical practice, patients&rsquo, tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin&rsquo, s solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, let-7e-5p, miR-423-3p, miR-92a-1-5p, miR-30d-5p, miR-155-5p, miR-200a-3p, and miR-429 were investigated in formalin and matched Bouin&rsquo, s solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin&rsquo, s-fixed specimens, but on average, higher Ct values and lower copies/&micro, L were found in Bouin&rsquo, s-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin&rsquo, s ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin&rsquo, s-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin&rsquo, s-fixed ones.

Highlights • Pathogens’ long distance dispersal might drive epidemics in agricultural landscapes. • We explored the relevance of long distance dispersal using network analysis. • We simulated insect-born outbreaks of Xylella fastidiosa on olives in Andalusia. • Probability of pathogen’s long distance dispersal dramatically affected outbreaks. • Limiting pathogen’s dispersal might be more effective than local control strategies., Outbreaks of a plant disease in a landscape can be meaningfully modelled using networks with nodes representing individual crop-fields, and edges representing potential infection pathways between them. Their spatial structure, which resembles that of a regular lattice, makes such networks fairly robust against epidemics. Yet, it is well-known how the addition of a few shortcuts can turn robust regular lattices into vulnerable ‘small world’ networks. Although the relevance of this phenomenon has been shown theoretically for networks with nodes corresponding to individual host plants, its real-world implications at a larger scale (i.e. in networks with nodes representing crop fields or other plantations) remain elusive. Focusing on realistic spatial networks connecting olive orchards in Andalusia (Southern Spain), the world’s leading olive producer, we show how even very small probabilities of long distance dispersal of infectious vectors result in a small-world effect that dramatically exacerbates a hypothetical outbreak of a disease targeting olive trees (loosely modelled on known epidemiological information on the bacterium Xylella fastidiosa, an important emerging threat for European agriculture). More specifically, we found that the probability of long distance vector dispersal has a disproportionately larger effect on epidemic dynamics compared to pathogen’s intrinsic infectivity, increasing total infected area by up to one order of magnitude (in the absence of quarantine). Furthermore, even a very small probability of long distance dispersal increased the effort needed to halt a hypothetical outbreak through quarantine by about 50% in respect to scenarios modelling local/short distance pathogen’s dispersal only. This highlights how identifying (and disrupting) long distance dispersal processes may be more efficacious to contain a plant disease epidemic than surveillance and intervention concentrated on local scale transmission processes.

Simple Summary Predator-prey relationships and competition shape interspecific coexistence in wildlife communities. So far, most published studies have focused on large carnivores and their prey, whereas little is known about medium and small-sized mammal communities. The European brown hare Lepus europaeus is a widespread species in Europe and is part of the diet of many birds of prey and mammalian carnivores of all sizes. Furthermore, competition with other herbivorous mammals at feeding sites has also been suggested. In an area in Central Italy, we have assessed spatiotemporal overlap among brown hare and its potential predators (red fox Vulpes vulpes, pine marten Martes martes, domestic cat Felis catus, and domestic dog Canis familiaris) and a competitor (roe deer Capreolus capreolus). We showed that, outside a fenced area excluding predators and competitors, brown hares become more nocturnal and more active on dark nights to limit encounters with predators, and that they adopt spatial partitioning to avoid competitors, as expected by ecological theory. Abstract Analysis of spatiotemporal partitioning is pivotal to shed light on interspecific coexistence. Most research effort has involved large-sized carnivores and their prey, whereas little attention has been devoted to lagomorphs. We assessed spatiotemporal overlap among the European brown hare Lepus europaeus and its potential competitors and predators through camera-trapping in an area in Central Italy. We estimated the interspecific patterns of the spatiotemporal activity rhythms of brown hares, its potential predators (the red fox Vulpes vulpes, the pine marten Martes martes, the domestic cat Felis catus, and the domestic dog Canis familiaris), and a competitor, the roe deer Capreolus capreolus. Brown hare activity was studied in natural conditions as well as in a fenced area that excluded terrestrial predators and competitors. Free-ranging hares developed a more nocturnal behavior to avoid diurnal predators (i.e., domestic carnivores and martens). Although high temporal overlap was observed between free-ranging brown hares and both red foxes (82%) and roe deer (81%), hares avoided fox by being more active on darkest nights, as well as avoided roe deer through spatial partitioning. We suggest that hares may adapt their spatiotemporal behavior to avoid potential predators and competitors.

