Discharge of organic micropollutants (OMPs) from urban wastewater treatment plants (WWTPs) poses a growing environmental concern, as effluents contain complex mixtures of pharmaceuticals, pesticides, and per- and polyfluoroalkyl substances (PFAS). This study evaluated nonthermal dielectric barrier discharge (DBD) plasma as a reagent-free advanced oxidation process (AOP) for OMP removal and ecotoxicity reduction in unspiked tertiary treated wastewater. Of 343 targeted compounds, 67 were detected; 15 of 28 quantified compounds exceeded the limit of quantification prior to treatment. At 16 kV, 30 min of plasma treatment achieved >90% removal of most quantified OMPs, including clarithromycin, carbamazepine, diclofenac, and venlafaxine, with a median electrical energy per order (EEO) of 4.8 kWh m−3 order−1. Extending treatment to 90 min yielded no proportional improvement in removal efficiency. PFAS, particularly perfluorooctanoic acid (PFOA), were recalcitrant to DBD treatment under the conditions tested.The untreated effluent had an EC10 of 6.9 ± 1.1 relative enrichment factor (REF), whereas the EC10 exceeded 100 REF after treatment, corresponding to >93% reduction in cyanobacterial growth inhibition. Four antibiotics and herbicides explained 63.8% of the observed toxicity, with clarithromycin contributing 52.6%. All four compounds fell below detection or quantification limits after treatment. By integrating native OMP mixtures, energy demand, and biological effects, a more environmentally relevant picture of DBD plasma performance is provided than by chemical removal data alone. Broader validation across trophic levels and endpoints is necessary before conclusions on ecological safety or full-scale readiness can be drawn.

Pharmaceutical residues in freshwater systems constitute a growing environmental problem. An important point source of these pharmaceuticals is hospital wastewater. The characterization of hospital wastewater is challenging because of its complex nature. Pharmaceuticals and their metabolites display a large variety of physicochemical properties, while matrix compounds create additional complexity. Innovative analytical approaches are hence needed to characterize these challenging samples. A promising technique is online comprehensive two-dimensional liquid chromatography (LC × LC), combining two orthogonal separation modes to increase the separation power significantly. Because of the many optimization parameters involved, method development in online LC × LC is complicated. It is difficult to predict which combinations will result in the highest peak capacity for a specific sample. In this work, different separation systems are evaluated for the online LC × LC analysis of pharmaceuticals, using an in-house developed Python-based 2D combination selector (PCS) tool. Practical peak capacities of different combinations, determined using an orthogonality score based on 12 different orthogonality metrics and predicted peak capacities, are used to select promising LC × LC conditions, including reversed-phase (RPLC) and hydrophilic interaction (HILIC) LC. Three promising combinations are further optimized, with special focus on their mobile phase incompatibility. To deal with these incompatibility issues, both active solvent modulation and flow splitting are investigated. After optimization, the RPLC × RPLC method displays the best 2D-peak shapes and highest effective peak capacity (1877) in line with predictions made by the PCS tool, highlighting its effectiveness for online LC × LC method development. The RPLC × RPLC method is successfully applied to identify 36 pharmaceuticals of various classes in real hospital wastewater.

OBJECTIVE:Drug-resistant epilepsy (DRE) affects >25 million people worldwide and is often associated with neuroinflammation. Increasing evidence links deficiency or malfunctioning of the enzyme phosphoglycerate dehydrogenase (PHGDH), which converts 3-phosphoglycerate to generate serine and the neurotransmitter glycine, with (drug-resistant) epilepsy. Moreover, PHGDH, which is primarily expressed in astrocytes within the brain, has been identified as a critical enzyme in driving macrophage polarization toward an anti-inflammatory state. Hence, PHGDH activators may be beneficial for treating DRE by exhibiting both antiseizure and anti-inflammatory activity. The objective of this study was to identify such PHGDH activators. METHODS:We screened a drug repurposing library for PHGDH activators and assessed their antiseizure and anti-inflammatory properties using various zebrafish and mouse epilepsy models and explored the mechanistic consequences of activating PHGDH in a cell line, in astrocytes, and in zebrafish heads. Finally, we assessed the efficacy of clioquinol as add-on treatment in three severe DRE patients in a clinical open pilot proof-of-concept study. RESULTS:We identified haloquinolines from a drug repurposing library as potent activators of PHGDH. The most promising haloquinoline clioquinol can increase the catalytic activity of PHGDH up to 2.5-fold, thereby increasing de novo glycine biosynthesis and resulting in reduced glutamate levels. Moreover, we show that clioquinol has PHGDH-dependent antiseizure activity as well as anti-inflammatory properties in vivo using various zebrafish and mouse epilepsy models. Finally, we demonstrate the efficacy of clioquinol as add-on treatment in severe DRE patients; two patients showed a 37%-47% reduction in seizure frequency, and all three patients noted a positive impact on quality of life and seizure severity. SIGNIFICANCE:Increasing activity of PHGDH is a promising new approach to treat DRE.

