Dephosphorylation of the pre-initiation complex is critical for origin firing.

In eukaryotes, cyclin-dependent kinase (CDK) ensures that the genome is duplicated exactly once by inhibiting helicase loading factors before activating origin firing. CDK activates origin firing by phosphorylating two substrates, Sld2 and Sld3, forming a transient and limiting intermediate-the pre-initiation complex (pre-IC). Here, we show in the budding yeast Saccharomyces cerevisiae that the CDK phosphorylations of Sld3 and Sld2 are rapidly turned over during S phase by the PP2A and PP4 phosphatases. PP2ARts1 targets Sld3 specifically through an Rts1-interaction motif, and this targeted dephosphorylation is important for origin firing genome-wide, for formation of the pre-IC at origins and for ensuring that Sld3 is dephosphorylated in G1 phase. PP2ARts1 promotes replication in vitro, and we show that targeted Sld3 dephosphorylation is critical for viability. Together, these studies demonstrate that phosphatases enforce the correct ordering of replication factor phosphorylation and in addition to kinases are also key drivers of replication initiation.

Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

Are Fish Populations at Risk? Metformin Disrupts Zebrafish Development and Reproductive Processes at Chronic Environmentally Relevant Concentrations.

The antidiabetic drug Metformin (MET), one of the most prevalent pharmaceuticals in the environment, is currently detected in surface waters in the range of ng/L to low µg/L. As current knowledge regarding the long-term effects of environmentally relevant concentrations of MET in nontarget organisms is limited, the present study aimed at investigating the generational effects of MET, in concentrations ranging from 390 to 14 423 ng/L in the model organism Danio rerio (up to 9 mpf), including the effects on its nonexposed offspring (until 60 dpf). We integrate several apical end points, i.e., embryonic development, survival, growth, and reproduction, with qRT-PCR and RNA-seq analyses to provide additional insights into the mode of action of MET. Reproductive-related parameters in the first generation were particularly sensitive to MET. MET parental exposure impacted critical molecular processes involved in the metabolism of zebrafish males, which in turn affected steroid hormone biosynthesis and upregulated male vtg1 expression by 99.78- to 155.47-fold at 390 and 14 432 MET treatment, respectively, pointing to an estrogenic effect. These findings can potentially explain the significant decrease in the fertilization rate and the increase of unactivated eggs. Nonexposed offspring was also affected by parental MET exposure, impacting its survival and growth. Altogether, these results suggest that MET, at environmentally relevant concentrations, severely affects several biological processes in zebrafish, supporting the urgent need to revise the proposed Predicted No-Effect Concentration (PNEC) and the Environmental Quality Standard (EQS) for MET.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

γδ-T cells promote IFN-γ-dependent Plasmodium pathogenesis upon liver-stage infection.

Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αß-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αß-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection.

Novel Organic Salts Based on Mefloquine: Synthesis, Solubility, Permeability, and In Vitro Activity against Mycobacterium tuberculosis.

The development of novel pharmaceutical tools to efficiently tackle tuberculosis is the order of the day due to the rapid development of resistant strains of Mycobacterium tuberculosis. Herein, we report novel potential formulations of a repurposed drug, the antimalarial mefloquine (MFL), which was combined with organic anions as chemical adjuvants. Eight mefloquine organic salts were obtained by ion metathesis reaction between mefloquine hydrochloride ([MFLH][Cl]) and several organic acid sodium salts in high yields. One of the salts, mefloquine mesylate ([MFLH][MsO]), presented increased water solubility in comparison with [MFLH][Cl]. Moreover, all salts with the exception of mefloquine docusate ([MFLH][AOT]) showed improved permeability and diffusion through synthetic membranes. Finally, in vitro activity studies against Mycobacterium tuberculosis revealed that these ionic formulations exhibited up to 1.5-times lower MIC values when compared with [MFLH][Cl], particularly mefloquine camphorsulfonates ([MFLH][(1R)-CSA], [MFLH][(1S)-CSA]) and mefloquine HEPES ([MFLH][HEPES]).

