Inner Ear and Muscle Developmental Defects in Smpx-Deficient Zebrafish Embryos.

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.

Phosphorylation of H3-Thr3 by Haspin Is Required for Primary Cilia Regulation.

Primary cilia are commonly found on most quiescent, terminally differentiated cells and play a major role in the regulation of the cell cycle, cell motility, sensing, and cell-cell communication. Alterations in ciliogenesis and cilia maintenance are causative of several human diseases, collectively known as ciliopathies. A key determinant of primary cilia is the histone deacetylase HDAC6, which regulates their length and resorption and whose distribution is regulated by the death inducer-obliterator 3 (Dido3). Here, we report that the atypical protein kinase Haspin is a key regulator of cilia dynamics. Cells defective in Haspin activity exhibit longer primary cilia and a strong delay in cilia resorption upon cell cycle reentry. We show that Haspin is active in quiescent cells, where it phosphorylates threonine 3 of histone H3, a known mitotic Haspin substrate. Forcing Dido3 detachment from the chromatin prevents Haspin inhibition from impacting cilia dynamics, suggesting that Haspin activity is required for the relocalization of Dido3-HDAC6 to the basal body. Exploiting the zebrafish model, we confirmed the physiological relevance of this mechanism. Our observations shed light on a novel player, Haspin, in the mechanisms that govern the determination of cilia length and the homeostasis of mature cilia.

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity.

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable-and easily accessible-tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

NF-YAl drives EMT in Claudinlow tumours.

NF-Y is a trimeric transcription factor whose binding site -the CCAAT box- is enriched in cancer-promoting genes. The regulatory subunit, the sequence-specificity conferring NF-YA, comes in two major isoforms, NF-YA long (NF-YAl) and short (NF-YAs). Extensive expression analysis in epithelial cancers determined two features: widespread overexpression and changes in NF-YAl/NF-YAs ratios (NF-YAr) in tumours with EMT features. We performed wet and in silico experiments to explore the role of the isoforms in breast -BRCA- and gastric -STAD- cancers. We generated clones of two Claudinlow BRCA lines SUM159PT and BT549 ablated of exon-3, thus shifting expression from NF-YAl to NF-YAs. Edited clones show normal growth but reduced migratory capacities in vitro and ability to metastatize in vivo. Using TCGA, including upon deconvolution of scRNA-seq data, we formalize the clinical importance of high NF-YAr, associated to EMT genes and cell populations. We derive a novel, prognostic 158 genes signature common to BRCA and STAD Claudinlow tumours. Finally, we identify splicing factors associated to high NF-YAr, validating RBFOX2 as promoting expression of NF-YAl. These data bring three relevant results: (i) the definition and clinical implications of NF-YAr and the 158 genes signature in Claudinlow tumours; (ii) genetic evidence of 28 amino acids in NF-YAl with EMT-promoting capacity; (iii) the definition of selected splicing factors associated to NF-YA isoforms.

The Pick-and-Roll in Basketball From Deep Interviews of Elite Coaches: A Mixed Method Approach From Polar Coordinate Analysis.

Pick-and-roll is the most widespread cooperative action among high-level basketball teams and the most applied strategy by coaches to gain an advantage over the rival team. During pick-and-roll, opposing teams perform antagonistic actions based on goals that are expressed in offensive and defensive tactics. The aim of this study is to examine the approaches of high-level coaches on the offensive and defensive dynamics emerging in matches of a basketball elite team during an entire season of the Spanish Asociación de Clubes de Baloncesto (ACB) league. To this end, we used a mixed-methods approach based on systematic observation of verbatim transcripts of interviews conducted with six high-level coaches about the pick-and-roll dynamics that emerged in matches of the Unicaja Málaga team during an entire season of the ACB league. The observational design was nomothetic, punctual, and multidimensional. The choice of this methodology is justified since we developed an ad hoc indirect observation tool to evaluate the coaches' perspective on this dynamic. Once the intra-observer reliability of the instrument was confirmed, we performed a polar coordinate analysis to identify the significant relationships between the coaches' evaluations and the offensive and defensive pick-and-roll elements that supported such verbal behaviors. The results highlight the presence of various offensive and defensive aspects of pick-and-roll (n = 2224) emerging in the Unicaja team that were significantly associated with positive and negative evaluations of the coaches. The interview confirms that coach 1 and his staff were less confident in options that pick-and-roll offer, which is also reflected in the record of screens made and simulated, than coach 3. This study shows that the application of mixed methods, by analysis of the polar coordinate of the coding carried out on responses of a systematized interview, has proven to be an effective strategy in obtaining relevant information on the expert knowledge of the elite coaches on the influence of pick-and-roll on tactical actions in basketball.

