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Background: Although zebrafish are increasingly utilized in biomedicine for CNS disease modelling and drug discovery, this generates big data necessitating objective, precise and reproducible analyses. The artificial intelligence (AI) applications have empowered automated image recognition and video-tracking to ensure more efficient behavioral testing., New Method: Capitalizing on several AI tools that most recently became available, here we present a novel open-access AI-driven platform to analyze tracks of adult zebrafish collected from in vivo neuropharmacological experiments. For this, we trained the AI system to distinguish zebrafish behavioral patterns following systemic treatment with several well-studied psychoactive drugs - nicotine, caffeine and ethanol., Results: Experiment 1 showed the ability of the AI system to distinguish nicotine and caffeine with 75 % and ethanol with 88 % probability and high (81 %) accuracy following a post-training exposure to these drugs. Experiment 2 further validated our system with additional, previously unexposed compounds (cholinergic arecoline and varenicline, and serotonergic fluoxetine), used as positive and negative controls, respectively., Comparison With Existing Methods: The present study introduces a novel open-access AI-driven approach to analyze locomotor activity of adult zebrafish., Conclusions: Taken together, these findings support the value of custom-made AI tools for unlocking full potential of zebrafish CNS drug research by monitoring, processing and interpreting the results of in vivo experiments., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes and global collaborative studies offer a unique solution. This study is the first global collaboration using the zebrafish ( Danio rerio ) novel tank test, a behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show females exhibit more anxiety-like behavior than males, underscoring sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (e.g., rotifers), mitigated anxiety-like behaviors. Environmental stressors, like noise and transportation, significantly impacted behavior. We recommend standardizing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds, and minimizing stressors to improve zebrafish behavioral study reliability., Competing Interests: Competing Interest Statement: The authors declare no competing interests.

Cognitive decline is commonly seen both in normal aging and in neurodegenerative and neuropsychiatric diseases. Various experimental animal models represent a valuable tool to study brain cognitive processes and their deficits. Equally important is the search for novel drugs to treat cognitive deficits and improve cognitions. Complementing rodent and clinical findings, studies utilizing zebrafish (Danio rerio) are rapidly gaining popularity in translational cognitive research and neuroactive drug screening. Here, we discuss the value of zebrafish models and assays for screening nootropic (cognitive enhancer) drugs and the discovery of novel nootropics. We also discuss the existing challenges, and outline future directions of research in this field., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Highly prevalent in laboratory rodents, 'social' hetero-grooming behavior is translationally relevant to modeling a wide range of neuropsychiatric disorders. Here, we comprehensively evaluated all known to date mouse genes linked to aberrant hetero-grooming phenotype, and applied bioinformatics tools to construct a network of their established protein-protein interactions (PPI). We next identified several distinct molecular clusters within this complex network, including neuronal differentiation, cytoskeletal, WNT-signaling and synapsins-associated pathways. Using additional bioinformatics analyses, we further identified 'central' (hub) proteins within these molecular clusters, likely key for mouse hetero-grooming behavior. Overall, a more comprehensive characterization of intricate molecular pathways linked to aberrant rodent grooming may markedly advance our understanding of underlying cellular mechanisms and related neurological disorders, eventually helping discover novel targets for their pharmacological or gene therapy interventions., (Copyright © 2024 IBRO. Published by Elsevier Inc. All rights reserved.)

Cerebral cortex is found only in mammals and is particularly prominent and developed in humans. Various rodent models with fully or partially ablated cortex are commonly used to probe the role of cortex in brain functions and its multiple subcortical projections, including pallium, thalamus and the limbic system. Various rodent models are traditionally used to study the role of cortex in brain functions. A small teleost fish, the zebrafish (Danio rerio), has gained popularity in neuroscience research, and albeit (like other fishes) lacking cortex, its brain performs well some key functions (e.g., memory, consciousness and motivation) with complex, context-specific and well-defined behaviors. Can rodent and zebrafish models help generate insights into the role of cortex in brain functions, and dissect its cortex-specific (vs. non-cortical) functions? To address this conceptual question, here we evaluate brain functionality in intact vs. decorticated rodents and further compare it in the zebrafish, a naturally occurring acortical species. Overall, comparing cortical and acortical rodent models with naturally acortical zebrafish reveals both distinct and overlapping contributions of neocortex and 'precortical' zebrafish telencephalic regions to higher brain functions. Albeit morphologically different, mammalian neocortex and fish pallium may possess more functional similarities than it is presently recognized, calling for further integrative research utilizing both cortical and decorticated/acortical vertebrate model organisms., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Potently affecting human and animal brain and behavior, hallucinogenic drugs have recently emerged as potentially promising agents in psychopharmacotherapy. Complementing laboratory rodents, the zebrafish ( Danio rerio ) is a powerful model organism for screening neuroactive drugs, including hallucinogens. Here, we tested four novel N -benzyl-2-phenylethylamine (NBPEA) derivatives with 2,4- and 3,4-dimethoxy substitutions in the phenethylamine moiety and the -F, -Cl, and -OCF 3 substitutions in the ortho position of the phenyl ring of the N -benzyl moiety (34H-NBF, 34H-NBCl, 24H-NBOMe(F), and 34H-NBOMe(F)), assessing their behavioral and neurochemical effects following chronic 14 day treatment in adult zebrafish. While the novel tank test behavioral data indicate anxiolytic-like effects of 24H-NBOMe(F) and 34H-NBOMe(F), neurochemical analyses reveal reduced brain norepinephrine by all four drugs, and (except 34H-NBCl) - reduced dopamine and serotonin levels. We also found reduced turnover rates for all three brain monoamines but unaltered levels of their respective metabolites. Collectively, these findings further our understanding of complex central behavioral and neurochemical effects of chronically administered novel NBPEAs and highlight the potential of zebrafish as a model for preclinical screening of small psychoactive molecules.

