Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

Effects of Shrimp Shell-Derived Chitosan on Growth, Immunity, Intestinal Morphology, and Gene Expression of Nile Tilapia (Oreochromis niloticus) Reared in a Biofloc System.

Chitosan (CH) shows great potential as an immunostimulatory feed additive in aquaculture. This study evaluates the effects of varying dietary CH levels on the growth, immunity, intestinal morphology, and antioxidant status of Nile tilapia (Oreochromis niloticus) reared in a biofloc system. Tilapia fingerlings (mean weight 13.54 ± 0.05 g) were fed diets supplemented with 0 (CH0), 5 (CH5), 10 (CH10), 20 (CH20), and 40 (CH40) mL·kg-1 of CH for 8 weeks. Parameters were assessed after 4 and 8 weeks. Their final weight was not affected by CH supplementation, but CH at 10 mL·kg-1 significantly improved weight gain (WG) and specific growth rate (SGR) compared to the control (p < 0.05) at 8 weeks. Skin mucus lysozyme and peroxidase activities were lower in the chitosan-treated groups at weeks 4 and 8. Intestinal villi length and width were enhanced by 10 and 20 mL·kg-1 CH compared to the control. However, 40 mL·kg-1 CH caused detrimental impacts on the villi and muscular layer. CH supplementation, especially 5-10 mL·kg-1, increased liver and intestinal expressions of interleukin 1 (IL-1), interleukin 8 (IL-8), LPS-binding protein (LBP), glutathione reductase (GSR), glutathione peroxidase (GPX), and glutathione S-transferase (GST-α) compared to the control group. Overall, dietary CH at 10 mL·kg-1 can effectively promote growth, intestinal morphology, innate immunity, and antioxidant capacity in Nile tilapia fingerlings reared in biofloc systems.

Zebrafish xenograft as a tool for the study of colorectal cancer: a review.

Colorectal cancer (CRC) is the second leading cause of cancer-related death, mostly due to metastatic disease and the fact that many patients already show signs of metastasis at the time of first diagnosis. Current CRC therapies negatively impact patients' quality of life and have little to no effect on combating the tumor once the dissemination has started. Danio rerio (zebrafish) is a popular animal model utilized in cancer research. One of its main advantages is the ease of xenograft transplantation due to the fact that zebrafish larvae lack the adaptative immune system, guaranteeing the impossibility of rejection. In this review, we have presented the many works that choose zebrafish xenograft as a tool for the study of CRC, highlighting the methods used as well as the promising new therapeutic molecules that have been identified due to this animal model.

Migraine chronification as an allostatic disorder: a proof-of-concept study.

OBJECTIVE: The underpinning biologics of migraine chronification are not well understood. We aim to investigate the role of the cumulative burden of stress, namely the allostatic load, in migraine chronification. METHODS: This was a cross-sectional study. The allostatic load was measured with a composite multi-system score (BALI: Bologna Allostatic Load Index), evaluating 20 biomarkers representing four physiological systems: immune, metabolic, cardiovascular, and neuroendocrinological systems. BALI score was subdivided into high score and low score based on the distribution in controls. Migraine patients were included and subclassified into low-frequency episodic migraine group (low-EM group), high-frequency episodic migraine group (high-EM group), and chronic migraine group (CM group). RESULTS: The distribution of BALI high-score increased in parallel with headache attacks monthly frequency: 16% in low-EM group (n = 10), 24% in high-EM group (n = 12), and 40% in CM group (n = 21) (p = 0.017). In a multivariable analysis, the odds ratio of having a high-score BALI in CM patients (vs. low-EM patients) was 2.78 (95% CI 1.07-7.22; p = 0.036). Individual BALI biomarkers values which were significantly different among migraine subgroups included systolic blood pressure (p = 0.018), diastolic blood pressure (p < 0.001), and heart rate (p = 0.019). CONCLUSION: Our study substantiates this emerging concept of migraine chronification as an allostatic disorder.

To be born twin: effects on long-term neurodevelopment of very preterm infants-a cohort study.