The zebrafish (Danio rerio) embryo-larval contact assay combined with biochemical biomarkers and swimming performance in sewage sludge and hydrochar hazard assessment.

Hydrothermal carbonization is considered a powerful technology to convert sewage sludge (SS) into a valuable carbonaceous solid known as hydrochar (HC). Up to now criteria for landfill application of SS and HC are based only on physicochemical properties and levels of pollutant residues. Nevertheless, to ensure their safe environmental applications it is mandatory to develop biosensors which can provide relevant information on their toxic potential for natural ecosystems. Therefore, this study aimed to assess the suitability of a contact assay using zebrafish embryo/larvae combined with sub-lethal end-points to evaluate the hazard associated with SS and related HC exposure. A suite of biomarkers was also applied on larvae, related to detoxification and oxidative stress as the activity of Ethoxyresorufin-O-deethylase, glutathione-S-transferase, and catalase, the content of reactive oxygen species and the behavioral assay using the DanioVision™ chamber. Legacy priority pollutants were also measured either in SS and HC tested samples and in contact waters. The exposure to SS caused higher lethality compared to HC. No significant changes in the activity of oxidative stress markers was observed upon exposure to both matrices. The behavioral test showed a hypoactivity condition in larvae exposed to both SS and HC with the effects of SS stronger than HC. Chemical analysis revealed the presence of trace elements and halogenated compounds in either SS, HC. Heavy metals were also released in contact waters, while volatile hydrocarbons (C6-C10) and halogenated compounds resulted below LOD (<0.05 µ L-1). Our study highlights the suitability of zebrafish embryotoxicity test, coupled with behavioral traits, as screening tool for assessing potential risks, associated with the landfill application of both SS and HC, for aquatic wildlife.

The Action of Verbal and Non-verbal Communication in the Therapeutic Alliance Construction: A Mixed Methods Approach to Assess the Initial Interactions With Depressed Patients.

In psychodynamic psychotherapy, verbal (structures and intents) and non-verbal (voice and interruptions) dimensions of communication intertwine conveying information and determining the mutual regulation between therapist and patient through conversational sequences. The communication components interplay is the foundation for building the therapeutic alliance, a relational dimension that predicts a psychotherapy outcome and change, influenced by patient-therapist exchanges from the initial stages of their encounter. Depressed patients present specific verbal and non-verbal communication and show difficulties in developing and maintaining the therapeutic alliance. Based on the reviewed literature, the main aim of this study was to analyze how the action of specific communicative modes, implemented by the therapist and depressed patients, affect the reciprocal construction of the early therapeutic alliance by each participant during the mutual regulation processes. We employed a mixed methods approach based on a systematic observation of communication and alliance ruptures and repairs within the audio recordings and verbatim transcripts of 20 psychotherapy sessions (6,232 speaking turns) with seven depressed patients. The observational design was nomothetic, follow-up, and multidimensional. The choice of methodology is justified because we developed a comprehensive procedure that integrates an ad hoc indirect observation system (the Communicative Modes Analysis System in Psychotherapy), analyzing verbal and non-verbal communication, and an observational tool with deductive categories (the Collaborative Interactions Scale-Revised), assessing the therapeutic alliance construction. Once we confirmed the intra-and inter-observer reliability for the ad hoc system and the inter-rater reliability for the tool with deductive (or theoretical) categories, we performed descriptive statistics (to describe quantitatively communicative modes and alliance ruptures and repairs), lag sequential analysis (to detect stable patterns in communication-alliance interactions), and polar coordinate analysis (to identify significant relationships between communicative modes and alliance ruptures and repairs). Results confirm that the therapist's verbal (asking and exploring) and non-verbal (elaborating and cooperatively interrupting) modes and the depressed patients' verbal (asserting and exploring) and non-verbal (expressing emotions and cooperatively interrupting) modes determine stable patterns and significant associations with collaborative behaviors connected to the reciprocal construction of alliance by each participant. All this may provide professionals with useful information to increase the psychotherapy effectiveness with depressed patients.