Rodent self-grooming is an important complex behavior, and its deficits are translationally relevant to a wide range of neuropsychiatric disorders. Here, we analyzed a comprehensive dataset of 227 genes whose mutations are known to evoke aberrant self-grooming in mice. Using these genes, we constructed the network of their established protein-protein interactions (PPI), yielding several distinct molecular clusters related to postsynaptic density, the Wnt signaling, transcription factors, neuronal cell cycle, NOS neurotransmission, microtubule regulation, neuronal differentiation/trafficking, neurodevelopment and mitochondrial function. Utilizing further bioinformatics analyses, we also identified novel central ('hub') proteins within these clusters, whose genes may also be implicated in aberrant self-grooming and other repetitive behaviors in general. Untangling complex molecular pathways of this important behavior using in silico approaches contributes to our understanding of related neurological disorders, and may suggest novel potential targets for their pharmacological or gene therapy., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Human neocortex controls and integrates cognition, emotions, perception and complex behaviors. Aberrant cortical development can be triggered by multiple genetic and environmental factors, causing cortical malformations. Animal models, especially rodents, are a valuable tool to probe molecular and physiological mechanisms of cortical malformations. Complementing rodent studies, the zebrafish (Danio rerio) is an important model organism in biomedicine. Although the zebrafish (like other fishes) lacks neocortex, here we argue that this species can still be used to model various aspects and brain phenomena related to human cortical malformations. We also discuss novel perspectives in this field, covering both advantages and limitations of using mammalian and zebrafish models in cortical malformation research. Summarizing mounting evidence, we also highlight the importance of translationally-relevant insights into the pathogenesis of cortical malformations from animal models, and discuss future strategies of research in the field., Competing Interests: Declaration of Competing Interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The research project used the facilities and equipment of the Resource Fund of Applied Genetics MIPT (ID 075-15-2021-684)., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Delirium is an acute neuropsychiatric condition characterized by impaired behavior and cognition. Although the syndrome has been known for millennia, its CNS mechanisms and risk factors remain poorly understood. Experimental animal models, especially rodent-based, are commonly used to probe various pathogenetic aspects of delirium. Complementing rodents, the zebrafish (Danio rerio) emerges as a promising novel model organism to study delirium. Zebrafish demonstrate high genetic and physiological homology to mammals, easy maintenance, robust behaviors in various sensitive behavioral tests, and the potential to screen for pharmacological agents relevant to delirium. Here, we critically discuss recent developments in the field, and emphasize the developing utility of zebrafish models for translational studies of delirium and deliriant drugs. Overall, the zebrafish represents a valuable and promising aquatic model species whose use may help understand delirium etiology, as well as develop novel therapies for this severely debilitating disorder., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In recent decades, metal-containing nanocomposites have attracted considerable attention from researchers. In this work, for the first time, a detailed analysis of the preparation of reactive indicator papers (RIPs) based on silver-containing nanocomposites derived from silver fumarate was carried out. Thermolysis products are silver-containing nanocomposites containing silver nanoparticles uniformly distributed in a stabilizing carbon matrix. The study of the optical properties of silver-containing nanocomposites made it possible to outline the prospects for their application in chemical analysis. RIPs were made by impregnating a cellulose carrier with synthesized silver fumarate-derived nanocomposites, which change their color when interacting with chlorine vapor. This made it possible to propose a method for the determination of chloride ions with preliminary oxidation to molecular chlorine, which is then separated from the solution by gas extraction. The subsequent detection of the active zone of RIPs using colorimetry makes it possible to identify mathematical dependences of color coordinates on the concentration of chloride ions. The red (R) color coordinate in the RGB (red-green-blue) system was chosen as the most sensitive and promising analytical signal. Calibration plots of exponential and linear form and their equations are presented. The limit of detection is 0.036 mg/L, the limits of quantification are 0.15-2.4 mg/L, and the time of a single determination is 25 min. The prospects of the developed technique have been successfully shown in the example of the analysis of the natural waters of the Don River, pharmaceuticals, and food products.

Background: The dopamine transporter (DAT) is the main regulator of dopamine concentration in the extrasynaptic space. The pharmacological inhibition of the DAT results in a wide spectrum of behavioral manifestations, which have been identified so far in a limited number of species, mostly in rodents., Aim: Here, we used another well-recognized model organism, the zebrafish ( Danio rerio ), to explore the behavioral effects of GBR 12909, a highly-affine selective DAT blocker., Methods: We evaluated zebrafish locomotion, novelty-related exploration, spatial cognition, and social phenotypes in the novel tank, habituation and shoaling tests, following acute 20-min water immersion in GBR 12909., Results: Our findings show hypolocomotion, anxiety-like state, and impaired spatial cognition in fish acutely treated with GBR 12909. This behavioral profile generally parallels that of the DAT knockout rodents and zebrafish, and it overlaps with behavioral effects of other DAT-inhibiting drugs of abuse, such as cocaine and D-amphetamine., Conclusion: Collectively, our data support the utility of zebrafish in translational studies on DAT targeting neuropharmacology and strongly implicate DAT aberration as an important mechanisms involved in neurological and psychiatric diseases.

Depression and schizophrenia are two highly prevalent and severely debilitating neuropsychiatric disorders. Both conventional antidepressant and antipsychotic pharmacotherapies are often inefficient clinically, causing multiple side effects and serious patient compliance problems. Collectively, this calls for the development of novel drug targets for treating depressed and schizophrenic patients. Here, we discuss recent translational advances, research tools and approaches, aiming to facilitate innovative drug discovery in this field. Providing a comprehensive overview of current antidepressants and antipsychotic drugs, we also outline potential novel molecular targets for treating depression and schizophrenia. We also critically evaluate multiple translational challenges and summarize various open questions, in order to foster further integrative cross-discipline research into antidepressant and antipsychotic drug development.