Assessment of Water Quality Parameters and their Seasonal Behaviour in a Portuguese Water Supply System: a 6-year Monitoring Study.

Water quality monitoring is a fundamental tool in the management of freshwater resources. The purpose of monitoring is to provide meaningful quality data for local action planning and catchment-wide decision making. The assessment of water quality is crucial to guarantee the efficient operation of the Water Treatment Plants (WTPs), promoting health conditions and contributing for a more sustainable urban water cycle. In accordance, the objective of this study was to evaluate key target chemical and microbiological water quality parameters, some of them already monitored within Portuguese/EU legal framework and others still not regulated, but with environmental and human heath relevance. A local monitoring database model, using a 6-year period (from 2014 to 2019) of water quality data, regarding water samples collected on representative sampling locations covering the freshwater abstraction sites, conventional WTPs and distribution network was assessed. This work provides new knowledge regarding occurrence and seasonal behaviour for both microbiological and chemical water quality parameters, essential to understand/manage the water supply system. Additionally, relationships between the target variables were also assessed. Particularly, strong correlations were identified between TOC and THMs formation at distribution network (r = 0.69; p ≤ 0.001); nitrates were the water quality parameter that revealed the best correlation between surface water source and treated water (r = 0.81; p ≤ 0.001), suggesting that treatment yield/performance is dependent on surface water load. The local and continuous monitoring of water systems are crucial to implement new approaches to guarantee the best quality of drinking water throughout the supply system.

Functional, biochemical and molecular impact of sediment plumes from deep-sea mining on Mytilus galloprovincialis under hyperbaric conditions.

The deep-sea is the biggest ecosystem in the world and despite the extreme conditions that characterize it, is highly biodiverse and complex. Deep-sea mining has been foreseen as a potential and concerning new stressor, and among the deep-sea mining associated stressors, sediment plumes, likely to be released into the water column as a side effect of mining, can reach habitats within a radius of more than a hundred kilometers. The present study examined the effects of suspended sediments of different grain sizes (63-125 µm, 125-250 µm and 250-500 µm) in the model species Mytilus galloprovincialis, at 4 bar, as a proxy to address the potential effects of sediment plumes, in the water column, with different grain sizes under high pressure conditions. Functional (filtration rate - FR), biochemical (catalase - CAT, glutathione s-transferase - GST, lipid peroxidation - LPO) and molecular (gene expression of [actin (ACTN), glutathione S-transferase alpha (GSTA), superoxide dismutase 2 (SOD2), catalase (CAT), heat shock protein 60 (HSP60), cytochrome c oxidase (COI) and DNA mismatch repair protein (MSH6)]) endpoints were studied in juvenile organisms. The FR decreased significantly for all tested grain size ranges, with a more severe effect for the particles with a diameter between 63 and 125 µm. In addition to the FR, significant changes were also observed for all tested biomarkers. Gene expression was significantly downregulated for CAT and ACTN. Overall, this study demonstrated that the smaller sized particles are the ones leading to more severe effects. Given their high dispersion potential and longer suspension periods under mining operation scenarios, particular attention should be given to the release of sediment plumes that may affect deep-sea environments and the water column. It is, therefore, vital to create standards and guidelines for sustainable mining practices.

The anti-lipidemic drug simvastatin modifies epigenetic biomarkers in the amphipod Gammarus locusta.

The adverse effects of certain environmental chemicals have been recently associated with the modulation of the epigenome. Although changes in the epigenetic signature have yet to be integrated into hazard and risk assessment, they are interesting candidates to link environmental exposures and altered phenotypes, since these changes may be passed across multiple non-exposed generations. Here, we addressed the effects of simvastatin (SIM), one of the most prescribed pharmaceuticals in the world, on epigenetic regulation using the amphipod Gammarus locusta as a proxy, to support its integration into hazard and environmental risk assessment. SIM is a known modulator of the epigenome in mammalian cell lines and has been reported to impact G. locusta ecological endpoints at environmentally relevant levels. G. locusta juveniles were exposed to three SIM environmentally relevant concentrations (0.32, 1.6 and 8 µg L-1) for 15 days. Gene transcription levels of selected epigenetic regulators, i.e., dnmt1, dmap1, usp7, kat5 and uhrf1 were assessed, along with the quantification of DNA methylation levels and evaluation of key ecological endpoints: survival and growth. Exposure to 0.32 and 8 µg L-1 SIM induced significant downregulation of DNA methyltransferase 1 (dnmt1), concomitant with global DNA hypomethylation and growth impacts. Overall, this work is the first to validate the basal expression of key epigenetic regulators in a keystone marine crustacean, supporting the integration of epigenetic biomarkers into hazard assessment frameworks.