Objective: To examine the effect of twin birth on long-term neurodevelopmental outcomes in a cohort of Italian preterm infants with very low birth weight. Study design: We performed a retrospective cohort study on children born in a tertiary care centre. We included children born between 1 January 2007 and 31 December 2013 with a gestational age (GA) of ≤32 weeks and birth weight of <1,500 g. The infants born from twin pregnancies complicated by twin-to-twin transfusion syndrome and from higher-order multiple pregnancies were excluded. The children were evaluated both at 2 years corrected age and 5 years chronological age with Griffiths mental development scales revised (GMDS-R). The linear mixed effects models were used to study the effect of being a twin vs. being a singleton on GMDS-R scores, adjusting for GA, being born small for gestational age, sex, length of NICU stay, socio-economic status, and comorbidity score (CS) calculated as the sum of the weights associated with each of the major morbidities of the infants. Results: A total of 301 children were included in the study, of which 189 (62.8%) were singletons and 112 (37.2%) were twins; 23 out of 112 twins were monochorionic (MC). No statistically significant differences were observed between twins and singletons in terms of mean general quotient and subscales at both 2 and 5 years. No effect of chorionicity was found when comparing scores of MC and dichorionic twins vs. singletons; however, after adjusting for the CS, the MC twins showed lower scores in the hearing and language and performance subscales at 5 years. Conclusion: Overall, in our cohort of children born very preterm, twin infants were not at higher risk of neurodevelopmental impairment compared with singletons at pre-school age.

In-hospital growth and long-term neurodevelopmental outcomes of very low birth weight infants.

Background and Objectives: Very low birth weight infants (VLBW) are at risk for adverse growth and neurodevelopmental outcomes. We aimed to evaluate the association between growth during Neonatal Intensive Care Unit (NICU) stay and long-term neurodevelopmental outcomes in a cohort of preterm VLBW newborns. Methods: We conducted a longitudinal observational study in the Follow-up Service of our Clinic from January 2014 to April 2017. All preterm VLBW infants born at our hospital and enrolled in our follow-up program were considered eligible for the study. The neurodevelopmental assessment was performed using the Griffiths Mental Development Scales at 12 and 24 months corrected age. Results: Study population included 172 subjects (47.1% males) with a mean gestational age of 29 weeks and a mean birth weight of 1,117 g. A unitarian Δz-score increase in head circumference from birth to discharge was associated with a 1.6-point increase in General Quotient at 24 months corrected age. An association with subscales C and D was also found. Likewise, an increase in length Δz-score was associated with better 24-month subscale C scores although not reaching statistical significance. No relationship with the outcome at 24 months was found for weight gain. Conclusions: Growth during NICU stay appears to be related to a more favorable neurodevelopmental outcome at 24 months corrected age, especially in the hearing and language domain (subscale C). The longitudinal evaluation of auxological parameters during hospitalization can contribute to the identification of subjects at risk for adverse neurodevelopmental outcomes in the first years of life.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Quantity discrimination in newly hatched zebrafish suggests hardwired numerical abilities.

An intriguing hypothesis to explain the ubiquity of numerical abilities is that all vertebrates are born with hardwired neuronal networks for processing numbers. To date, only studies on human foetuses have clearly supported this hypothesis. Zebrafish hatch 48-72 h after fertilisation with an embryonic nervous system, providing a unique opportunity for investigating this hypothesis. Here, we demonstrated that zebrafish larvae exposed to vertical bars at birth acquired an attraction for bar stimuli and we developed a numerical discrimination task based on this preference. When tested with a series of discriminations of increasing difficulty (1vs.4, 1vs.3, 1vs.2, and 2vs.4 bars), zebrafish larvae reliably selected the greater numerosity. The preference was significant when stimuli were matched for surface area, luminance, density, and convex hull, thereby suggesting a true capacity to process numerical information. Converging results from two phylogenetically distant species suggests that numerical abilities might be a hallmark feature of vertebrates' brains.

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.