The Rift Valley fever virus (RVFV) causes haemorrhagic fever, encephalitis, and permanent blindness and has been listed by the WHO as a priority pathogen. To study RVFV pathogenesis and identify small-molecule antivirals, we established a novel In Vivo model using zebrafish larvae. Pericardial injection of RVFV resulted in ~4 log10 viral RNA copies/larva, which was inhibited by the antiviral 2′-fluoro-2′-deoxycytidine. The optical transparency of the larvae allowed detection of RVFVeGFP in the liver and sensory nervous system, including the optic tectum and retina, but not the brain or spinal cord. Thus, RVFV-induced blindness likely occurs due to direct damage to the eye and peripheral neurons, rather than the brain. Treatment with the JAK-inhibitor ruxolitinib, as well as knockout of stat1a but not stat1b, enhanced RVFV replication to ~6 log10 viral RNA copies/larva and ultra-bright livers, although without dissemination to sensory neurons or the eye, thereby confirming the critical role of stat1 in RVFV pathogenesis.

Human norovirus (HuNoV) accounts for over 700 million cases of gastroenteritis annually. Episodes of HuNoV disease are characterized by vomiting and diarrhea as the two most prominent symptoms. Despite its prevalence, our understanding of the pathophysiological mechanisms triggered upon HuNoV infection is limited, mainly due to a lack of suitable animal models. Our aim was to use the recent HuNoV zebrafish larvae model to study the effect of HuNoV infection on intestinal motility and investigate whether one viral protein could act as an enterotoxin, as seen with rotavirus. We studied whether HuNoV infection affects the contraction frequency of the intestinal bulb and the posterior intestine as well as the transit time. Infection of larvae, following injection of a HuNoV GII.4-containing stool sample in the yolk, resulted in an increased contraction frequency in the intestinal bulb. A comparable effect was observed in serotonin-treated larvae, corresponding to the natural function of serotonin. The higher replication efficacy of HuNoV GII.4 likely explains why they have a more marked effect on gut motility, when compared to other genotypes. Additionally, transit time of fluorescent food was prolonged in HuNoV GII.4 infected larvae, suggesting a loss of coordination in bowel movements upon infection. To identify the proteins responsible for the effect, individual HuNoV non-structural proteins and virus-like particles (VLPs) were injected intraperitoneally (ip). VLPs carrying VP1/VP2, but not those with only VP1, induced increased contraction frequencies in the intestinal bulb in a dose-dependent manner. In conclusion, our findings suggest that the viral capsid and potentially the minor capsid protein VP2 play a crucial role in the aetiology of symptoms associated with HuNoV, potentially acting as a viral enterotoxin. This work contributes to the understanding of the pathophysiological mechanisms in HuNoV-induced disease and further attests zebrafish as a valuable HuNoV disease model.