Expression pattern of the small muscle protein, X-linked (smpx) gene during zebrafish embryonic and larval developmental stages.

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in both cardiac and skeletal muscles, as well as in fetal inner ears, with suggested roles as mechanotransductor. Recently, several mutations in the SMPX gene have been associated with X-chromosomal progressive deafness in human. However, very little information is known concerning the roles of SMPX, and no in-vivo models are currently available. Therefore, we characterized the zebrafish ortholog of SMPX to pave the way towards the establishment of a biotool for future functional studies. Despite the genome duplication occurred in the ancestry of teleosts, zebrafish retain only one copy of smpx which shares a high degree of similarity with the mammalian counterpart in terms of genomic organization, syntenic map, and encoded protein. RT-PCR, as well as whole-mount in-situ hybridization and immunofluorescence analyses, revealed that smpx is expressed in several embryonic areas starting from the 4-somite stage. Specifically, smpx mRNA marked the Kupffer's vesicle (KV), the somites, the myocardium, the hair cells of the anterior and the posterior macula of the inner ear, the pronephric ducts, and the muscles of the branchial arches, eyes and pectoral fins. According to our data, zebrafish smpx expression pattern closely resembles that observed in mouse and human, supporting the notion that zebrafish might represent a suitable in-vivo model to disclose the cellular and molecular mechanisms underlying the involvement of SMPX in development and disease.

The Communicative Modes Analysis System in Psychotherapy From Mixed Methods Framework: Introducing a New Observation System for Classifying Verbal and Non-verbal Communication.

Communication represents the core of psychotherapy. The dynamic interaction between verbal and non-verbal components during patient-therapist exchanges, indeed, promotes the co-construction of meanings bringing about change within a process of reciprocal influence of participants. Our paper aims to illustrate the building of a new observational instrument of the therapeutic discourse, the Communicative Modes Analysis System in Psychotherapy (CMASP), and its reliability study from Mixed Methods framework. The CMASP is a single classification system analyzing the communication features within therapeutic exchanges. Born to overcome the limits of traditional psychotherapy research which considers verbal and non-verbal dimensions of communication as in polar opposition, the CMASP building was based on the performative function derived from the Speech Act Theory. We used this function as a comprehensive theorization to interpret the communication components in psychotherapy as an integrated and interacting system. In fact, the instrument detects and classifies, at the overall and dimension level, the verbal and extra-linguistic components of psychotherapeutic communication implemented by the therapist and patients in the form of communicative modes. From the observational methodology framework, it was built an instrument able to record and analyze verbal, vocal and interruption behaviors by combining elements of qualitative and quantitative research approaches. The sample consisted of 30 psychotherapy audio recordings and verbatim transcripts of psychotherapy sessions (for a total of 8327 speaking turns). Four main dimensions were elaborated (Verbal Mode-Structural Form, Verbal Mode-Communicative Intent, Vocal Mode, and Interruption Mode) according to the agency role of communication components. The instrument is a field format combined with category systems. For each dimension, we built a category system that is exhaustive and mutually exclusive. From all dimensions, we have a total of 33 categories. Intra-and inter-judge reliability among four independent judges was computed on a total of 503 speaking turns coded through Cohen's κ and Krippendorff's canonical agreement coefficients (Cc), respectively. The CMASP showed high intra-and inter-judge agreement at the global, dimensional, and categorical level providing researchers and professionals with a single and flexible classification system, able to give multiple and concurrent information about the psychotherapy process.

