Basin scale variability of Ostreopsis spp. blooms provides evidence of effectiveness of an integrated sampling approach.

Ostreopsis spp. blooms have been occurring in the last two decades in the Mediterranean Sea in association with a variety of biotic and abiotic substrata (macroalgae, seagrasses, benthic invertebrates, sand, pebbles and rocks). Cells proliferate attached to the surfaces through mucilaginous trichocysts, which lump together microalgal cells, and can also be found in the plankton and on floating aggregates: such tychoplanktonic behavior makes the quantitative assessment of blooms more difficult than planktonic or benthic ones. Different techniques have been so far applied for quantifying cell abundances of benthic microalgae for research, monitoring and risk assessment purposes. In this context, the Benthic Dinoflagellates Integrator (BEDI), a non-destructive quantification method for benthic dinoflagellate abundances, was developed and tested within the EU ENPI-CBCMED project M3-HABs. This device allows mechanical detachment of cells without collecting the benthic substrate, providing an integrated assessment of both epiphytic and planktonic cells, i.e. of the number of cells potentially made available in the water volume from "resuspension" which could have harmful effects on other organisms (including humans). The present study confirms the effectiveness of the BEDI sampling device across different environments across the Mediterranean Sea and constitutes the first large-scale study of Ostreopsis spp. blooms magnitude in function of different macro- and meso­habitat features across the basin.

Tropical bloom-forming mesoalgae Cladophoropsis sp. and Laurencia sp.-responses to ammonium enrichment and a simulated heatwave.

Algal blooms are increasing worldwide, driven by elevated nutrient inputs. However, it is still unknown how tropical benthic algae will respond to heatwaves, which are expected to be more frequent under global warming. In the present study, a multifactorial experiment was carried out to investigate the potential synergistic effects of increased ammonium inputs (25 µM, control at 2.5 µM) and a heatwave (31°C, control at 25°C) on the growth and physiology (e.g., ammonium uptake, nutrient assimilation, photosynthetic performance, and pigment concentrations) of two bloom-forming algal species, Cladophoropsis sp. and Laurencia sp. Both algae positively responded to elevated ammonium concentrations with higher growth and chlorophyll a and lutein concentrations. Increased temperature was generally a less important driver, interacting with elevated ammonium by decreasing the algaes' %N content and N:P ratios. Interestingly, this stress response was not captured by the photosynthetic yield (Fv/Fm) nor by the carbon assimilation (%C), which increased for both algae at higher temperatures. The negative effects of higher temperature were, however, buffered by nutrient inputs, showing an antagonistic response in the combined treatment for the concentration of VAZ (violaxanthin, antheraxanthin, zeaxanthin) and thalli growth. Ammonium uptake was initially higher for Cladophoropsis sp. and increased for Laurencia sp. over experimental time, showing an acclimation capacity even in a short time interval. This experiment shows that both algae benefited from increased ammonium pulses and were able to overcome the otherwise detrimental stress of increasingly emerging temperature anomalies, which provide them a strong competitive advantage and might support their further expansions in tropical marine systems.

Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes.

Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.

Towards the outwelling hypothesis in a Patagonian estuary: First support from lipid markers and bacterial communities.

Biogeochemical markers in combination with bacterial community composition were studied at two contrasting stations at the Río Negro (RN) estuary to assess the outwelling hypothesis in the Argentinian Patagonia. Inorganic nutrients and dissolved organic matter were exported clearly during the last hours of the ebb at the station Wetland. Moreover, a considerable outwelling of polyunsaturated fatty acids (PUFA), particulates and microalgae was inferred by this combined approach. The exported 22:6(n-3) and 20:5(n-3) contributed very likely to sustain higher trophic levels in the coasts of the Southwest Atlantic. The stable isotopes did not evidence clearly the outwelling; nevertheless, the combination of δ13C with fatty acid bacterial markers indicated organic matter degradation in the sediments. The dominance of Desulfobacterales and Desulfuromonadales suggested sulphate reduction in the sediments, a key mechanism for nutrient outwelling in salt marshes. Marivivens and other Rhodobacterales (Alphaproteobacteria) in the suspended particulate matter were clear indicators of the nutrient outwelling. The colonization of particles according to the island biogeography theory was a good hypothesis to explain the lower bacterial biodiversity at the wetland. The copiotrophic conditions of the RN estuary and particularly at the wetland were deduced also by the dynamic of some Actinobacteria, Bacteroidia and Gammaproteobacteria. This high-resolution snapshot combining isotopic, lipid and bacterial markers offers key pioneer insights into biogeochemical and ecological processes of the RN estuary.

Competition between electrostatic interactions and halogen bonding in the protein-ligand system: structural and thermodynamic studies of 5,6-dibromobenzotriazole-hCK2α complexes.