Drug-resistant epilepsy (DRE) affects over 25 million people worldwide and is associated with neuroinflammation. We identified haloquinolines from a drug repurposing library as potent activators of phosphoglycerate dehydrogenase (PHGDH) enzyme, which converts 3-phosphoglycerate to generate serine and the neurotransmitter glycine, and steers anti-inflammatory responses. The most promising haloquinoline clioquinol can increase the catalytic activity of PHGDH up to 2.5-fold, thereby increasing de novo glycine biosynthesis and resulting in reduced glutamate levels. Moreover, we show that clioquinol has PHGDH-dependent antiseizure activity as well as anti-inflammatory properties in vivo using various zebrafish and mouse epilepsy models. Finally, we demonstrate the efficacy of clioquinol as add-on treatment in severe DRE patients. Therefore, increasing activity of PHGDH is a promising new approach to treat DRE. ### Competing Interest Statement MM has served on advisory board for Merck and has received speaker honoraria from Merck and Biogen. Her institution receives funding from Merck, Australian National Health Medical Research Council, Brain Foundation, Charles and Sylvia Viertel Foundation, and MS Research Australia. LVDB is head of the Scientific Advisory Board of Augustine Therapeutics (Leuven, Belgium) and is part of the Investment Advisory Board of Droia Ventures (Meise, Belgium). LL has received grants as well as speaker/consultant honoraria from Zogenix (now part of UCB Pharma), LivaNova, UCB Pharma, Shire, Eisai, Novartis, Takeda/Ovid. All other authors declare that they have no competing interests. ### Clinical Trial EUDRACT 2020-004511-27 ### Funding Statement KU Leuven, IOFm/05/022 (KT) KU Leuven, C3/22/016 (KT) KU Leuven, IOFm/16/003 (AN) KU Leuven, C14/17/107; C14/22/132 (LVDB) National Health and Medical Research Council of Australia APP1141347 (BDS) US Department of Defense W81XWH2010848 (BDS) and W81XWH-21-1-0927 (IA) Fonds Wetenschappelijk Onderzoek (FWO), 1174523N (CG) and 11PGF24N (BF) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee Research UZ Leuven/KU Leuven gave ethical approval for this study (Nr S64647). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Diclofenac (DCF) is an environmentally persistent, nonsteroidal anti-inflammatory drug (NSAID) with thyroid disrupting properties. Electrochemical advanced oxidation processes (eAOPs) can efficiently remove NSAIDs from wastewater. However, eAOPs can generate transformation products (TPs) with unknown chemical and biological characteristics. In this study, DCF was electrochemically degraded using a boron-doped diamond anode. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was used to analyze the TPs of DCF and elucidate its potential degradation pathways. The biological impact of DCF and its TPs was evaluated using the Xenopus Eleutheroembryo Thyroid Assay, employing a transgenic amphibian model to assess thyroid axis activity. As DCF degradation progressed, in vivo thyroid activity transitioned from anti-thyroid in non-treated samples to pro-thyroid in intermediately treated samples, implying the emergence of thyroid-active TPs with distinct modes of action compared to DCF. Molecular docking analysis revealed that certain TPs bind to the thyroid receptor, potentially triggering thyroid hormone-like responses. Moreover, acute toxicity occurred in intermediately degraded samples, indicating the generation of TPs exhibiting higher toxicity than DCF. Both acute toxicity and thyroid effects were mitigated with a prolonged degradation time. This study highlights the importance of integrating in vivo bioassays in the environmental risk assessment of novel degradation processes.

Human noroviruses (HuNoVs) are a major cause of diarrheal disease, yet critical aspects of their biology, including cellular tropism, remain unclear. Although research has traditionally focused on the intestinal epithelium, the hypothesis that HuNoV infects macrophages has been recurrently discussed and is investigated here using a zebrafish larval model. Through single-cell RNA sequencing of dissected zebrafish intestines, we unbiasedly identified macrophages as host cells for HuNoV replication, with all three open reading frames mapped to individual macrophages. Notably, HuNoV preferentially infects actively phagocytosing inflammatory macrophages. HuNoV capsid proteins and double-stranded RNA colocalized within intestinal macrophages of infected zebrafish larvae, and the negative-strand RNA intermediate was detected within FACS-sorted macrophages. Flow cytometry confirmed viral replication within these macrophages, constituting approximately 23% of HuNoV’s host cells. Identifying macrophages as host cells prompts a reevaluation of their role in HuNoV pathogenesis, offering new directions for understanding and controlling this infection.