Cognitive and Behavioral Development of 9-Year-Old Children After Maternal Cancer During Pregnancy: A Prospective Multicenter Cohort Study.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.This multicenter cohort study reports on the long-term effects of prenatal exposure to maternal cancer and its treatment on cognitive and behavioral outcomes in 9-year-old children. In total, 151 children (mean age, 9.3 years; range, 7.8-10.6 years) were assessed using a neurocognitive test battery and parent-report behavioral questionnaires. During pregnancy, 109 children (72.2%) were exposed to chemotherapy (only or in combination with other treatment modalities), 18 (11.9%) to surgery only, 16 (10.6%) to radiotherapy, one to trastuzumab, and 16 (10.6%) were not exposed to oncologic treatment. Mean cognitive and behavioral outcomes were within normal ranges. Gestational age at birth showed a positive association with Full Scale Intelligence Quotient (FSIQ), with the average FSIQ score increasing by 1.6 points for each week increase in gestational age (95% CI, 0.7 to 2.5; P < .001). No difference in FSIQ was found between treatment types (F[4,140] = 0.45, P = .776). In children prenatally exposed to chemotherapy, no associations were found between FSIQ and chemotherapeutic agent, exposure level, or timing during pregnancy. These results indicate a reassuring follow-up during the critical maturational period of late childhood, when complex functions develop and rely on the integrity of early brain development. However, associations were observed with preterm birth, maternal death, and maternal education.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer.

Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide's (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated. Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation. To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells. AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA's beneficial effects. In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies.

Development of a new scoring method in the neurofunctional assessment of preterm infants.

Infants born preterm are at high risk of presenting neurodevelopmental delay. The Neurofunctional Assessment (NFA) describes infants' neurodevelopment through the evaluation of six different domains. This study aimed to evaluate how, in a cohort of preterm infants, each NFA domain assessed at 3 months of corrected age (CA) was associated with neurodevelopment at 2 years of CA using the Griffiths Mental Developmental Scales Extended Revised (GMDS-ER). In addition, by introducing the NFA complexity score (CS), the study aimed to define a threshold that can help clinicians discriminate infants at higher risk of later neurodevelopmental delay. We conducted an observational, longitudinal study including 211 preterm infants. At 3 months of CA, infants who had normal scores in each domain showed a significantly higher GMDS-ER global quotient (GQ) at 2 years of CA. In addition, linear model results showed a significant negative relationship between the NFA CS and 2-year GMDS-ER GQ (estimate: - 0.27; 95% CI - 0.35, - 0.20; p value < 0.001). Each 10-point increase in the NFA CS was associated with an average 2.7-point decrease in the GMDS GQ. These results highlight how the NFA domains and NFA CS are compelling instruments for the early identification of children at risk for long-term adverse outcomes.

Serotonin transporter gene methylation and emotional regulation in preschool children born preterm: A longitudinal evaluation of the role of negative emotionality in infancy.

The aim of the study was to assess the contribution of negative emotionality at 3 months (T1) and serotonin transporter gene (SLC6A4) DNA methylation at 4.5 years of age (T2) to emotion regulation in pre-schoolers born very preterm and full-term. Forty one children (n = 21 born very preterm, n = 20 born full-term) participated in the study. Fretful behavior was assessed at T1 in response to the Face-to-FaceStill-Face (FFSF) paradigm. At T2, SLC6A4 DNA methylation was analyzed and emotion regulation was assessed using an observational procedure (i.e., the Pre-schooler Regulation of Emotional Stress, PRES). The very preterm group displayed higher emotion dysregulation during the PRES Reactivity phase than the full-term group. Higher levels of fretful behavior at 3 months were associated with greater emotional distress only for very preterm children with higher methylation at T2. No significant associations emerged in the full-term group. Despite current findings cannot be generalized owing to the relatively small sample size, this work provides preliminary longitudinal evidence about the link between negative emotionality during infancy, stress-linked epigenetic status at 4.5 years and emotion dysregulation in preschoolers born preterm.

El propósito del estudio fue evaluar la contribución de la emocionalidad negativa a los 3 meses (T1) y la metilación del ADN en el gen transportador de la serotonina (SLC6A4) a los 4 años y medio de edad (T2) a la regulación de la emoción en prescolares nacidos muy antes de la gestación completa o de gestación completa. Cuarenta y un niños (n = 21 nacidos muy antes de la gestación completa, n = 20 nacidos de gestación completa) participaron en el estudio. El comportamiento irritable se evaluó a T1 como respuesta al Cara-a-Cara del paradigma de la Cara Inmóvil (FFSF). A T2, se analizó la metilación de ADN SLC6A4 y se evaluó la regulación de la emoción usando un procedimiento de observación (v.g. La Regulación del Estrés Emocional del Prescolar, PRES). El grupo nacido muy antes de la gestación completa mostró una más alta desregulación durante la fase de Reactividad PRES que el grupo nacido de gestación completa. Los niveles más altos de comportamiento irritable a los 3 meses se asociaron con una mayor angustia emocional solamente para los niños nacidos muy antes de la gestación completa con más alta metilación al T2. Ninguna asociación significativa surgió del grupo nacido de gestación completa. A pesar de que los actuales resultados no se pueden generalizar debido al tamaño relativamente pequeño del grupo muestra, este trabajo ofrece aporta evidencia longitudinal preliminar acerca de la conexión entre la emocionalidad negativa durante la infancia, el estado epigenético relacionado con el estrés a los 4 años y medio y la desregulación de la emoción en prescolares nacidos antes de la completa gestación.