CK2 is a member of the CMGC group of eukaryotic protein kinases and a cancer drug target. It can be efficiently inhibited by halogenated benzotriazoles and benzimidazoles. Depending on the scaffold, substitution pattern, and pH, these compounds are either neutral or anionic. Their binding poses are dictated by a hydrophobic effect (desolvation) and a tug of war between a salt bridge/hydrogen bond (to K68) and halogen bonding (to E114 and V116 backbone oxygens). Here, we test the idea that binding poses might be controllable by pH for ligands with near-neutral pKa, using the conditionally anionic 5,6-DBBt and constitutively anionic TBBt as our models. We characterize the binding by low-volume Differential Scanning Fluorimetry (nanoDSF), Isothermal Calorimetry (ITC), Hydrogen/Deuterium eXchange (HDX), and X-ray crystallography (MX). The data indicate that the ligand pose away from the hinge dominates for the entire tested pH range (5.5-8.5). The insensitivity of the binding mode to pH is attributed to the perturbation of ligand pKa upon binding that keeps it anionic in the ligand binding pocket at all tested pH values. However, a minor population of the ligand, detectable only by HDX, shifts towards the hinge in acidic conditions. Our findings demonstrate that electrostatic (ionic) interactions predominate over halogen bonding.

Effect of Narrowband UV-B Irradiation on the Growth Performance of House Crickets.

Indoor co-cultivation systems can answer to the need for sustainable and resilient food production systems. Rearing organisms under light-emitting diodes (LEDs) irradiation provides the possibility to control and shape the emitted light spectra. UV-B-irradiation (280-315 nm) can positively affect the nutritional composition of different plants and other organisms, whereas information on edible insects is scarce. To evaluate the potential effect of the photosynthetically active radiation (PAR) and LED-emitting LEDs on the rearing and nutritional quality of edible insects, house crickets (Acheta domesticus) were reared from the age of 21 days under controlled LED spectra, with an additional UV-B (0.08 W/m2) dose of 1.15 KJm2 d-1 (illuminated over a period for 4 h per day) for 34 days. UV-B exposure showed no harm to the weight of the crickets and significantly increased their survival by ca. 10% under narrowband UV-B treatment. The nutritional composition including proteins, fat and chitin contents of the insects was not affected by the UV-B light and reached values of 60.03 ± 10.41, 22.38 ± 2.12 and 9.33 ± 1.21%, respectively, under the LED irradiation. Therefore, house crickets can grow under LED irradiation with a positive effect of narrowband UV-B application on their survival.

Insights into the bacterial community composition of farmed Caulerpa lentillifera: A comparison between contrasting health states.

The bacterial communities of Caulerpa lentillifera were studied during an outbreak of an unknown disease in a sea grape farm from Vietnam. Clear differences between healthy and diseased cases were observed at the order, genus, and Operational Taxonomic Unit (OTU) level. A richer diversity was detected in the diseased thalli of C. lentillifera, as well as the dominance of the orders Flavobacteriales (phylum Bacteroidetes) and Phycisphaerales (Planctomycetes). Aquibacter, Winogradskyella, and other OTUs of the family Flavobacteriaceae were hypothesized as detrimental bacteria, this family comprises some well-known seaweed pathogens. Phycisphaera together with other Planctomycetes and Woeseia were probably saprophytes of C. lentillifera. The Rhodobacteraceae and Rhodovulum dominated the bacterial community composition of healthy C. lentillifera. The likely beneficial role of Bradyrhizobium, Paracoccus, and Brevundimonas strains on nutrient cycling and phytohormone production was discussed. The bleaching of diseased C. lentillifera might not only be associated with pathogens but also with an oxidative response. This study offers pioneering insights on the co-occurrence of C. lentillifera-attached bacteria, potential detrimental or beneficial microbes, and a baseline for understanding the C. lentillifera holobiont. Further applied and basic research is urgently needed on C. lentillifera microbiome, shotgun metagenomic, metatranscriptomic, and metabolomic studies as well as bioactivity assays are recommended.

Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.

The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.

Benthic Dinoflagellate Integrator (BEDI): A new method for the quantification of Benthic Harmful Algal Blooms.