In this study, hazardous wastes including fluff, dust, and scrubbing sludge were sampled in 2019 from two metal shredding facilities located in Wallonia, Belgium. To assess the extent of the contamination, a global approach combining chemical and biological techniques was used, to better reflect the risks to health and the environment. The samples investigated induced significant in vitro aryl hydrocarbon receptor (AhR) agonistic bioactivities and estrogenic receptor (ERα) (ant)agonistic bioactivities in the respective CALUX (chemical activated luciferase gene expression) bioassays. The mutagenicity of the samples was investigated with the bacterial reverse gene mutation test using the Salmonella typhimurium TA98 and TA100 strains. Except for the sludge sample (site 3), all samples induced a mutagenic response in the TA98 strain (± S9 metabolic fraction) whereas in the TA100 strain (+ S9 metabolic fraction), only the sludge sample (site 2) showed a clear mutagenic effect. The in vivo toxicity/teratogenicity of the shredder wastes was further evaluated with zebrafish embryos. Except for the dust sample (site 2), all samples were found to be teratogenic as they returned teratogenic indexes (TIs) > 1. The high levels of contamination, the mutagenicity, and the teratogenicity of these shredder wastes raise significant concerns about their potential negative impacts on both human health and environment.

A library of surfactants derived from natural products was synthesized. Bioderived surfactants exhibited diverse pH-dependent foaming properties, CMC values and generally low aquatic toxicity.

Conventional water treatment methods are not efficient in eliminating endocrine disrupting compounds (EDCs) in wastewater. Electrochemical Advanced Oxidation Processes (eAOPs) offer a promising alternative, as they electro-generate highly reactive species that oxidize EDCs. However, these processes produce a wide spectrum of transformation products (TPs) with unknown chemical and biological properties. Therefore, a comprehensive chemical and biological evaluation of these remediation technologies is necessary before they can be safely applied in real-life situations. In this study, 17 & alpha;-ethinylestradiol (EE2), a persistent estrogen, was electrochemically degraded using a boron doped diamond anode with sodium sulfate (Na2SO4) and sodium chloride (NaCl) as supporting electrolytes. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the quantification of EE2 and the identification of TPs. Estrogenic activity was assessed using a transgenic medaka fish line. At optimal operating conditions, EE2 removal reached over 99.9% after 120 min and 2 min, using Na2SO4 and NaCl, respectively. The combined EE2 quantification and in vivo estrogenic assessment demonstrated the overall estrogenic activity was consistently reduced with the degradation of EE2, but not completely eradicated. The identification and time monitoring of TPs showed that the radical agents readily oxidized the phenolic A-ring of EE2, leading to the generation of hydroxylated and/or halogenated TPs and ring-opening products. eAOP revealed to be a promising technique for the removal of EE2 from water. However, caution should be exercised with respect to the generation of potentially toxic TPs.

Two-dimensional liquid chromatography (2D-LC) is becoming increasingly popular for the analysis of complex samples, which is partly due to the recent introduction of commercial 2D-LC systems. To deal with the mobile phase incompatibility between highly orthogonal retention mechanisms, such as hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC), several strategies have been introduced over the years. One of these strategies is active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension before transfer to the second dimension. This strategy has gained a lot of attention and holds great potential, however, no clear guidelines are currently in place for its use. Therefore, this study aims to investigate how the ASM process can be optimized when using highly incompatible LC combinations, such as HILIC and RPLC, in a simplified selective comprehensive 2D-LC set-up (sHILIC x RPLC) to suggest guidelines for future users. Using a representative sample, the dilution factor (DF), the duration of the ASM phase, the filling percentage of the sample loops, and their unloading configuration are investigated and optimized. It is observed that a DF of 10 with an optimal ASM phase duration, a sample loop filling of maximum 25%, and an unloading configuration in backflush mode, result in the best peak shapes, intensities, and recoveries for early eluting compounds, while keeping the total analysis time minimal. Based on these results, some general recommendations are made that could also be applied in other 2D-LC modes, such as comprehensive 2D-LC (LC x LC), heart-cutting 2D-LC (LC-LC), and other chromatographic combinations with mobile phase incompatibility issues.