Epilepsy is a highly prevalent, severely debilitating neurological disorder characterized by seizures and neuronal hyperactivity due to an imbalanced neurotransmission. As genetic factors play a key role in epilepsy and its treatment, various genetic and genomic technologies continue to dissect the genetic causes of this disorder. However, the exact pathogenesis of epilepsy is not fully understood, necessitating further translational studies of this condition. Here, we applied a computational in silico approach to generate a comprehensive network of molecular pathways involved in epilepsy, based on known human candidate epilepsy genes and their established molecular interactors. Clustering the resulting network identified potential key interactors that may contribute to the development of epilepsy, and revealed functional molecular pathways associated with this disorder, including those related to neuronal hyperactivity, cytoskeletal and mitochondrial function, and metabolism. While traditional antiepileptic drugs often target single mechanisms associated with epilepsy, recent studies suggest targeting downstream pathways as an alternative efficient strategy. However, many potential downstream pathways have not yet been considered as promising targets for antiepileptic treatment. Our study calls for further research into the complexity of molecular mechanisms underlying epilepsy, aiming to develop more effective treatments targeting novel putative downstream pathways of this disorder.

Psychiatric disorders are highly prevalent brain pathologies that represent an urgent, unmet biomedical problem. Since reliable clinical diagnoses are essential for the treatment of psychiatric disorders, their animal models with robust, relevant behavioral and physiological endpoints become necessary. Zebrafish ( Danio rerio ) display well-defined, complex behaviors in major neurobehavioral domains which are evolutionarily conserved and strikingly parallel to those seen in rodents and humans. Although zebrafish are increasingly often used to model psychiatric disorders, there are also multiple challenges with such models as well. The field may therefore benefit from a balanced, disease-oriented discussion that considers the clinical prevalence, the pathological complexity, and societal importance of the disorders in question, and the extent of its detalization in zebrafish central nervous system (CNS) studies. Here, we critically discuss the use of zebrafish for modeling human psychiatric disorders in general, and highlight the topics for further in-depth consideration, in order to foster and (re)focus translational biological neuroscience research utilizing zebrafish. Recent developments in molecular biology research utilizing this model species have also been summarized here, collectively calling for a wider use of zebrafish in translational CNS disease modeling.

Antimicrobial drugs represent a diverse group of widely utilized antibiotic, antifungal, antiparasitic and antiviral agents. Their growing use and clinical importance necessitate our improved understanding of physiological effects of antimicrobial drugs, including their potential effects on the central nervous system (CNS), at molecular, cellular, and behavioral levels. In addition, antimicrobial drugs can alter the composition of gut microbiota, and hence affect the gut-microbiota-brain axis, further modulating brain and behavioral processes. Complementing rodent studies, the zebrafish ( Danio rerio ) emerges as a powerful model system for screening various antimicrobial drugs, including probing their putative CNS effects. Here, we critically discuss recent evidence on the effects of antimicrobial drugs on brain and behavior in zebrafish, and outline future related lines of research using this aquatic model organism.

The mammalian target of rapamycin (mTOR) is an important molecular regulator of cell growth and proliferation. Brain mTOR activity plays a crucial role in synaptic plasticity, cell development, migration and proliferation, as well as memory storage, protein synthesis, autophagy, ion channel expression and axonal regeneration. Aberrant mTOR signaling causes a diverse group of neurological disorders, termed 'mTORopathies'. Typically arising from mutations within the mTOR signaling pathway, these disorders are characterized by cortical malformations and other neuromorphological abnormalities that usually co-occur with severe, often treatment-resistant, epilepsy. Here, we discuss recent advances and current challenges in developing experimental models of mTOR-dependent epilepsy and other related mTORopathies, including using zebrafish models for studying these disorders, as well as outline future directions of research in this field.

Mounting evidence links psychiatric disorders to central and systemic inflammation. Experimental (animal) models of psychiatric disorders are important tools for translational biopsychiatry research and CNS drug discovery. Current experimental models, most typically involving rodents, continue to reveal shared fundamental pathological pathways and biomarkers underlying the pathogenetic link between brain illnesses and neuroinflammation. Recent data also show that various proinflammatory factors can alter brain neurochemistry, modulating the levels of neurohormones and neurotrophins in neurons and microglia. The role of "active" glia in releasing a wide range of proinflammatory cytokines also implicates glial cells in various psychiatric disorders. Here, we discuss recent animal inflammation-related models of psychiatric disorders, focusing on their translational perspectives and the use of some novel promising model organisms (zebrafish), to better understand the evolutionally conservative role of inflammation in neuropsychiatric conditions., (© 2023. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

At present, conjugated thermolysis of metal-containing monomers is widely used as single-source precursors to obtain new metal- and metal oxide-containing nanocomposites. In this study, a detailed analysis of the main stages of conjugated thermolysis of silver itaconate was carried out. The obtained nanocomposites containing silver nanoparticles are evenly distributed in a stabilizing carbon matrix. The structural characteristics and properties of the resulting nanomaterials were studied using X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS). We have developed a method of test analysis of chlorides using paper modified with the obtained silver-containing nanocomposites. The analysis technique is based on the in situ conversion of chlorides to molecular chlorine, its dynamic release, and colorimetric detection using NP-modified paper test strips. A simple installation device is described that allows this combination to be realized. The proposed approach seems promising for nanoparticle-based determinations of other analytes that can be converted into volatile derivatives.

Channelopathies are a large group of systemic disorders whose pathogenesis is associated with dysfunctional ion channels. Aberrant transmembrane transport of K + , Na + , Ca 2+ and Cl - by these channels in the brain induces central nervous system (CNS) channelopathies, most commonly including epilepsy, but also migraine, as well as various movement and psychiatric disorders. Animal models are a useful tool for studying pathogenesis of a wide range of brain disorders, including channelopathies. Complementing multiple well-established rodent models, the zebrafish ( Danio rerio ) has become a popular translational model organism for neurobiology, psychopharmacology and toxicology research, and for probing mechanisms underlying CNS pathogenesis. Here, we discuss current prospects and challenges of developing genetic, pharmacological and other experimental models of major CNS channelopathies based on zebrafish.