Developmental and environmental signaling networks often converge during plant growth in response to changing conditions. Stress-induced hormones, such as jasmonates (JAs), can influence growth by crosstalk with other signals like brassinosteroids (BRs) and ethylene (ET). Nevertheless, it is unclear how avoidance of an abiotic stress triggers local changes in development as a response. It is known that stress hormones like JAs/ET and BRs can regulate the division rate of cells from the first asymmetric cell divisions (ACDs) in meristems, suggesting that stem cell activation may take part in developmental changes as a stress-avoidance-induced response. The root system is a prime responder to stress conditions in soil. Together with the primary root and lateral roots (LRs), adventitious roots (ARs) are necessary for survival in numerous plant species. AR and LR formation is affected by soil pollution, causing substantial root architecture changes by either depressing or enhancing rooting as a stress avoidance/survival response. Here, a detailed overview of the crosstalk between JAs, ET, BRs, and the stress mediator nitric oxide (NO) in auxin-induced AR and LR formation, with/without cadmium and arsenic, is presented. Interactions essential in achieving a balance between growth and adaptation to Cd and As soil pollution to ensure survival are reviewed here in the model species Arabidopsis and rice.

Simple Summary Mountains are ideal natural laboratories to study how biodiversity is influenced by environmental characteristics because climate varies rapidly from lowlands to high elevations. Scientists have investigated how the number of species varies with elevation for the most disparate plant and animal groups worldwide. However, species richness is only one aspect of biodiversity that does not consider species abundances. The so-called Hill numbers are a unified family of mathematical indices that express biodiversity in terms of both richness and abundance. We used Hill numbers to investigate how dung beetle diversity varies along an elevational gradient in a Mediterranean mountain. We found that scarabaeids were the most abundant dung beetle group. These insects construct subterranean nests protecting their offspring from desiccation during warm and dry summer climatic conditions. Additionally, in accordance with their preference for open habitats, we found that dung beetles are more abundant and diversified in grasslands than in woodlands. In the woodlands, diversity increased with elevation because of tree thinning, whereas in the grasslands, diversity decreased because of increasingly harsher environmental conditions. This indicates a trade-off in the beetle response to elevation between the positive effects of increasing the availability of suitable habitats and the worsening of environmental conditions. Abstract Most studies of biodiversity–elevational patterns do not take species abundance into consideration. Hill numbers are a unified family of indices that use species abundance and allow a complete characterization of species assemblages through diversity profiles. Studies on dung beetle responses to elevation were essentially based on species richness and produced inconsistent results because of the non-distinction between different habitats and the use of gradients dispersed over wide areas. We analyzed dung beetle diversity in a Mediterranean mountain (central Italy) for different habitats (woodlands vs. grasslands) and taxonomic groups (scarabaeids and aphodiids). Scarabaeids were the most abundant. Since scarabaeids are able to construct subterranean nests, this indicates that the warm and dry summer climatic conditions of high elevations favor species capable of protecting their larvae from desiccation. Dung beetles were more abundant and diversified in grasslands than in woodlands, which is consistent with their preference for open habitats. In the woodlands, diversity increased with increasing elevation because of increasing tree thinning, whereas, in the grasslands, diversity decreased with elevation because of increasingly harsher environmental conditions. These results indicate a trade-off in the beetle response to elevation between the positive effects of increasing the availability of more suitable habitats and the decrease of optimal environmental conditions.

Genetic structure may be highly variable across seabird species, and particularly among those that are distributed over large geographical areas. The Adélie penguin (Pygoscelis adeliae) is a numerically dominant Antarctic seabird that is considered to be a key species in coastal ecosystems. Since the Last Glacial Maximum, penguin colonization of the Antarctic coastline occurred at varying geographical and temporal scales, contributing to an incomplete understanding of how modern colonies relate to each other at local or regional scales. We assessed the population genetic structure of Adélie penguins (n = 86 individuals) from three adjacent colonies along the Victoria Land coast using molecular genetic markers (i.e. seven microsatellite loci isolated through next-generation sequencing). Our results indicate meta-population dynamics and possibly relationships with habitat quality. A generally low genetic diversity (Nei's index: 0.322–0.667) was observed within each colony, in contrast to significant genetic heterogeneity among colonies (pairwise FST = 0.071–0.148), indicating that populations were genetically structured. Accordingly, an assignment test correctly placed individuals within the respective colonies from which they were sampled. The presence of inter-colony genetic differentiation contrasts with previous studies on this species that showed a lack of genetic structure, possibly due to higher juvenile or adult dispersal. Our sampled colonies were not panmictic and suggest a lower migration rate, which may reflect relatively stable environmental conditions in the Ross Sea compared to other regions of Antarctica, where the ocean climate is warming.

