Expansion of a neural crest gene signature following ectopic MYCN expression in sympathoadrenal lineage cells in vivo.

Neural crest cells (NCC) are multipotent migratory stem cells that originate from the neural tube during early vertebrate embryogenesis. NCCs give rise to a variety of cell types within the developing organism, including neurons and glia of the sympathetic nervous system. It has been suggested that failure in correct NCC differentiation leads to several diseases, including neuroblastoma (NB). During normal NCC development, MYCN is transiently expressed to promote NCC migration, and its downregulation precedes neuronal differentiation. Overexpression of MYCN has been linked to high-risk and aggressive NB progression. For this reason, understanding the effect overexpression of this oncogene has on the development of NCC-derived sympathoadrenal progenitors (SAP), which later give rise to sympathetic nerves, will help elucidate the developmental mechanisms that may prime the onset of NB. Here, we found that overexpressing human EGFP-MYCN within SAP lineage cells in zebrafish led to the transient formation of an abnormal SAP population, which displayed expanded and elevated expression of NCC markers while paradoxically also co-expressing SAP and neuronal differentiation markers. The aberrant NCC signature was corroborated with in vivo time-lapse confocal imaging in zebrafish larvae, which revealed transient expansion of sox10 reporter expression in MYCN overexpressing SAPs during the early stages of SAP development. In these aberrant MYCN overexpressing SAP cells, we also found evidence of dampened BMP signaling activity, indicating that BMP signaling disruption occurs following elevated MYCN expression. Furthermore, we discovered that pharmacological inhibition of BMP signaling was sufficient to create an aberrant NCC gene signature in SAP cells, phenocopying MYCN overexpression. Together, our results suggest that MYCN overexpression in SAPs disrupts their differentiation by eliciting abnormal NCC gene expression programs, and dampening BMP signaling response, having developmental implications for the priming of NB in vivo.

Caspase-3 cleaved tau impairs mitochondrial function through the opening of the mitochondrial permeability transition pore.

Mitochondrial dysfunction is a significant factor in the development of Alzheimer's disease (AD). Previous studies have demonstrated that the expression of tau cleaved at Asp421 by caspase-3 leads to mitochondrial abnormalities and bioenergetic impairment. However, the underlying mechanism behind these alterations and their impact on neuronal function remains unknown. To investigate the mechanism behind mitochondrial dysfunction caused by this tau form, we used transient transfection and pharmacological approaches in immortalized cortical neurons and mouse primary hippocampal neurons. We assessed mitochondrial morphology and bioenergetics function after expression of full-length tau and caspase-3-cleaved tau. We also evaluated the mitochondrial permeability transition pore (mPTP) opening and its conformation as a possible mechanism to explain mitochondrial impairment induced by caspase-3 cleaved tau. Our studies showed that pharmacological inhibition of mPTP by cyclosporine A (CsA) prevented all mitochondrial length and bioenergetics abnormalities in neuronal cells expressing caspase-3 cleaved tau. Neuronal cells expressing caspase-3-cleaved tau showed sustained mPTP opening which is mostly dependent on cyclophilin D (CypD) protein expression. Moreover, the impairment of mitochondrial length and bioenergetics induced by caspase-3-cleaved tau were prevented in hippocampal neurons obtained from CypD knock-out mice. Interestingly, previous studies using these mice showed a prevention of mPTP opening and a reduction of mitochondrial failure and neurodegeneration induced by AD. Therefore, our findings showed that caspase-3-cleaved tau negatively impacts mitochondrial bioenergetics through mPTP activation, highlighting the importance of this channel and its regulatory protein, CypD, in the neuronal damage induced by tau pathology in AD.

In toto imaging of early enteric nervous system development reveals that gut colonization is tied to proliferation downstream of Ret.

The enteric nervous system is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and these are pertinent subjects for study due to their relevance to the human congenital disease Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in wild type and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs localized along the gut, and these ENCCs failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation-dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs. These results suggest that Ret signaling may regulate maintenance of a stem state in ENCCs.

A protocol for whole-mount immuno-coupled hybridization chain reaction (WICHCR) in zebrafish embryos and larvae.

Characterizing mRNA and protein expression with temporal and spatial resolution is a valuable component of nearly every developmental study. Here, we describe a protocol that combines in situ hybridization chain reaction (HCR) and immunofluorescence, allowing for the detection of mRNAs and proteins simultaneously, in zebrafish embryos and larvae. This protocol expands the flexibility of multiplexed HCR by coupling it with traditional immunofluorescence detection. For complete details on the use and execution of this protocol, please refer to Choi et al. (2010, 2016, 2018) and Howard et al. (2021).

CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain-of-function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti-tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.

An atlas of neural crest lineages along the posterior developing zebrafish at single-cell resolution.