Depression is a widespread and severely debilitating neuropsychiatric disorder whose key clinical symptoms include low mood, anhedonia and despair (the inability or unwillingness to overcome stressors). Experimental animal models are widely used to improve our mechanistic understanding of depression pathogenesis, and to develop novel antidepressant therapies. In rodents, various experimental models of 'behavioral despair' have already been developed and rigorously validated. Complementing rodent studies, the zebrafish (Danio rerio) is emerging as a powerful model organism to assess pathobiological mechanisms of depression and other related affective disorders. Here, we critically discuss the developing potential and important translational implications of zebrafish models for studying despair and its mechanisms, and the utility of such aquatic models for antidepressant drug screening., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hallucinogenic drugs potently affect brain and behavior and have also recently emerged as potentially promising agents in pharmacotherapy. Complementing laboratory rodents, the zebrafish ( Danio rerio ) is a powerful animal model organism for screening neuroactive drugs, including hallucinogens. Here, we test a battery of ten novel N -benzyl-2-phenylethylamine (NBPEA) derivatives with the 2,4- and 3,4-dimethoxy substitutions in the phenethylamine moiety and the -OCH 3 , -OCF 3 , -F, -Cl, and -Br substitutions in the ortho position of the phenyl ring of the N -benzyl moiety, assessing their acute behavioral and neurochemical effects in the adult zebrafish. Overall, substitutions in the Overall, substitutions in the N -benzyl moiety modulate locomotion, and substitutions in the phenethylamine moiety alter zebrafish anxiety-like behavior, also affecting the brain serotonin and/or dopamine turnover. The 24H-NBOMe(F) and 34H-NBOMe(F) treatment also reduced zebrafish despair-like behavior. Computational analyses of zebrafish behavioral data by artificial intelligence identified several distinct clusters for these agents, including anxiogenic/hypolocomotor (24H-NBF, 24H-NBOMe, and 34H-NBF), behaviorally inert (34H-NBBr, 34H-NBCl, and 34H-NBOMe), anxiogenic/hallucinogenic-like (24H-NBBr, 24H-NBCl, and 24H-NBOMe(F)), and anxiolytic/hallucinogenic-like (34H-NBOMe(F)) drugs. Our computational analyses also revealed phenotypic similarity of the behavioral activity of some NBPEAs to that of selected conventional serotonergic and antiglutamatergic hallucinogens. In silico functional molecular activity modeling further supported the overlap of the drug targets for NBPEAs tested here and the conventional serotonergic and antiglutamatergic hallucinogens. Overall, these findings suggest potent neuroactive properties of several novel synthetic NBPEAs, detected in a sensitive in vivo vertebrate model system, the zebrafish, raising the possibility of their potential clinical use and abuse.

Background: Cognitive deficits represent an urgent biomedical problem, and are commonly reduced by nootropic drugs. Animal models, including both rodents and zebrafish, offer a valuable tool for studying cognitive phenotypes and screening novel nootropics. Beta-alanine and its derivatives have recently been proposed to exert nootropic activity., Aims: This study aimed to characterize putative nootropic profile of a novel β-alanine analogue, 1,3-diaminopropane (MB-005), in adult zebrafish., Methods: Nootropic profile of MB-005 was assessed in adult zebrafish in the novel tank and conditioned place aversion (CPA) tests acutely, and in cued-learning plus-maze (PMT) tests chronically., Results/outcomes: MB-005 did not alter zebrafish anxiety-like behavior or monoamine neurochemistry acutely, improved short-term memory in the CPA test, but impaired cognitive performance in both CPA and PMT tests chronically., Conclusions/interpretation: This study reveals high sensitivity of zebrafish cognitive phenotypes to MB-005, suggesting it as a potential novel cognitive enhancer acutely, but raises concerns over its cognitive (and, possibly, other) side-effects chronically.

Long-term recurrent stress is a common cause of neuropsychiatric disorders. Animal models are widely used to study the pathogenesis of stress-related psychiatric disorders. The zebrafish (Danio rerio) is emerging as a powerful tool to study chronic stress and its mechanisms. Here, we developed a prolonged 11-week chronic unpredictable stress (PCUS) model in zebrafish to more fully mimic chronic stress in human populations. We also examined behavioral and neurochemical alterations in zebrafish, and attempted to modulate these states by 3-week treatment with an antidepressant fluoxetine, a neuroprotective omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), a pro-inflammatory endotoxin lipopolysaccharide (LPS), and their combinations. Overall, PCUS induced severe anxiety and elevated norepinephrine levels, whereas fluoxetine (alone or combined with other agents) corrected most of these behavioral deficits. While EPA and LPS alone had little effects on the zebrafish PCUS-induced anxiety behavior, both fluoxetine (alone or in combination) and EPA restored norepinephrine levels, whereas LPS + EPA increased dopamine levels. As these data support the validity of PCUS as an effective tool to study stress-related pathologies in zebrafish, further research is needed into the ability of various conventional and novel treatments to modulate behavioral and neurochemical biomarkers of chronic stress in this model organism., (© 2021. The Author(s).)