Elevational gradients offer special opportunities to investigate the relative role of intraspecific and interspecific trait variations in relation to stress gradients. We used an altitudinal gradient in the Mediterranean (Mt Velino, Central Italy) to study (1) how community-weighted means (CWM) and nonweighted means (CM) vary with elevation for plant height, specific leaf area, and seed mass, and (2) how variation patterns differ for inter- and intraspecific functional variability. We tested (1) if elevation influences community functional composition on the basis of the adaptive value of plant traits and (2) if the latter shows intraspecific variations according to the species’ ability to cope with local conditions. We found that different traits showed different patterns, which can be linked to the function they express. Differences between communities were influenced more by differences between their traits (CM) than by the relative species coverage (CWM). Both highest and lowest elevations were the most selective due to their particularly severe climatic conditions. Intermediate elevations were the most favorable thanks to less constraining climatic conditions. Interspecific trait variability was the most relevant component, indicating a low plant ability to cope with environmental variations through phenotypic plasticity.

Sexing weakly/criptically dimorphic birds requires invasive techniques or molecular analysis. Alternatively, sexing can be based upon morphometric analysis, which remains invaluable in remote field conditions such as Polar regions. Nevertheless, discriminatory power may be affected by methodological issues hampering comparison between/within studies, e.g. considering alternative analytical techniques or measurements taken by different researchers. We investigated the sexing potential of bill length, bill depth and body mass in adult Adélie penguins Pygoscelis adeliae of known sex. We performed discriminant analysis on a large dataset of measurements (237 males; 264 females) taken from penguins marked between 1994 and 2001, at Edmonson Point (Ross Sea, Antarctica). In a second step, we accounted for measurements collected by different researchers through permuted discriminant analysis. We found moderate support for sex discrimination through bill measurements (77%). Considering body mass did not improve classification power substantially (78.2%), possibly because of confounding inter/intra-annual oscillations in body condition. Discriminant rate decreased when controlling the researcher effect (72.7% and 71.4%, respectively). Results were also confirmed by logistic (mixed) models. Simulations showed that reduction in sample size markedly increased uncertainty in classification power. Differences with previous studies achieving a slightly greater classification power for sexing Adélie penguins through morphometrics could be related to (i) our higher sample size and/or (ii) geographical, population-specific differences, e.g. lower degree of sexual dimorphism. Our work emphasises key-factors influencing performance of morphometric sexing in avian species, advocating population-specific validation with large sample size as well as the necessity to account for the researcher effect prior to apply morphometric sexing.

Previous studies on Escherichia coli demonstrated that sub-minimum inhibitory concentration (MIC) of fluoroquinolones induced the SOS response, increasing drug tolerance. We characterized the transcriptional response to moxifloxacin in Mycobacterium tuberculosis. Reference strain H37Rv was treated with moxifloxacin and gene expression studied by qRT-PCR. Five SOS regulon genes, recA, lexA, dnaE2, Rv3074 and Rv3776, were induced in a dose- and time-dependent manner. A range of moxifloxacin concentrations induced recA, with a peak observed at 2 &times, MIC (0.25 &mu, g/mL) after 16 h. Another seven SOS responses and three DNA repair genes were significantly induced by moxifloxacin. Induction of recA by moxifloxacin was higher in log-phase than in early- and stationary-phase cells, and absent in dormant bacilli. Furthermore, in an H37Rv fluoroquinolone-resistant mutant carrying the D94G mutation in the gyrA gene, the SOS response was induced at drug concentrations higher than the mutant MIC value. The 2 &times, MIC of moxifloxacin determined no significant changes in gene expression in a panel of 32 genes, except for up-regulation of the relK toxin and of Rv3290c and Rv2517c, two persistence-related genes. Overall, our data show that activation of the SOS response by moxifloxacin, a likely link to increased mutation rate and persister formation, is time, dose, physiological state and, possibly, MIC dependent.