Environmental concentration of fluoxetine disturbs larvae behavior and increases the defense response at molecular level in zebrafish (Danio rerio).

Fluoxetine (FLX) is one of the main antidepressants used worldwide. After human use, FLX enters the aquatic ecosystems, where it has commonly detected in the high ng/L concentration range. Several investigations have shown that exposure to different concentrations of FLX caused different adverse effects towards a number of aquatic species. However, the information on the onset and the relationship between molecular and behavioral FLX-induced effects remains scant. The aim of this study was to assess the effects induced by two FLX concentrations, namely 50 ng/L and 500 ng/L, on swimming activity of zebrafish (Danio rerio) larvae at 96-h post-fertilization (hpf) and to investigate if such behavioral effects were related to modulation of the expression of oxidative stress-related (sod1, sod2, cat, gpxa, and gst), stress- and anxiety-related (oxtl, prl2, npy, and ucn3l) genes, and genes encoding for the transporters of the main neurotransmitters (slc6a3, slc6a4a, slc6a4b, slc6a11). Fluoxetine exposure altered the swimming behavior of larvae, as shown by the reduction of the distance traveled by treated larvae in response to an external stimulus. Such behavioral change was related, at molecular level, to an enhanced expression of sod1, cat, and gpxa, suggesting an overproduction of pro-oxidant molecules. In addition, FLX modulated the expression of oxtl, slc6a4a, slc6a4b, and slc6a11, suggesting its capability to affect anxiety- and neurotransmitter-related genes.

Environmental concentrations of triclosan activate cellular defence mechanism and generate cytotoxicity on zebrafish (Danio rerio) embryos.

Triclosan (TCS, 5­chloro­2­(2,4­dichlorophenoxy) phenol) is becoming a major surface waters pollutant worldwide at concentrations ranging from ng L-1 to µg L-1. Up to now, the adverse effects on aquatic organisms have been investigated at concentrations higher than the environmental ones, and the pathways underlying the observed toxicity are still not completely understood. Therefore, the aim of this study was to investigate the toxic effects of TCS at environmental concentrations on zebrafish embryos up to 120 hours post fertilization (hpf). The experimental design was planned considering both the quantity and the exposure time for the effects on the embryos, exposing them to two different concentrations (0.1 µg L-1, 1 µg L-1) of TCS, for 24 h (from 96 to 120 hpf) and for 120 h (from 0 to 120 hpf). A suite of biomarkers was applied to measure the induction of embryos defence system, the possible increase of oxidative stress and the DNA damage. We measured the activity of glutathione­S­transferase (GST), P­glycoprotein efflux and ethoxyresorufin­o­deethylase (EROD), the level of ROS, the oxidative damage through the Protein Carbonyl Content (PCC) and the activity of antioxidant enzymes. The genetic damage was evaluated through DNA Diffusion Assay, Micronucleus test (MN test), and Comet test. The results showed a clear response of embryos defence mechanism, through the induction of P-gp efflux functionality and the activity of detoxifying/antioxidant enzymes, preventing the onset of oxidative damage. Moreover, the significant increase of cell necrosis highlighted a strong cytotoxic potential for TCS. The overall results obtained with environmental concentrations and both exposure time, underline the critical risk associated to the presence of TCS in the aquatic environment.

Evaluation of the infiltration of polystyrene nanobeads in zebrafish embryo tissues after short-term exposure and the related biochemical and behavioural effects.