Le but de cette étude était d'évaluer la contribution de l'émotivité négative à 3 mois (T1) et du gène vecteur de la sérotonine (SLC6A4) méthylation de l'ADN à l'âge de 4,5 ans (T2) à la régulation de l'émotion chez les enfants d'âge préscolaire nés très prématurés et à plein terme. Quarante et un enfant (n = 21 nés très prématurés, n = 20 nés à plein terme) ont participé à l'étude. Le comportement agité a été évalué au T1 en réponse au paradigme face-à-face visage inexpressif (abrégé FFSF en anglais). Au T2, la méthylation de l'ADN SLC6A4 a été analysée et la régulation de l'émotion a été évaluée en utilisant un protocole d'observation (à savoir, la Régulation du Stress Emotionnel de l'Enfant d'Age Préscolaire, abrégé en anglais PRES). Le groupe très prématuré a fait état d'une dysrégulation de l'émotion plus élevée durant la phase de Réactivité PRES que le groupe né à plein terme. Des niveaux plus élevés de comportement agité à 3 mois étaient liés à une détresse émotionnelle plus grande uniquement pour les enfants très prématurés avec une méthylation plus élevée au T2. Aucune association importante n'a émergé dans le groupe à plein terme. En dépit du fait que les résultats actuels ne peuvent pas être généralisés à cause de la taille relativement petite de l'échantillon, ce travail offre des preuves longitudinales préliminaires sur le lien entre l'émotivité négative durant la petite enfant, le statut épigénétique lié au stress à 4,5 ans et la dysrégulation de l'émotion chez les enfants d'âge préscolaires nés avant terme.

epg5 knockout leads to the impairment of reproductive success and courtship behaviour in a zebrafish model of autophagy-related diseases.

BACKGROUND: Dysregulation of the autophagic flux is linked to a wide array of human diseases, and recent findings highlighted the central role of autophagy in reproduction, as well as an association between impairment of autophagy and behavioural disorders. Here we deepened on the possible multilevel link between impairment of the autophagic processes and reproduction at both the physiological and the behavioural level in a zebrafish mutant model. METHODS: Using a KO epg5 zebrafish line we analysed male breeding success, fertility rate, offspring survival, ejaculate quality, sperm and testes morphology, and courtship behaviour. To this aim physiological, histological, ultrastructural and behavioural analyses on epg5+/+ and mutant epg5-/- males coupled to WT females were applied. RESULTS: We observed an impairment of male reproductive performance in mutant epg5-/- males that showed a lower breeding success with a reduced mean number of eggs spawned by their WT female partners. The spermatogenesis and the ability to produce fertilising ejaculates were not drastically impaired in our mutant males, whereas we observed a reduction of their courtship behaviour that might contribute to explain their lower overall reproductive success. CONCLUSION: Collectively our findings corroborate the hypothesis of a multilevel link between the autophagic process and reproduction. Moreover, by giving a first glimpse on behavioural disorders associated to epg5 KO in model zebrafish, our results open the way to more extensive behavioural analyses, also beyond the reproductive events, that might serve as new tools for the molecular screening of autophagy-related multisystemic and neurodegenerative diseases.

Learning and visual discrimination in newly hatched zebrafish.

With the exception of humans, early cognitive development has been thoroughly investigated only in precocial species, well developed at birth and with a broad behavioral and cognitive repertoire. We investigated another highly altricial species, the zebrafish, Danio rerio, whose embryonic development is very rapid (< 72 h). The hatchlings' nervous system is poorly developed, and their cognitive capacities are largely unknown. Larvae trained at 8 days post fertilization rapidly learned to associate a visual pattern with a food reward, showing significant performance at 10 days post fertilization. We exploited this ability to study hatchlings' discrimination learning capacities. Larvae rapidly and accurately learned color and shape discriminations. They also discriminated a figure from its mirror image and from its 90°-rotated version, although with lower performance. Our study revealed impressive similarities in learning and visual discrimination capacities between newborn and adult zebrafish, despite their enormous differences in brain size and degree of development.