Despite the potential negative human health, ecological and economic impact, the ecology of harmful benthic dinoflagellate blooms remains largely unknown. This is probably due to the complex interactions among biotic and abiotic drivers that influence blooms, but also to the difficulty in quantifying cell abundance in a comparable way over large spatial and temporal scales. One of the recognized priorities for bHABs (benthic Harmful Algal Blooms) assessment is developing and standardizing methods that can provide comparable data. In this context, the Benthic Dinoflagellates Integrator (BEDI), a new non-destructive quantification method for benthic dinoflagellate abundances, has been developed and tested within the present study. The rationale behind the BEDI standard assessment method is that mechanical resuspension of cells enables the quantification of abundances as cells per unit of seabed surface area (i.e. cellsmm-2) or as Potentially Resuspended cells per unit of volume (PRcellsml-1), by integrating both cells in the biofilm and those in the surrounding water. Estimations of Ostreopsis performed with BEDI method are independent of the substratum (i.e. macroalgal species) or the dominant ecosystem (i.e. algal forests or turfs, seagrass beds, coral reefs) and potentially allow the comparison of benthic dinoflagellate blooms over broad temporal and spatial scales. The first application of the BEDI method, presented in this study, gave encouraging results: the characterization of blooms of Ostreopsis cf. ovata at three sites in the NW Mediterranean Sea is consistent with results derived from the other commonly applied methods. Quantification of the ratio between abundances of cells in the biofilm and in the surrounding water was calculated for the first time per unit of seabed surface area, demonstrating that the highest abundances of cells (the stock), and therefore the associated risk for human health, are in the biofilm. For risk assessment purposes, conversion values for commonly used monitoring alert thresholds of Mediterranean Ostreopsis blooms are provided.

Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics.

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.

Biogeochemical and hydrological drivers of the dynamics of Vibrio species in two Patagonian estuaries.

The ecology of the most relevant Vibrio species for human health and their relation to water quality and biogeochemistry were studied in two estuaries in Argentinian Patagonia. Vibrio cholerae and Vibrio parahaemolyticus were reported in >29% of cases at the Río Colorado and Río Negro estuaries. Neither the pandemic serogroups of Vibrio cholerae O1, Vibrio cholerae O139 nor the cholera toxin gene were detected in this study. However, several strains of V. cholerae (not O1 or O139) are able to cause human disease or acquire pathogenic genes by horizontal transfer. Vibrio vulnificus was detected only in three instances in the microplankton fraction of the Río Negro estuary. The higher salinity in the Río Colorado estuary and in marine stations at both estuaries favours an abundance of culturable Vibrio. The extreme peaks for ammonium, heterotrophic bacteria and faecal coliforms in the Río Negro estuary supported a marked impact on sewage discharge. Generally, the more pathogenic strains of Vibrio have a faecal origin. Salinity, pH, ammonium, chlorophyll a, silicate and carbon/nitrogen ratio of suspended organic particulates were the primary factors explaining the distribution of culturable bacteria after distance-based linear models. Several effects of dissolved organic carbon on bacterial distribution are inferred. Global change is expected to increase the trophic state and the salinisation of Patagonian estuaries. Consequently, the distribution and abundance of Vibrio species is projected to increase under future changing baselines. Adaptation strategies should contribute to sustaining good water quality to buffer climate- and anthropogenic- driven impacts.

Sampling of Ostreopsis cf. ovata using artificial substrates: Optimization of methods for the monitoring of benthic harmful algal blooms.

In the framework of monitoring of benthic harmful algal blooms (BHABs), the most commonly reported sampling strategy is based on the collection of macrophytes. However, this methodology has some inherent problems. A potential alternative method uses artificial substrates that collect resuspended benthic cells. The current study defines main improvements in this technique, through the use of fiberglass screens during a bloom of Ostreopsis cf. ovata. A novel set-up for the deployment of artificial substrates in the field was tested, using an easy clip-in system that helped restrain substrates perpendicular to the water flow. An experiment was run in order to compare the cell collection efficiency of different mesh sizes of fiberglass screens and results suggested an optimal porosity of 1-3mm. The present study goes further on showing artificial substrates, such as fiberglass screens, as efficient tools for the monitoring and mitigation of BHABs.

Global change effects on biogeochemical processes of Argentinian estuaries: an overview of vulnerabilities and ecohydrological adaptive outlooks.

The aims of this work are to provide an overview of the current stresses of estuaries in Argentina and to propose adaptation strategies from an ecohydrological approach. Several Argentinian estuaries are impacted by pollutants, derived mainly from sewage discharge and agricultural or industrial activities. Anthropogenic impacts are expected to rise with increasing human population. Climate-driven warmer temperature and hydrological changes will alter stratification, residence time, oxygen content, salinity, pollutant distribution, organism physiology and ecology, and nutrient dynamics. Good water quality is essential in enhancing estuarine ecological resilience to disturbances brought on by global change. The preservation, restoration, and creation of wetlands will help to protect the coast from erosion, increase sediment accretion rates, and improve water quality by removing excess nutrients and pollutants. The capacity of hydrologic basin ecosystems to absorb human and natural impacts can be improved through holistic management, which should consider social vulnerability in complex human-natural systems.