Recently, two-dimensional liquid chromatography (2D-LC) has become a popular approach to analyze complex samples. This is partly due to the introduction of commercial 2D-LC systems. In the past, 2D-LC was carried out on in-house developed setups, typically consisting of several switching valves and sample loops as the interface between the two dimensions. Commercial systems usually offer different 2D-LC modes in combination with specialized software to operate the instrument and analyze the data. This makes them highly user-friendly, however, at an increased cost compared to in-house developed setups. This study aims to make a comparison between an in-house developed 2D-LC setup and a commercially available 2D-LC instrument. The comparison is made based on experimental differences, in addition to more general differences, including cost price, flexibility, and ease of operation. Special attention is also paid to the different strategies to deal with the mobile phase incompatibility between the highly orthogonal separation mechanisms considered in this work: hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC). For the commercial 2D-LC instrument, this is done using active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension column before transfer to the second dimension (2D) column. For the in-house developed setup, a combination of restriction capillaries and a trap column is used. Using a sample of 28 compounds with a large polarity range, peak shapes and recoveries of the 2D-chromatograms are compared for both setups. For early eluting compounds, the selective comprehensive approach, currently only possible on the commercial 2D-LC instrument, results in the best peak shapes and recoveries, however, at the cost of an increased analysis time. In general, depending on the analytical goal (single heart-cut versus full-comprehensive 2D-LC), an in-house developed system can be satisfactory for the analysis of specific target compounds/samples. For more complex problems, it can be interesting to use a more specialized commercial 2D-LC instrument. Overall, this comparison study provides advice for analytical scientists, who are considering to use 2D-LC, on the type of equipment to consider, depending on the needs of their particular applications.

Antimicrobial resistance can be considered a hidden global pandemic and research must be reinforced for the discovery of new antibiotics. The spirotetronate class of polyketides, with more than 100 bioactive compounds described to date, has recently grown with the discovery of phocoenamicins, compounds displaying different antibiotic activities. Three marine Micromonospora strains (CA-214671, CA-214658 and CA-218877), identified as phocoenamicins producers, were chosen to scale up their production and LC/HRMS analyses proved that EtOAc extracts from their culture broths produce several structurally related compounds not disclosed before. Herein, we report the production, isolation and structural elucidation of two new phocoenamicins, phocoenamicins D and E (1–2), along with the known phocoenamicin, phocoenamicins B and C (3–5), as well as maklamicin (7) and maklamicin B (6), the latter being reported for the first time as a natural product. All the isolated compounds were tested against various human pathogens and revealed diverse strong to negligible activity against methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis H37Ra, Enterococcus faecium and Enterococcus faecalis. Their cell viability was also evaluated against the human liver adenocarcinoma cell line (Hep G2), demonstrating weak or no cytotoxicity. Lastly, the safety of the major compounds obtained, phocoenamicin (3), phocoenamicin B (4) and maklamicin (7), was tested against zebrafish eleuthero embryos and all of them displayed no toxicity up to a concentration of 25 μM.

Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by pathogenic variants in TSC1 and TSC2 genes. TSC patients present with seizures and brain abnormalities such as tubers and subependymal giant cells astrocytoma (SEGA). Despite common molecular and clinical features, the severity of the disease varies greatly, even intrafamilially. The second hit hypothesis suggests that an additional, inactivating mutation in the remaining functional allele causes a more severe phenotype and therefore explains the phenotypic variability. Recently, second hit mutations have been detected frequently in mTORopathies. To investigate the pathophysiological effects of second hit mutations, several mouse models have been developed. Here, we opted for a double mutant zebrafish model that carries a LOF mutation both in the tsc2 and the depdc5 gene. To the best of our knowledge, this is the first time a second-hit model has been studied in zebrafish. Significantly, the DEP domain-containing protein 5 (DEPDC5) gene has an important role in the regulation of mTORC1, and the combination of a germline TSC2 and somatic DEPDC5 mutation has been described in a TSC patient with intractable epilepsy. Our depdc5 ( -/- )x tsc2 ( -/- ) double mutant zebrafish line displayed greatly increased levels of mammalian target of rapamycin (mTORC1) activity, augmented seizure susceptibility, and early lethality which could be rescued by rapamycin. Histological analysis of the brain revealed ventricular dilatation in the tsc2 and double homozygotes. RNA-sequencing showed a linear relation between the number of differentially expressed genes (DEGs) and the degree of mTORC1 hyperactivity. Enrichment analysis of their transcriptomes revealed that many genes associated with neurological developmental processes were downregulated and mitochondrial genes were upregulated. In particular, the transcriptome of human SEGA lesions overlapped strongly with the double homozygous zebrafish larvae. The data highlight the clinical relevance of the depdc5 (-/-) x tsc2 (-/-) double mutant zebrafish larvae that showed a more severe phenotype compared to the single mutants. Finally, analysis of gene-drug interactions identified interesting pharmacological targets for SEGA, underscoring the value of our small zebrafish vertebrate model for future drug discovery efforts.