One of the current main challenges faced by the scientific community is concerning the fate and toxicity of plastics, due to both the well-known threats made by larger plastic items spreading in ecosystems and their fragmentation into micro- and nanoparticles. Since the chemical and physical characteristics of these smaller plastic fragments are markedly different with respect to their bulk product, the potential toxicological effects in the environment need to be deeply investigated. To partially fill this gap of knowledge, the aim of this study was to evaluate the polystyrene nanobead intake in the tissues of zebrafish (Danio rerio) embryos and their related toxicity. Embryos at 72 h post fertilization (hpf) were exposed for 48 h to 0.5 µm fluorescent polystyrene nanobeads at a concentration of 1 mg L-1. Confocal microscopy was employed to investigate nanoplastic ingestion and tissue infiltration, while potential sub-lethal effects were evaluated by measuring several endpoints, which covered the adverse effects at the molecular (protein carbonylation), cellular (P-glycoprotein, activity of several antioxidant/detoxifying enzymes) and organism levels by evaluating of possible changes in the embryos' swimming behaviour. Imaging observations clearly highlighted the nanoplastics' uptake, showing nanobeads not only in the digestive tract, but also migrating to other tissues through the gut epithelium. Biomarker analyses revealed a significant decrease in cyclooxygenase activity and an induction of superoxide dismutase. The behavioural test highlighted a significant (p < 0.05) variation in the turn angle between the control and exposed embryos. This study points out the capability of nanoplastics to infiltrate zebrafish embryo tissues, even after a short exposure, thus suggesting the need for deeper investigations following longer exposure times, and highlighting the potential of nanoplastics to cause toxicological effects on freshwater organisms, at the organism level.

Crucial role of zebrafish prox1 in hypothalamic catecholaminergic neurons development.

BACKGROUND: Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development. RESULTS: Here we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium. CONCLUSION: Our findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.

Exposure to cocaine and its main metabolites altered the protein profile of zebrafish embryos.

Illicit drugs have been identified as emerging aquatic pollutants because of their widespread presence in freshwaters and potential toxicity towards aquatic organisms. Among illicit drug residues, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly detected in freshwaters worldwide at concentration that can induce diverse adverse effects to non-target organisms. However, the information of toxicity and mechanisms of action (MoA) of these drugs, mainly of COC metabolites, to aquatic species is still fragmentary and inadequate. Thus, this study was aimed at investigating the toxicity of two concentrations (0.3 and 1.0 µg/L) of COC, BE and EME similar to those found in aquatic ecosystems on zebrafish (Danio rerio) embryos at 96 h post fertilization through a functional proteomics approach. Exposure to COC and both its metabolites significantly altered the protein profile of zebrafish embryos, modulating the expression of diverse proteins belonging to different functional classes, including cytoskeleton, eye constituents, lipid transport, lipid and energy metabolism, and stress response. Expression of vitellogenins and crystallins was modulated by COC and both its main metabolites, while only BE and EME altered proteins related to lipid and energy metabolism, as well as to oxidative stress response. Our data confirmed the potential toxicity of low concentrations of COC, BE and EME, and helped to shed light on their MoA on an aquatic vertebrate during early developmental period.

The interactions of fullerene C60 and Benzo(α)pyrene influence their bioavailability and toxicity to zebrafish embryos.

This study aimed to assess the toxicological consequences related to the interaction of fullerene nanoparticles (C60) and Benzo(α)pyrene (B(α)P) on zebrafish embryos, which were exposed to C60 and B(α)P alone and to C60 doped with B(α)P. The uptake of pollutants into their tissues and intra-cellular localization were investigated by immunofluorescence and electron microscopy. A set of biomarkers of genotoxicity and oxidative stress, as well as functional proteomics analysis were applied to assess the toxic effects due to C60 interaction with B(α)P. The carrier role of C60 for B(α)P was observed, however adsorption on C60 did not affect the accumulation and localization of B(α)P in the embryos. Instead, C60 doped with B(α)P resulted more prone to sedimentation and less bioavailable for the embryos compared to C60 alone. As for toxicity, our results suggested that C60 alone elicited oxidative stress in embryos and a down-regulation of proteins involved in energetic metabolism. The C60 + B(α)P induced cellular response mechanisms similar to B(α)P alone, but generating greater cellular damages in the exposed embryos.