3,4-Dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is a selective μ-opioid receptor agonist originally synthesized as a prospective analgesic drug. Several times more potent than morphine, U-47700 has high abuse potential and may cause clinical neurotoxicity, euphoria, respiratory depression and occasional mortality. U-47700 also evokes analgesia, sedation and euphoria-like states in both humans and rodents. Despite the growing use and abuse of U-47700, its psychopharmacological and toxicological profiles in vivo remain poorly understood. The zebrafish (Danio rerio) is rapidly becoming a popular aquatic model organism for central nervous system (CNS) disease modeling and drug discovery. Here, we examine acute (1, 5, 10, 25 and 50 mg/L for 20-min) and chronic (0.1, 0.5 and 1 mg/L for 14 days) effects of U-47700 in adult zebrafish. Overall, we found overt sedation evoked in fish by acute, and hyperlocomotion with an anxiolytic-like action by chronic, drug treatments. Acute treatment with 1 and 10 mg/L U-47700 also resulted in detectable amounts of this drug in the brain samples, supporting its permeability through the blood-brain barrier. Collectively, these findings emphasize complex dose- and treatment-dependent CNS effects of U-47700 following its acute and chronic administration. Our study also supports high sensitivity of zebrafish to U-47700, and suggests these aquatic models as promising in-vivo screens for probing potential CNS effects evoked by novel synthetic opioid drugs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Stress-related neuropsychiatric disorders are widespread, debilitating and often treatment-resistant illnesses that represent an urgent unmet biomedical problem. Animal models of these disorders are widely used to study stress pathogenesis. A more recent and historically less utilized model organism, the zebrafish (Danio rerio), is a valuable tool in stress neuroscience research. Utilizing the 5-week chronic unpredictable stress (CUS) model, here we examined brain transcriptomic profiles and complex dynamic behavioral stress responses, as well as neurochemical alterations in adult zebrafish and their correction by chronic antidepressant, fluoxetine, treatment. Overall, CUS induced complex neurochemical and behavioral alterations in zebrafish, including stable anxiety-like behaviors and serotonin metabolism deficits. Chronic fluoxetine (0.1 mg/L for 11 days) rescued most of the observed behavioral and neurochemical responses. Finally, whole-genome brain transcriptomic analyses revealed altered expression of various CNS genes (partially rescued by chronic fluoxetine), including inflammation-, ubiquitin- and arrestin-related genes. Collectively, this supports zebrafish as a valuable translational tool to study stress-related pathogenesis, whose anxiety and serotonergic deficits parallel rodent and clinical studies, and genomic analyses implicate neuroinflammation, structural neuronal remodeling and arrestin/ubiquitin pathways in both stress pathogenesis and its potential therapy.

Anticholinergic drugs based on tropane alkaloids, including atropine, scopolamine, and hyoscyamine, have been used for various medicinal and toxic purposes for millennia. These drugs are competitive antagonists of acetylcholine muscarinic (M-) receptors that potently modulate the central nervous system (CNS). Currently used clinically to treat vomiting, nausea, and bradycardia, as well as alongside other anesthetics to avoid vagal inhibition, these drugs also evoke potent psychotropic effects, including characteristic delirium-like states with hallucinations, altered mood, and cognitive deficits. Given the growing clinical importance of anti-M deliriant hallucinogens, here we discuss their use and abuse, clinical importance, and the growing value in preclinical (experimental) animal models relevant to modeling CNS functions and dysfunctions.

Alpha-pyrrolidinopentiophenone (α-PVP) is a synthetic cathinone which exerts robust mental and physiological effects clinically, as well as causes aberrant stereotypic behaviors and altered locomotion in rodents. Given the rich spectrum of pharmacological activity of α-PVP in rodents and humans, as well as its high abuse potential, further studies are needed to better understand the pharmacology and toxicology of this drug. The zebrafish (Danio rerio) is a relatively novel model organism in neuropharmacology and toxicology research. Here, we characterize behavioral effects of α-PVP in adult zebrafish following its acute (1, 5, 25 and 50 mg/L for 20 min) and chronic (1, 5 and 10 mg/L for 7 days) treatments. Overall, acute exposure to α-PVP evoked psychostimulant (but not anxiolytic-like) effects in zebrafish novel tank test, with characteristic stereotypic 'side-to-side' bottom swimming at 5, 25 and 50 mg/L. The high-performance liquid chromatography/high-resolution mass spectrometry (HPLC/HRMS) analyses of zebrafish brains showed detectable levels of α-PVP following its acute administration, likely underlying the observed behavioral effects. Although acute 2-day discontinuation of chronic 7-day α-PVP at 1, 5 and 10 mg/L produced no effects, hypolocomotion occurred after a 7-day chronic treatment and repeated withdrawal, resembling rodent effects of some chronic psychostimulants. Collectively, these findings support zebrafish sensitivity to α-PVP and show some parallels with its effects in mammals and humans. This study also suggests that aquatic models based on zebrafish can help further examine the CNS effects evoked by α-PVP and screen for related synthetic new psychoactive drugs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Flakka (alpha-pyrrolidinovalerophenone, α-PVP) is a new psychoactive substance, chemically close to cathinone, the primary psychoactive alkaloid of khat ( Catha edulis). Like other synthetic cathinones, α-PVP is a potent inhibitor of the dopamine and norepinephrine transporters. Its robust clinical effects include hallucinations, arousal, aggression/violence, and euphoria. In animal models, α-PVP evokes hyperlocomotion and aberrant/stereotypic behaviors. Here, we discuss the history, synthesis, pharmacological mechanisms, metabolism, abuse potential, and societal impact of α-PVP. Today, α-PVP is a tightly controlled substance, currently banned in the United States and other countries worldwide. However, the growing abuse and complex central nervous system (CNS) effects of α-PVP remain poorly understood, necessitating further pharmacological and pharmacogenetic studies of this drug. Its interesting pharmacological profile (co-inhibition of dopamine and norepinephrine, but not serotonin, transporters) also calls for further studies of α-PVP in animal models, to dissect serotonergic from other monoaminergic mechanisms of action of drugs of abuse. Finally, screening α-PVP and related compounds in vivo may foster discovery of new CNS drugs, including developing novel CNS drugs and identifying their molecular targets.