A major treatment modality of epilepsy consists of pharmacological interventions using small-molecule drugs that reduce the incidence and severity of seizures. In the last decades, efforts to discover these antiseizure medications (ASMs) have been dominated by a phenotypic approach using rodent seizure and epilepsy models. These efforts have resulted in the marketing of numerous small molecules that effectively treat the symptoms of the disease.1 Unfortunately, a significant number of patients fail to achieve seizure freedom due to the occurrence of drug-resistant epilepsy (DRE), even when these ASMs are administered in combination.2 In an effort to find new molecules with an improved clinical efficacy profile, different research groups have taken different paths. An interesting approach is based on identifying compounds of interest using connectivity mapping (CMap, Broad Institute),3 a database that provides the ability to compare the transcriptional profile of a disease with the gene expression signatures elicited by treatment of mammalian cells with these compounds. Highly similar or opposing expression signatures are termed connected and suggest disease- and compound-related physiological effects on mammalian cells. Thus, by querying a disease-related gene expression profile against the CMap database, one can find compounds with an expression profile that is opposite to the profile found for the disease, and that by reversing the genetic background of the disease, may improve the disease-related symptomatology. Importantly, the database also includes approved medicinal products, which offers the possibility to bring repurposed therapeutics to the market, reducing the costs associated with traditional de novo drug development. In previous work, a similar CMap-based strategy was used to identify compounds that could revert the diseased transcriptional profile of human tissue from epilepsy-affected brains. Next, a selection of compounds was functionally tested in a behavioral assay with zebrafish larvae and was found to suppress PTZ-induced seizures.4 In the present follow-up study, Morgan Sturgeon and coworkers discovered that naltrexone, a compound previously found in the C-Map query, was able to decrease the abnormal locomotion of PTZ-treated zebrafish larvae and homozygous scn1Lab mutant (scn1Lab−/−) zebrafish.5 The latter mutant fish represent a model of Dravet syndrome (DS), a severe and highly drug-resistant developmental and epileptic encephalopathy, in the majority of cases due to SCN1A gene mutations, with onset of recurrent seizures during the first year of life of patients.6 Of note, the zebrafish DS model was previously used to identify potential treatment options for Dravet syndrome7 and to further decipher the mechanism of action of fenfluramine,8 a therapeutic that together with cannabidiol is now considered second-line treatment for DS patients.9, 10 In addition to the effects observed in the zebrafish models, naltrexone was also able to reduce in vitro neocortical seizure-like events in brain slices of adult WT mice, and to decrease in vivo the number and duration of PTZ-induced convulsive seizures in mice. The results of the current study therefore confirm the possibility of using certain opioids as ASMs, at least in specific conditions. Naltrexone is presently used in the clinic for treating alcohol use disorder and manage opioid abuse.11, 12 The compound is considered a nonselective opioid receptor antagonist, although there is a significantly higher affinity for μ-receptors than for κ- and δ-receptors (Ki: 0.89 nM, 20.2 nM and 64.1 nM, respectively).13 As a result, naltrexone has been found to produce a nearly complete blockade of μ-receptors in humans at a dose of 50 mg/day, compared with 20% to 35% blockage of δ-receptors.12 In previous studies, naltrexone has demonstrated antiseizure but also proseizure effects at different doses using different animal models.5 It is therefore tempting to think that the conflicting profile of the compound largely depends on the amount of different opioid receptors occupied, in combination with their relative distribution across different areas of the brain and the epileptic foci involved. In the current study, the homozygous scn1Lab mutant zebrafish larvae were exposed by immersion to 75 μM naltrexone at 7dpf (days post-fertilization), but it is presently not known how much compound entered the brain, and what the affinity is for the zebrafish's individual opioid receptors. Future work could address these pending issues with the goal of further deciphering the mechanistic background of the results and translating the present findings to the clinic. In conclusion, the data show that a combined CMap-zebrafish drug discovery strategy can be successfully applied to find antiseizure hits and identify novel pharmacological applications in the field of epilepsy. I have no conflicts of interests to disclose. I confirm that I have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