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Environmental concentrations of cocaine and its main metabolites modulated antioxidant response and caused cyto-genotoxic effects in zebrafish embryo cells.

Illicit drugs have been recently identified as a serious environmental problem because of the growing evidence regarding their occurrence in aquatic environment and potential toxicity towards non-target organisms. Among them, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly measured in freshwaters worldwide at levels that might cause diverse sub-lethal effects to aquatic organisms. Thus, the present study was aimed at investigating the potential adverse effects induced by the exposure to environmental concentrations (0.04, 0.4, 4 and 40 nM) of COC, BE, and EME on zebrafish (Danio rerio) embryos at 96 h post fertilization. Cytotoxicity was assessed by the Trypan Blue exclusion method, while primary and fixed genetic damages were evaluated by the Single Cell Gel Electrophoresis (SCGE) assay, and the DNA diffusion assay together with the Micronucleus test, respectively. The involvement of oxidative stress in the mechanism of action (MoA) of all tested drugs was assessed by measuring the activity of defense enzymes (SOD, CAT, GPx, and GST) and the expression of their encoding genes. Exposure to COC and both metabolites significantly reduced cell viability, increased DNA fragmentation and promoted the onset of apoptotic cells and micronuclei in zebrafish embryos. Results from oxidative stress-related endpoints and gene expression suggested that the observed genotoxicity may be caused by an overproduction of free radicals that imbalanced the oxidative status of embryos. The integration of biomarker responses into a synthetic index showed that at each tested concentration, BE and EME had a similar toxicity and were both more toxic than COC. Our data confirmed the potential toxicity of environmental concentrations of COC, BE, and EME, suggesting the need of further in-depth studies to shed light on their MoA and long-term toxicity towards non-target aquatic species.

Differential regulation of the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons.

BACKGROUND: The homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development. RESULTS: Here, we identified two otp orthologues in zebrafish (otp1 and otp2) and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO) (anterior alar plate) and the posterior tuberculum (PT) (posterior basal plate). The latter structure is characterized by Tyrosine Hydroxylase (TH)-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA) neurons. Disruptions of Hedgehog (HH) and Fibroblast Growth Factor (FGF) pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF. CONCLUSION: In this study, we pinpoint the evolutionary importance of otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates.

Intronless WNT10B-short variant underlies new recurrent allele-specific rearrangement in acute myeloid leukaemia.

Defects in the control of Wnt signaling have emerged as a recurrent mechanism involved in cancer pathogenesis and acute myeloid leukaemia (AML), including the hematopoietic regeneration-associated WNT10B in AC133bright leukaemia cells, although the existence of a specific mechanism remains unproven. We have obtained evidences for a recurrent rearrangement, which involved the WNT10B locus (WNT10BR) within intron 1 (IVS1) and flanked at the 5' by non-human sequences whose origin remains to be elucidated; it also expressed a transcript variant (WNT10BIVS1) which was mainly detected in a cohort of patients with intermediate/unfavorable risk AML. We also identified in two separate cases, affected by AML and breast cancer respectively, a genomic transposable short form of human WNT10B (ht-WNT10B). The intronless ht-WNT10B resembles a long non-coding RNA (lncRNA), which suggests its involvement in a non-random microhomology-mediated recombination generating the rearranged WNT10BR. Furthermore, our studies supports an autocrine activation primed by the formation of WNT10B-FZD4/5 complexes in the breast cancer MCF7 cells that express the WNT10BIVS1. Chemical interference of WNT-ligands production by the porcupine inhibitor IWP-2 achieved a dose-dependent suppression of the WNT10B-FZD4/5 interactions. These results present the first evidence for a recurrent rearrangement promoted by a mobile ht-WNT10B oncogene, as a relevant mechanism for Wnt involvement in human cancer.