Cartilaginous fishes offer unique insights into the evolution of the nuclear receptor gene repertoire in gnathostomes.

Nuclear receptors (NRs) are key transcription factors that originated in the common ancestor of metazoans. The vast majority of NRs are triggered by binding to either endogenous (e.g. retinoic acid) or exogenous (e.g. xenobiotics) ligands, and their evolution and expansion is tightly linked to the function of endocrine systems. Importantly, they represent classic targets of physiological exploitation by endocrine disrupting chemicals. The NR gene repertoire in different lineages has been shaped by gene loss, duplication and mutation, denoting a dynamic evolutionary route. As the earliest diverging class of gnathostomes (jawed vertebrates), cartilaginous fishes offer an exceptional opportunity to address the early diversification of NR gene families and the evolution of the endocrine system in jawed vertebrates. Here we provide an exhaustive analysis into the NR gene composition in five elasmobranch (sharks and rays) and two holocephalan (chimaeras) species. For this purpose, we generated also a low coverage draft genome assembly of the chimaera small-eyed rabbitfish, Hydrolagus affinis. We show that cartilaginous fish retain an archetypal NR gene repertoire, similar to that of mammals and coincident with the two rounds of whole genome duplication that occurred in the gnathostome ancestor. Furthermore, novel gene members of the non-canonical NR0B receptors were found in the genomes of this lineage. Our findings provide an essential view into the early diversification of NRs in gnathostomes, paving the way for functional studies.

Chronic exposure to environmentally relevant levels of simvastatin disrupts zebrafish brain gene signaling involved in energy metabolism.

Simvastatin (SIM), a hypocholesterolaemic drug belonging to the statins group, is a widely prescribed pharmaceutical for prevention of cardiovascular diseases. Several studies showed that lipophilic statins, as SIM, cross the blood-brain barrier and interfere with the energy metabolism of the central nervous system in humans and mammalian models. In fish and other aquatic organisms, the effects of SIM on the brain energy metabolism are unknown, particularly following exposure to low environmentally relevant concentrations. Therefore, the present study aimed at investigating the influence of SIM on gene signaling pathways involved in brain energy metabolism of adult zebrafish (Danio rerio) following chronic exposure (90 days) to environmentally relevant SIM concentrations ranging from 8 ng/L to 1000 ng/L. Real-time PCR was used to determine the transcript levels of several genes involved in different pathways of the brain energy metabolism (glut1b, gapdh, acadm, accα, fasn, idh3a, cox4i1, and cox5aa). The findings here reported integrated well with ecological and biochemical responses obtained in a parallel study. Data demonstrated that SIM modulates transcription of key genes involved in the mitochondrial electron transport chain, in glucose transport and metabolism, in fatty acid synthesis and ß-oxidation. Further, SIM exposure led to a sex-dependent transcription profile for some of the studied genes. Overall, the present study demonstrated, for the first time, that SIM modulates gene regulation of key pathways involved in the energy metabolism in fish brain at environmentally relevant concentrations.

Does the antidepressant sertraline show chronic effects on aquatic invertebrates at environmentally relevant concentrations? A case study with the keystone amphipod, Gammarus locusta.