Aggression is a common agonistic behavior affecting social life and well-being of humans and animals. However, the underlying mechanisms of aggression remain poorly understood. For decades, studies of aggression have mostly focused on laboratory rodents. The growing importance of evolutionarily relevant, cross-species disease modeling necessitates novel model organisms to study aggression and its pathobiology. The zebrafish (Danio rerio) is rapidly becoming a new experimental model organism in neurobehavioral research. Zebrafish demonstrate high genetic and physiological homology with mammals, fully sequenced genome, ease of husbandry and testing, as well as rich, robust behavioral repertoire. As zebrafish present overt aggressive behaviors, here we focus on their behavioral models and discuss their utility in probing aggression neurobiology and its genetic, pharmacological and environmental modulation. We argue that zebrafish-based models represent an excellent translational tool to understand aggressive behaviors and related pathobiological brain mechanisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To detect and study diseases, research and clinical laboratories must quantify specific biomarkers in the plasma and urine of patients with precision, sensitivity, and cost-effectiveness. Newly developed techniques, such as particle-based immunoassays, must be validated in these terms against standard methods such as enzyme-linked immunosorbent assays (ELISAs). Here, we compare the performance of assays that use hollow polyelectrolyte microcapsules with assays based on solid plastic beads, and with standard microplate immunoassays. The polyelectrolyte microcapsules detect the disease biomarker beta-2 microglobulin with a fifty-fold increase in sensitivity than polystyrene (PS) beads. For sequence-specific nucleic acid detection, the oligonucleotide-coated microcapsules exhibit a two-fold lower increase in sensitivity over PS beads. The microcapsules also detect the presence of a monoclonal antibody in hybridoma supernatant at a fifty-six-fold increase in sensitivity compared to a microplate assay. Overall, polyelectrolyte microcapsule-based assays are more sensitive for the detection of protein and nucleic acid analytes than PS beads and microplate assays, and they are viable alternatives as a platform for the rapid quantitative detection of analytes at very low concentrations., Competing Interests: The authors have declared that no competing interests exist.

Antidepressant drugs are currently one of the most prescribed medications. In addition to treatment resistance and side effects of antidepressants, their clinical use is further complicated by antidepressant discontinuation syndrome (ADS). ADS is a common problem in patients following the interruption, dose reduction, or discontinuation of antidepressant drugs. Clinically, ADS resembles a classical drug withdrawal syndrome, albeit differing from it because antidepressants generally do not induce addiction. The growing clinical importance and prevalence of ADS necessitate novel experimental (animal) models of this disorder. Currently available preclinical models of ADS are mainly rodent-based, and study mostly serotonergic antidepressants and their combinations. Here, we systematically assess clinical ADS symptoms and discuss current trends and challenges in the field of experimental (animal) models of ADS. We also outline basic mechanisms underlying ADS pathobiology, evaluate its genetic, pharmacological and environmental determinants, and emphasize how using animal models may help generate important translational insights into human ADS condition, its prevention and therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amitriptyline is a commonly used tricyclic antidepressant (TCA) inhibiting serotonin and norepinephrine reuptake. The exact CNS action of TCAs remains poorly understood, necessitating new screening approaches and novel model organisms. Zebrafish (Danio rerio) are rapidly emerging as a promising tool for pharmacological research of antidepressants, including amitriptyline. Here, we examine the effects of chronic 2-week exposure to 10 and 50 μg/L amitriptyline on zebrafish behavior and monoamine neurotransmitters. Overall, the drug at 50 μg/L evoked pronounced anxiolytic-like effects in the novel tank test (assessed by more time in top, fewer transition and shorter latency to enter the top). Like other TCAs, amitriptyline reduced serotonin turnover, but also significantly elevated whole-brain norepinephrine and dopamine levels. The latter effect was not reported in this model previously, and accompanied higher brain expression of tyrosine hydroxylase (a rate-limiting enzyme of catecholamine biosynthesis), but unaltered expression of dopamine-β-hydroxylase and monoamine oxidase (the enzymes of dopamine metabolism). This response may underlie chronic amitriptyline action on dopamine and norepinephrine neurotransmission, and contribute to the complex CNS profile of this drug observed both clinically and in animal models. Collectively, these findings also confirm the important role of monoamine modulation in the regulation of anxiety-related behavior in zebrafish, and support the utility of this organism as a promising in-vivo model for CNS drug screening.

Tetracyanoethylene (TCNE) as one of the most versatile organic compounds is involved in various chemical reactions with electron transfer. Charge transfer complexes (CTCs) of a few antioxidants, nitrogen containing thioamides [pyrrolidine-2-thione (I), 1,3-H-imidazolidine-2-thione (II), 1,3-H-Imidazoline-2-thione (III), pyridine-2-thione (IV), 5-trifluoromethylpyridine-2-thione (V), 4-trifluoromethylpyrimidine-2-thione (VI), quinoline-2-thione (VII), 3,4,5,6-tetrahydropyrimidine-2-thione (VIII)] as π-donors and TCNE as π-acceptor were studied. The DFT PCM/UB3LYP/6-31++G(d,p) and SA-CASSCF quantum chemical calculations were used to study the structures and relative stabilities of these complexes in the ground and lowest excited electronic states. The formation of a weak molecular associates in the chloroform and acetonitrile solutions was confirmed by UV/vis and IR absorption spectroscopy. The stability constants and molar extinction coefficients were estimated by UV/vis spectroscopy. The highest stability in acetonitrile is found for associates formed by quinoline-2-thione and pyridine-2-thione with TCNE, the lowest one is found for CTC formed by imidazolidine-2-thione. Molecular associate formed by pyridine-2-thione and TCNE has the greatest stability in the chloroform solution. 5-Trifluoromethylpyridine-2-thione and 4-trifluoromethylpyrimidine-2-thione do not form CTC in CH 3 CN due to the presence of an electron acceptor group in the molecules. The molar extinction values of CTC vary within the range of 0.4 × 10 3 to 1.0 × 10 4 M -1 cm -1 . An analytical strategy of thioamides identification based on wavelength and intensity of CTCs absorption band has been suggested.