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

Neuroimaging appearance of hypothalamic hamartomas in monozygotic twins with Pallister-Hall syndrome: case report and review of the literature.

BACKGROUND: Pallister-Hall syndrome (OMIM #146510) is a rare autosomal dominant condition caused by a mutation in the GLI3 gene. The cardinal feature of Pallister-Hall syndrome is the presence of hypothalamic hamartomas, which may manifest with seizures, panhypopituitarism and visual impairment. In Pallister-Hall syndrome, dysplastic histogenetic processes responsible for hypothalamic hamartomas are thought to disrupt early craniofacial development. The clinical presentation of Pallister-Hall syndrome may include: characteristic facies (low-set and posteriorly angulated ears, short nose with flat nasal bridge), cleft palate and uvula, bifid epiglottis and laryngotracheal cleft, limb anomalies (e.g., polysyndactyly, short limbs and nail dysplasia), anal atresia, genitourinary abnormalities and congenital heart defects. CASE PRESENTATION: We report the case of two monochorionic diamniotic twins diagnosed with Pallister-Hall syndrome during the neonatal period, after the identification of a hypothalamic hamartoma on day 1 by cerebral ultrasound scan, later confirmed by brain magnetic resonance imaging. Cerebral ultrasound and magnetic resonance imaging presentations were identical in both twins. DISCUSSION AND CONCLUSIONS: We review previously published cases (four reports) of hypothalamic hamartomas identified via cerebral ultrasound and compare reported ultrasonographic features. Main differential diagnoses based on cerebral ultrasound findings are discussed. Full description of typical magnetic resonance imaging appearance is also provided. This is the first case reported in the literature of monochorionic diamniotic twins affected by genetically confirmed Pallister-Hall syndrome with identical hypothalamic hamartomas at cerebral ultrasound and magnetic resonance imaging. Moreover, this paper adds to the existing literature on the sonographic appearance of hypothalamic hamartomas. Considering the consistency in hypothalamic hamartomas' sonographic appearance, we support the use of cerebral ultrasound as a first-line neuroimaging modality in case of clinical suspicion of Pallister-Hall syndrome.

Red blood cell transfusions in preterm newborns and neurodevelopmental outcomes at 2 and 5 years of age.

BACKGROUND: Red blood cell (RBC) transfusion is often considered a life-saving measure in preterm neonates. However, it has been associated with detrimental effects on short-term morbidities and, recently, on brain development. The aim of the present study was to evaluate the association between RBC and long-term neurodevelopmental outcome in a cohort of preterm infants. MATERIALS AND METHODS: This retrospective cohort study was carried out in the period 2007-2013. Preterm infants with a gestational age (GA) ≤ 32 weeks and birthweight (BW) <1,500 g were included. Infants underwent Griffiths assessment at 24±6 months corrected age (CA) and at 5±1 years of age. We used a multivariate regression model to assess the association of RBC transfusions and long-term neurodevelopment after controlling for GA, being small for GA, major neonatal morbidities, and socio-economic status. We also evaluated the impact of early RBC administration (within the first 28 days of life) compared to those performed after the first month of life. RESULTS: We enrolled 644 preterm infants, among whom 54.3% were transfused during their stay in the neonatal intensive care unit (NICU). In infants with a longitudinal follow-up evaluation (n=360), each RBC transfusion was independently associated with a reduction in the Griffiths General Quotient (GQ) by -0.96 (p=0.002) at 24 months CA. Early RBC administration had the biggest impact, especially in children without brain lesions, where the reduction in Griffiths GQ for each additional transfusion was -2.12 (p=0.001) at 24 months CA and -1.31 (p=0.006) at 5 years of age, respectively. DISCUSSION: In preterm infants, RBC transfusions are associated with long-term neurodevelopmental outcome, with a cumulative effect. Early RBC administration is associated with a greater reduction in Griffiths scores. The impact of RBC transfusion on neurodevelopment is greater at 24 months CA, but persists, although to a lesser degree, at 5 years of age.