The increasing use of Sertraline (SER) as antidepressant and its consequent presence in the aquatic environment is raising concern about the chronic effects of this pharmaceutical to aquatic organisms. As the current concentrations of SER in surface waters are typically in the low ng/L range, acute toxicity is unlikely to occur. However, prolonged exposure to low concentrations of SER may lead to sub-lethal effects in aquatic organisms, including alterations in important physiological functions like growth, reproduction, behaviour, and also in key biochemical processes, such as those associated with neurotransmission and redox balance. To test this hypothesis, we selected the amphipod Gammarus locusta, a keystone species used in ecotoxicological hazard assessment. In the present study, juveniles' G. locusta from a permanent laboratory culture were chronically exposed to low concentrations of SER (8-1000 ng/L) in a bioassay that lasted for 48 days, allowing for a life-cycle study including effects on reproduction. At the lowest SER concentrations with environmental relevance (8, 40 and 200 ng/L) we detected no significant changes in key ecological endpoints such as survival, growth, reproduction and movement behaviour, or in any of the biochemical markers analysed. However, at 1000 ng/L SER (a concentration one order of magnitude higher than the levels reported in aquatic environments) females showed a significant increase in movement versus control, whereas no activity changes were observed in males. Overall, these findings indicate that G. locusta females are potentially more susceptible to the chronic effects of SER. Moreover, the current environmental SER concentrations are unlikely to affect amphipod's ecological endpoints because only SER concentrations higher than the levels reported in aquatic environments produced effects on the behaviour of G. locusta females. However, the increasing consumption of SER, highlights the importance of monitoring its chronic risk to the aquatic wildlife.

Evolutionary Plasticity in Detoxification Gene Modules: The Preservation and Loss of the Pregnane X Receptor in Chondrichthyes Lineages.

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.

Retention of fatty acyl desaturase 1 (fads1) in Elopomorpha and Cyclostomata provides novel insights into the evolution of long-chain polyunsaturated fatty acid biosynthesis in vertebrates.

BACKGROUND: Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes. RESULTS: We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. CONCLUSIONS: We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes.

Evolutionary Exploitation of Vertebrate Peroxisome Proliferator-Activated Receptor γ by Organotins.

Globally persistent man-made chemicals display ever-growing ecosystemic consequences, a hallmark of the Anthropocene epoch. In this context, the assessment of how lineage-specific gene repertoires influence organism sensitivity toward endocrine disruptors is a central question in toxicology. A striking example highlights the role of a group of compounds known as obesogens. In mammals, most examples involve the modulation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). To address the structural and biological determinants of PPARγ exploitation by a model obesogen, tributyltin (TBT), in chordates, we employed comparative genomics, transactivation and ligand binding assays, homology modeling, and site-directed-mutagenesis. We show that the emergence of multiple PPARs (α, ß and γ) in vertebrate ancestry coincides with the acquisition of TBT agonist affinity, as can be deduced from the conserved transactivation and binding affinity of the chondrichthyan and mammalian PPARγ. The amphioxus single-copy PPAR is irresponsive to TBT; as well as the investigated teleosts, this is a probable consequence of a specific mutational remodeling of the ligand binding pocket. Our findings endorse the modulatory ability of man-made chemicals and suggest an evolutionarily diverse setting, with impacts for environmental risk assessment.

Chronic effects of triclocarban in the amphipod Gammarus locusta: Behavioural and biochemical impairment.

Triclocarban (TCC), a common antimicrobial agent widely used in many household and personal care products, has been widely detected in aquatic ecosystems worldwide. Due to its high lipophilicity and persistence in the aquatic ecosystems, TCC is of emerging environmental concern. Despite the frequently reported detection of TCC in the environment and significant uncertainties about its long term effects on aquatic ecosystems, few studies have addressed the chronic effects of TCC in aquatic organisms at ecologically relevant concentrations. Therefore, we aimed at testing a broad range of biological responses in the amphipod Gammarus locusta following a chronic (60 days) exposure to environmentally relevant concentrations of TCC (100, 500 and 2500ng/L). This work integrated biochemical markers of oxidative stress (catalase (CAT), glutathione-s-transferase (GST) and lipid peroxidation (LPO)) and neurotransmission (acetylcholinesterase (AChE)) with several key ecological endpoints, i.e. behaviour, survival, individual growth and reproduction. Significant alterations were observed in all biochemical markers. While AChE showed a dose-response curve (with a significant increased activity at a TCC concentration of 2500ng/L), oxidative stress markers did not follow a dose-response curve, with significant increase at 100 and/or 500ng/L and a decreased activity in the highest concentration (2500ng/L). The same effect was observed in the females' behavioural response, whereas males' behaviour was not affected by TCC exposure. The present study represents a first approach to characterize the hazard of TCC to crustaceans.