In wild ungulates, habitat choice usually is influenced by foraging constraints and predator avoidance, potentially leading to spatial variation of population density (e.g., greater densities in food rich or safer habitats). Assessing habitat-correlates of abundance and foraging activity in turn is important in understanding determinants of distribution. We assessed habitat correlates of presence, density, and rooting, for wild boar Sus scrofa, the most widespread ungulate in the world, in six protected areas of central Italy. We worked along an altitudinal gradient ranging from the coast to mountains, in late spring-summer 2019. We surveyed 617 sampling plots randomly placed onto study areas with tessellation stratified sampling, where we used fecal counts to estimate wild boar density and visually estimated the proportion of rooted area. Overall estimates of density and rooting (± standard error) varied from 3.5 ± 2.0 to 17.9 ± 5.4 individuals/km2 and from 1.4 ± 0.8% to 10.9 ± 1.1% of rooted ground. Density and rooting showed a moderate yet nonsignificant correlation across sites. Probability of presence, abundance, and rooting in sampling plots were higher in ecotone habitats (transition habitats between wooded and open areas). Topography did not influence boar presence or local abundance. Rooting increased with decreasing slope and rock cover, as well as increasing elevation, possibly due to soil supporting forage of higher nutritional quality. Our results support the importance of ecotone habitats for wild boar, emphasizing the role of these transitional areas in the period of nursing/weaning of offspring, as well as when crops are actively growing. Differences in overall estimates of rooting across study sites may depend on site-specific features (soil moisture and availability of alternative food resources). Future studies should test the correlation between inter-annual differences of rooting and changes in population density. Notwithstanding the latter, we identified significant ecological drivers of wild boar density and rooting activity.

Body size and body mass are key indicators of individual phenotypic quality and predictors of important life-history traits such as survival and reproductive success. In wild herbivores, individual responses to changing environmental conditions influence morphometric traits over temporal scales and between populations. However, little research has assessed joint effects of weather and land use on body size/mass at finer, intra-population scale. We used data collected on female and juvenile red deer Cervus elaphus shot over a 17-year period (2001–2017) along two sides of a mountainous ridge largely differing in land cover and habitat productivity, to investigate how fine-scale land use patterns and weather influenced multiple morphometric indicators of phenotypic quality. Accounting for weather, body mass of all sex/age classes increased with increasing proportion of cultivated areas in the landscape and, for young females and calves, that increase was stronger or occurred only in the “low-quality” site. Other biometric traits such as mandible length and hind foot length showed the same pattern in young and calves, suggesting that body mass/size reflects individual responses especially in the early life-stage. Accounting for land use, body mass of adult females and calves was enhanced by increasing rainfall and decreasing temperature in spring-summer, i.e. favourable conditions for vegetation growth. This result also supports late gestation- and lactation-mediated effects of vegetation productivity on offspring quality. Additionally, in male calves, body mass and several other traits increased with decreasing severity of the previous winter, suggesting that quality of male offspring - but not that of females - could depend on winter conditions experienced in utero, likely due to higher maternal costs. Our findings emphasise how land cover and weather jointly affect indicators of phenotypic quality in a large mammal, helping to predict size responses of herbivores under the ongoing climatic- and anthropogenic land use-changes.

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

Targeted Protein Silencing (TPS) is an elegant approach to investigate protein function and its role in the cellular landscape, overcoming limitations of genetic perturbation strategies. In contrast to CRISPR/Cas9 and RNA interference, these systems act in a reversible manner and reduce off-target effects. Several TPS have been developed and wisely improved, including compartment delocalization tools and protein degradation systems. In this review, we focus on Anchor-Away, deGradFP, auxin inducible degron (AID) and dTAG technologies, and discuss their recent applications and advances. Finally, we propose Nano-Grad, a novel nanobody-based protein degradation tool to specifically proteolyze endogenous tag-free target protein.

Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track Fusarium species even in the early stages of infection, can contribute to mycotoxins’ risk control. Among DNA-based technologies for Fusarium detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digital PCR (dPCR), a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. In our work, a panel of chip digital PCR assays was developed to quantify Fusarium graminearum, F.culmorum, F. sporotrichioides, F. poae and F. avenaceum. The primers/probes combinations were evaluated on pure fungal samples with cdPCR technique, in comparison with the qPCR approach. Moreover, the cdPCR assays were applied to quantify Fusarium in durum wheat and oat samples, naturally contaminated or spiked with fungal DNA. For a better evaluation of infection level in plants, duplex assays were developed, able to co-amplify both plant and fungal DNA. To the best of our knowledge, this is the first study directed to the application of digital PCR to Fusarium diagnosis in plants.

Graphical abstract Principle of SARS-CoV RNA synthesis detection by a fluorescence-based High Throughput Screening assay. During the enzymatic step, the RNA template was incubated with the SARS-CoV RTC, the nucleotides triphosphate and the compound to be tested (putative inhibitor). The polymerase activity released a double stranded RNA which was detected by adding a fluorescent intercalant agent (Picogreen®, ex: 480 nM, em: 530 nm). In presence of an inhibitor, the fluorescence signal decreased and is quantified. NTP: Nucleotides Tri-Phosphate., Highlights • A new non-radioactive SARS-CoV RNA polymerase assay was developed. • The robotized assay is suitable to identify RdRp inhibitors based on HTS. • A total of 1520 FDA-approved compounds were tested using the SARS-CoV-RTC assay., The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96 % aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug discovery against the SARS-CoV-2.

Pollination is the transfer of pollen grains from the stamens to the stigma, an essential requirement of sexual reproduction in flowering plants. Cross-pollination increases genetic diversity and is favored by selection in the majority of situations. Flowering plants have evolved a wide variety of traits that influence pollination success, including those involved in optimization of self-pollination, attraction of animal pollinators, and the effective use of wind pollination. In this review we discuss our current understanding of the molecular basis of the development and production of these various traits. We conclude that recent integration of molecular developmental studies with population genetic approaches is improving our understanding of how selection acts on key floral traits in taxonomically diverse species, and that further work in nonmodel systems promises to provide exciting insights in the years to come.

Although some studies have showed the effects of different crystalline structures of nTiO2 (anatase and rutile) and their applicability in several fields, few studies has analyzed the effect of coexposure with other environmental contaminants such as copper. Thus, the objective of this study was to evaluate if the coexposure to nTiO2 (nominal concentration of 1 mg/L; anatase or rutile) can increase the incorporation and toxic effect induced by Cu (nominal concentration of 56 μg/L) in different tissues of Linmoperna fortunei after 120 h of exposure. Our results showed that the coexposure increased the accumulation of Cu in the gills and adductor muscle independently of the crystalline form and can positively or negatively modulate the antioxidant system, depending on the tissue analyzed. However, exposure only to rutile nTiO2 induced damage in the adductor muscle evidenced by the infiltration of hemocytes in this tissue. Additionally, histomorphometric changes based on fractal dimension analysis showed that coexposure to both forms of nTiO2 induced damage in the same tissue. These results suggest that both crystalline forms exhibited toxicity depending on the analyzed tissue and that coexposure of nTiO2 with Cu may be harmful in L. fortunei, indicating that increased attention to the use and release of nTiO2 in the environment is needed to avoid deleterious effects in aquatic biota.

A broad diversity of sex-determining systems has evolved in eukaryotes. However, information on the mechanisms of sex determination for unicellular microalgae is limited, including for diatoms, key-players of ocean food webs. Here we report the identification of a mating type (MT) determining gene for the diatom Pseudo-nitzschia multistriata. By comparing the expression profile of the two MTs, we find five MT-biased genes, of which one, MRP3, is expressed exclusively in MT+ strains in a monoallelic manner. A short tandem repeat of specific length in the region upstream of MRP3 is consistently present in MT+ and absent in MT− strains. MRP3 overexpression in an MT− strain induces sex reversal: the transgenic MT− can mate with another MT− strain and displays altered regulation of the other MT-biased genes, indicating that they lie downstream. Our data show that a relatively simple genetic program is involved in defining the MT in P. multistriata., Little is known about how sex is determined in diatoms. Here, Russo et al. show that in the diatom Pseudo-nitzschia multistriata, the gene MRP3 regulates mating type and the expression of four other mating-type biased genes.

Background Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species. Electronic supplementary material The online version of this article (10.1186/s12870-018-1392-4) contains supplementary material, which is available to authorized users.