The need to develop novel antidepressants is an emerging problem in biomedicine. An aquatic vertebrate species, the zebrafish (Danio rerio) may serve as a useful in-vivo screen for CNS drugs, and displays high sensitivity to a wide range of antidepressants. Amitriptyline is a commonly used tricyclic antidepressant which acts primarily as a serotonin and noradrenaline reuptake inhibitor. Here, we characterize drug-induced behavioral and neurochemical responses in adult zebrafish following their acute exposure to amitriptyline. Overall, the drug at 1 and 5mg/L significantly increased time spent in top and shortened the latency to enter it, thereby paralleling recent reports on zebrafish 'serotonin toxicity-like behavior' caused by various serotonergic agents. The 10mg/L dose of the drug also significantly decreased top entries and maximal velocity and evoked overt ataxia, likely due to emerging non-specific toxic effects. Amitriptyline at 5 and 10mg/L also dose-dependently increased serotonin turnover, but not noradrenaline levels, in zebrafish whole-brain samples. Overall, zebrafish high sensitivity to acute effects of amitriptyline can help improve our understanding of psychopharmacological profiles of this compound and the related CNS drugs, and contributes further to the development of aquatic experimental models of human toxidromes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Over the last few decades, many scientists considered microalgae as promising actors for future biofuels because of the high lipid productivity inside their cells. Moreover, much attention has been paid to algal lipids as they can be used in biodiesel production. In this study, we optimized the different suitable conditions such as incubation time, incubation temperature, Dimethylesulfoxide and Nile red concentrations of the lipophilic fluorescence dye Nile red as an excellent and fast vital stain to detect and quantify intracellular lipids. This was achieved using the green alga Nannochloropsis salina. In addition, investigating the accumulation of lipid vesicles inside different isolated microalgal species as a response to temperature stress. Furthermore, the confocal laser scanning microscopy (LS510) for imaging and measuring the size and volume of the accumulated lipid vesicles was used.

Targeted delivery systems recognizing specific receptors are a key element in personalized medicine. Such systems allow the delivery of therapeutics to desired sites of the body, increasing their local concentration and thus reducing the side effects. In this study, we fabricate chemically cross-linked (PAH/PAA) 2 microcapsules coated with specific cell-targeting antibodies in random (via direct covalent coupling to the surface) or optimized (via supporting layer of protein A) orientation. We use these antibody-functionalized capsules to target major histocompatibility complex (MHC) class I receptors in living cells and quantify the efficiency of targeting by flow cytometry. We show for the first time the selective binding of polyelectrolyte microcapsules to MHC class I receptors, and confirm that targeting is allotype-specific. Remarkably, protein A assisted immobilization of antibodies enhances targeting efficiency by 40-50% over capsules with randomly attached antibodies. Moreover, biofunctionalized capsules reveal low levels of cytotoxicity and nonspecific binding, excluding the need of additional modification with poly(ethylene glycol). Thus, protein A coated (PAH/PAA) 2 microcapsules represent a unique example of universal targeting tools providing high potential for selective binding to a broad range of cell surface receptors.

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2K b , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2K b and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tiletamine is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist chemically related to ketamine and phencyclidine. A common veterinary anesthetic drug, tiletamine is currently a Schedule III controlled substance in USA. This compound exerts sedative effects in humans and animals, also having an abuse potential, toxicity and dissociative hallucinogenic properties clinically. However, the neurotropic profile of tiletamine remains poorly understood, necessitating novel models and in-vivo screens, including non-mammalian species. Zebrafish (Danio rerio) are rapidly becoming a popular model organism for screening various CNS drugs, including those acting at NMDA receptors. Here, we investigated acute behavioral effects of 1, 5 and 10mg/L of tiletamine on adult zebrafish. In the standard novel tank test, a 20-min immersion in 1mg/L of tiletamine produced no overt differences from control zebrafish (receiving 0.1% DMSO vehicle), except for reduced top entries. In contrast, tiletamine at 5 and 10mg/L exerted robust dose-dependent sedative effects in zebrafish (also darkening their skin coloration, similar to ketamine and PCP). Gas chromatography/mass spectrometry (GC/MS) analyses revealed no tiletamine peaks in control and 1mg/L groups, but detected tiletamine peaks in zebrafish brain samples at 5 and 10mg/L. Together, these findings demonstrate potent neurotropic effects of tiletamine in zebrafish, and their high sensitivity to this drug. Our findings also support the growing utility of fish-based aquatic screens for studying neuroactive properties of NMDA antagonists in-vivo., (Copyright © 2016. Published by Elsevier Inc.)

Heteroaromatic thiols and thiones attracted the attention of chemists, pharmacologists and biochemists because of participation in the interception of free radicals. For the first time offered independent and reliable methods for evaluating of the antioxidant activity of thioamides-derivatives of pyridine, quinoline, imidazole, triazole, tetrazole, pyrimidine, pyrrolidine and 7-mercapto-4-methylcoumarin -based on kinetic parameters of the thioamide reaction with chromogenic radical (rate constant, M(-1)min(-1) and time to decrease concentration of test free radical by 50%, TEC50, min) or thermodynamics of the thioamides reaction with molecular iodine (extent of thioamide conversion, %). To compare the antioxidant activity of thioamides and widely used standard-antioxidant Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) we have proposed to use a value of relative antioxidant activity constant. As it was established, the kinetics of interaction between the chromogenic radical and thioamides in the presence of an excess of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) is described by the kinetics of the pseudo first order with respect to the reacting components. A kinetic-spectrophotometric method for the quantification of heteroaromatic thioamides is elaborated and was tested in the analysis of urine. Thioamides were detected at concentrations of 1.53μgml(-1), RSD=4.6% (2-mercaptoimidazole, V), 2.08μgml(-1), RSD=1.8% (1-methylimidazoline-2-thione, VI), 1.45μgml(-1), RSD=4.3% (2-mercaptopyridine, IX).The proposed procedures show good precision and accuracy of the results obtained., (Copyright © 2015. Published by Elsevier B.V.)

The behavior of quinoline-2(1H)-thione, which is a potential antithyroid drug toward molecular iodine, was investigated. The ability of quinoline-2(1H)-thione to form the outer-sphere charge-transfer complex C9H7NS·I2 with iodine molecular in dilute chloroform solution has been studied by UV-vis spectroscopy (lgβ=3.85). The crystal structure of the new salt 2-(quinoline-2-yldisulfanyl)quinolinium triiodide - product of irreversible oxidation of quinoline-2(1H)-thione was determined by X-ray diffraction. The 2-(quinoline-2-yldisulfanyl)quinolinium cations form dimers through π-π-stacking interaction between quinoline rings. Strong intramolecular interactions are observed between iodine - sulfur atoms and iodine - hydrogen atoms with shortened contacts (less of sum of van der Waals contacts). It is noteworthy that two perfectly centrosymmetrical anions I3(-) form a very short contact I(3)⋯I(3') 3.7550(5) so we can state the formation of the dianion I(6)(2-). Therefore the formation and topology of polyiodide species depend on the characteristics, such as shape, size and charge, etc., of the counter cation, 2-(quinoline-2-yldisulfanyl)quinolinium, which is considered as templating agent., (Copyright © 2015. Published by Elsevier B.V.)