New pharmacological approaches that target orexin receptors (OXRs) are being developed to treat sleep disorders such as insomnia and narcolepsy, with fewer side effects than existing treatments. Orexins are neuropeptides that exert excitatory effects on postsynaptic neurons via the OXRs, and are important in regulating sleep/wake states. To date, there are three FDA-approved dual orexin receptor antagonists for the treatment of insomnia, and several small molecule oral OX2R (OXR type 2) agonists are in the pipeline for addressing the orexin deficiency in narcolepsy. To find new hypnotics and psychostimulants, rodents have been the model of choice, but they are costly and have substantially different sleep patterns to humans. As an alternative model, zebrafish larvae that like humans are diurnal and show peak daytime activity and rest at night offer several potential advantages including the ability for high throughput screening. To pharmacologically validate the use of a zebrafish model in the discovery of new compounds, we aimed in this study to evaluate the functionality of a set of known small molecule OX2R agonists and antagonists on human and zebrafish OXRs and to probe their effects on the behavior of zebrafish larvae. To this end, we developed an in vitro IP-One Homogeneous Time Resolved Fluorescence (HTRF) immunoassay, and in vivo locomotor assays that record the locomotor activity of zebrafish larvae under physiological light conditions as well as under dark-light triggers. We demonstrate that the functional IP-One test is a good predictor of biological activity in vivo. Moreover, the behavioral data show that a high-throughput assay that records the locomotor activity of zebrafish throughout the evening, night and morning is able to distinguish between OXR agonists and antagonists active on the zebrafish OXR. Conversely, a locomotor assay with alternating 30 min dark-light transitions throughout the day is not able to distinguish between the two sets of compounds, indicating the importance of circadian rhythm to their pharmacological activity. Overall, the results show that a functional IP-one test in combination with a behavioral assay using zebrafish is well-suited as a discovery platform to find novel compounds that target OXRs for the treatment of sleep disorders.

PharmaSea performed large-scale in vivo screening of marine natural product (MNP) extracts, using zebrafish embryos and larvae, to identify compounds with the potential to treat epilepsy. In this study, we report the discovery of two new antiseizure compounds, the 2,5-diketopiperazine halimide and its semi-synthetic analogue, plinabulin. Interestingly, these are both known microtubule destabilizing agents, and plinabulin could have the potential for drug repurposing, as it is already in clinical trials for the prevention of chemotherapy-induced neutropenia and treatment of non-small cell lung cancer. Both halimide and plinabulin were found to have antiseizure activity in the larval zebrafish pentylenetetrazole (PTZ) seizure model via automated locomotor analysis and non-invasive local field potential recordings. The efficacy of plinabulin was further characterized in animal models of drug-resistant seizures, i.e., the larval zebrafish ethyl ketopentenoate (EKP) seizure model and the mouse 6 Hz psychomotor seizure model. Plinabulin was observed to be highly effective against EKP-induced seizures, on the behavioral and electrophysiological level, and showed activity in the mouse model. These data suggest that plinabulin could be of interest for the treatment of drug-resistant seizures. Finally, the investigation of two functional analogues, colchicine and indibulin, which were observed to be inactive against EKP-induced seizures, suggests that microtubule depolymerization does not underpin plinabulin's antiseizure action.