Methyl-triclosan and triclosan impact embryonic development of Danio rerio and Paracentrotus lividus.

The presence of emerging pollutants in the environment is of major concern not only because of the potential negative impact in human health, but also due to the potential toxicity to non-target organisms. Within the personal and care products (PCPs), the disinfectant Triclosan (TCS) is one of the most concerning compounds. Once in the wastewater treatment plants (WWTPs), a small part of TCS can be biotransformed into a more persistent by-product: methyl-triclosan (M-TCS). Although several studies have focused on the occurrence of this compound in the water systems, the information on its toxicity to aquatic organisms is very limited. Here, we used embryo bioassays with two aquatic model animals to improve risk assessment of M-TCS; zebrafish (Danio rerio) embryo bioassays run up to 144 h post fertilization (hpf) and sea urchin (Paracentrotus lividus) up to 48 hpf, following established protocols. M-TCS and TCS exhibited similar toxicity to zebrafish with a NOEC of 160 µg/L. In contrast, M-TCS induced a delay in the development of the sea urchin larvae at all tested concentrations (1-1000 µg/L), whereas NOEC of TCS for P. lividus embryos was 40 µg/L. Overall, given the reported effects of M-TCS in the close range of environmentally relevant concentrations, and considering the low degradation rate and tendency to bioaccumulation (logKow: 5.2), further studies are warrant to better characterize the risk of this TCS metabolite to aquatic organisms.

Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease.

Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinson's Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP(+), assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP(+) induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP(+)-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP(+), however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.

Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban.

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants.

Recognition of bio-relevant dicarboxylate anions by an azacalix[2]arene[2]triazine derivative decorated with urea moieties.

A new dichloroazacalix[2]arene[2]triazine receptor (1) with two chiral urea binding moieties is reported. The binding affinity of this macrocycle was evaluated by (1)H NMR titrations in CDCl3 for the dicarboxylate anions oxalate (ox(2-)), malonate (mal(2-)), succinate (suc(2-)), glutarate (glu(2-)), diglycolate (dg(2-)), fumarate (fum(2-)), maleate (male(2-)), and (R,R)- and (S,S)-tartarate (tart(2-)) enantiomers. Among the first five linear anions, the higher association constants were calculated for the larger anions glu(2-) and dg(2-) and for the smallest anion ox(2-), with Kass values following the sequence dg(2-) > glu(2-) > ox(2-) > suc(2-) > mal(2-). Despite the high binding affinity 1 of for both tart(2-) enantiomers, no enantioselectivity was observed. By contrast, Kass for fum(2-) is ca. 8.9 times greater than that for male(2-), showing the selectivity of 1 for the trans isomer. These binding preferences were further elucidated by theoretical methods. Molecular dynamics simulations showed that the linear anions are lodged between both pendant arms and that each anion can assume two distinct binding poses, with one or two carboxylate groups establishing intermittent hydrogen bonds with both urea binding units. On the other hand, the recognition of male(2-) ensues in an alternative scenario, characterised by the interaction between a carboxylate group and a single urea binding unit, mirroring the lower experimental binding affinity relatively to fum(2-). A linear increase of the receptor's Nurea···Nurea and the anions' (-)O2C···CO2(-) distances versus experimental Kass was established for mal(2-), suc(2-), glu(2-) and dg(2-) associations, indicating that the match between these two distances determines the anion binding strength. The affinity for ox(2-) was associated with the most negative values of electrostatic potential positioned near carboxylate groups.