We have developed a nanoplasmonic-based approach to induce nanometer-sized local defects in the phospholipid membranes. Here, gold nanorods and nanoparticles having plasmon resonances in the near-infrared (NIR) spectral range are used as optical absorption centers in the lipid membrane. Defects optically induced by NIR-laser irradiation of gold nanoparticles are continuously monitored by high-precision ion conductance measurement. Localized laser-mediated heating of nanorods and nanoparticle aggregates cause either (a) transient nanopores in lipid membranes or (b) irreversible rupture of the membrane. To monitor transient opening and closing, an electrophysiological setup is assembled wherein a giant liposome is spread over a micrometer hole in a glass slide forming a single bilayer of high Ohmic resistance (so-called gigaseal), while laser light is coupled in and focused on the membrane. The energy associated with the localized heating is discussed and compared with typical elastic parameters in the lipid membranes. The method presented here provides a novel methodology for better understanding of transport across artificial or natural biological membranes.

Polyelectrolyte multilayer coatings based on poly(methacrylic acid) and poly-l-histidine were formed on anodized titanium surfaces with adsorbed bone morphogenetic protein 2 (BMP-2) or basic fibroblast growth factor (FGFb). These coatings are proposed for use on titanium implanted devices. Coatings were capable of sustained release of growth factor over 25 days, with BMP-2 and FGFb exhibiting approximately identical release profiles. Cell culture on growth factor-eluting surfaces was more effective for preosteoblasts on BMP-2-eluting surfaces than for fibroblasts on FGFb-eluting surfaces. Cell counts at all time points on BMP-2-eluting surfaces were significantly higher than for those on anodized titanium or polyelectrolyte surfaces that did not contain BMP-2. Alkaline phosphatase levels were significantly higher after 21 days on BMP-2-eluting surfaces, indicating increased bone growth.

The behavior of 8-mercaptoquinoline, which is a potential antithyroid drug toward molecular iodine was investigated. The ability of 8-mercaptoquinoline to form the outer-sphere charge-transfer complex C9H7NS·I2 with iodine molecular in dilute chloroform solution has been studied by UV/vis spectroscopy (lgβ=3.14). The crystal structure of the new salt 8-(quinoline-8-yldisulfonyl)quinolinium triiodide - product of irreversible oxidation of 8-mercaptoquinoline was determined by X-ray diffraction. Intramolecular hydrogen bond of N-H⋯N type is presented in the organic cation. The triiodide ion is the nearly centrosymmetrical anion. The 8-(quinoline-8-yldisulfanyl)quinolinium cations form dimers through π-π-stacking interaction between quinolinium rings. The reduced intramolecular interactions are observed between iodine - sulfur atoms and iodine-hydrogen atoms with shortened contacts (less of sum of van-der-waals contacts)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: Red blood cells (RBCs) and lipid-based carriers on the one hand and polymeric capsules on the other hand represent two of the most widely used carriers in drug delivery. Each class of these carriers has its own set of properties, specificity and advantages. Thorough comparative studies of such systems are reported here for the first time., Areas Covered: In this review, RBCs are described in comparison with synthetic polymeric drug delivery vehicles using polyelectrolyte multilayer capsules as an example. Lipid-based composition of the shell in the former case is particularly attractive due to their inherent biocompatibility and flexibility of the carriers. On the other hand, synthetic approaches to fabrication of polyelectrolyte multilayer capsules permit manipulation of the permeability of their shell as well as tuning their composition, mechanical properties, release methods and targeting., Expert Opinion: In conclusion, properties of RBCs and polyelectrolyte multilayer capsules are reported here highlighting similarities and differences in their preparation and applications. In addition, their advantages and disadvantages are discussed.

We report on the selective cell detachment from nanoengineered gold nanoparticle (AuNP) surfaces triggered by laser irradiation, which occurs in a nonthermal manner. The gold nanoparticle-based surfaces reveal good adhesion of NIH3T3 fibroblast cells. Patterning is achieved by lithographic microcontact printing, selective gold nanoparticle deposition, and by laser beam profiling. It is shown that the effectiveness of fibroblast cell detachment depends on the cell age, laser power, and AuNP patterning profile. Heat distribution and temperature rise around gold nanoparticle functionalized surfaces is modeled, revealing low heating of nanoparticles by laser illumination. The nonthermal photochemical mechanism of cell detachment due to production of reactive oxygen species under illumination of gold nanoparticles by green laser light is studied. We also demonstrate that cells migrate from unirradiated areas leading to their reattachment and surface recovery which is important for controlled spatial organization of cells in wound healing and tissue engineering. Research presented in this work is targeted at designing biointerfaces for cell cultures.

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.

Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface., (© 2011. Published by The Company of Biologists Ltd)

EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP), which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP) known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.

SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.

We studied the influence of the Suppressor of Underreplication (SuUR) gene expression on the intercalary heterochromatin (IH) regions of Drosophila melanogaster polytene chromosomes. We observed a strong positive correlation between increased SuUR expression, underreplication extent, amount of DNA truncation, and formation of ectopic contacts in IH regions. SuUR overexpression from heat shock-driven transgene results in the formation of partial chromosomal aberrations whose breakpoints map exclusively to the regions of intercalary and pericentric heterochromatin. It is important to note that all these effects are seen only if SuUR overexpression is induced during early stages of chromosome polytenization. Therefore, we developed the idea that ectopic pairing results from the joining of free DNA ends, which are formed as a consequence of underreplication.