Neural crest cells (NCCs) are vertebrate stem cells that give rise to various cell types throughout the developing body in early life. Here, we utilized single-cell transcriptomic analyses to delineate NCC-derivatives along the posterior developing vertebrate, zebrafish, during the late embryonic to early larval stage, a period when NCCs are actively differentiating into distinct cellular lineages. We identified several major NCC/NCC-derived cell-types including mesenchyme, neural crest, neural, neuronal, glial, and pigment, from which we resolved over three dozen cellular subtypes. We dissected gene expression signatures of pigment progenitors delineating into chromatophore lineages, mesenchyme cells, and enteric NCCs transforming into enteric neurons. Global analysis of NCC derivatives revealed they were demarcated by combinatorial hox gene codes, with distinct profiles within neuronal cells. From these analyses, we present a comprehensive cell-type atlas that can be utilized as a valuable resource for further mechanistic and evolutionary investigations of NCC differentiation.

Effects of Rivaroxaban on Platelet Aggregation.

Rivaroxaban is a direct oral anti-factor Xa anticoagulant. It has recently been suggested that rivaroxaban may affect platelet function in vitro; however, little is known about the clinical impact of this likely antiplatelet effect and whether this probable phenomenon is dose-dependent. Our aim was to determine whether rivaroxaban at 4 different doses inhibits direct platelet aggregation. We included adult patients of both sexes and who were allocated to one of the following groups depending on the prescribed daily dose of rivaroxaban: 5, 10, 15, and 20 mg. In 80 patients (20 patients/group), the percentage of platelet aggregation was determined by means of platelet aggregometry tests before and after rivaroxaban use. Basal samples were obtained before starting rivaroxaban and 1 month after treatment, both 2 and 24 hours after the last dose of the drug (12 hours after in the case of rivaroxaban 5 mg). We used 5 platelet agonists: adenosine diphosphate, epinephrine, arachidonic acid, collagen, and thrombin. There were no significant changes in the percentage of platelet aggregation before and after rivaroxaban use independently of the dose administered and the agonist used. Our results have clearly shown that rivaroxaban, even at a high dose, does not directly affect platelet aggregation.

Asthma prevalence, but not allergic rhinitis nor atopic dermatitis, is associated to exposure to dogs in adolescents

Background: Exposure to pets can be a predisposing factor in the development of certain diseases, including allergic diseases. Objective: We analyzed the role that exposure to indoor dogs and cats plays in the prevalence of allergic diseases. Methods: We examined the cross-sectional data of 1056 women and 936 men aged 15 to 18 years; these individuals were selected through stratified and cluster random sampling. We asked all participants about their exposure to indoor dogs and cats during the year that preceded our study. The prevalence of allergic diseases was determined through core questions taken from The International Study of Asthma and Allergies in Childhood questionnaire. Results: The prevalence was 12.7% (95% CI: 11.3%-14.2%) for asthma, 9.0% (95% CI: 7.8%-10.4%) for allergic rhinitis, and 5.2% (95% CI: 4.3%-6.2%) for atopic dermatitis. The multivariate analyses showed that exposure to indoor dogs, but not indoor cats, was associated with asthma prevalence (aOR 1.37; 95% CI: 1.03-1.83), as was male sex (aOR = 1.42; 95% CI: 1.08-1.86), a personal history of allergic rhinitis (aOR = 3.24; 95% CI: 2.25-4.66), and a maternal history of asthma (aOR = 3.06; 95% CI: 1.89-4.98). The population attributable risk for exposure to indoor dogs was 18%. Notably, neither allergic rhinitis nor atopic dermatitis was found to be associated with dog or cat exposure (p > 0.05). Conclusion: Exposure to dogs in late adolescence is a factor associated with asthma, although its contribution to the development of asthma should be investigated in new studies

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Asthma prevalence, but not allergic rhinitis nor atopic dermatitis, is associated to exposure to dogs in adolescents.

BACKGROUND: Exposure to pets can be a predisposing factor in the development of certain diseases, including allergic diseases. OBJECTIVE: We analyzed the role that exposure to indoor dogs and cats plays in the prevalence of allergic diseases. METHODS: We examined the cross-sectional data of 1056 women and 936 men aged 15 to 18 years; these individuals were selected through stratified and cluster random sampling. We asked all participants about their exposure to indoor dogs and cats during the year that preceded our study. The prevalence of allergic diseases was determined through core questions taken from The International Study of Asthma and Allergies in Childhood questionnaire. RESULTS: The prevalence was 12.7% (95% CI: 11.3%-14.2%) for asthma, 9.0% (95% CI: 7.8%-10.4%) for allergic rhinitis, and 5.2% (95% CI: 4.3%-6.2%) for atopic dermatitis. The multivariate analyses showed that exposure to indoor dogs, but not indoor cats, was associated with asthma prevalence (aOR 1.37; 95% CI: 1.03-1.83), as was male sex (aOR=1.42; 95% CI: 1.08-1.86), a personal history of allergic rhinitis (aOR=3.24; 95% CI: 2.25-4.66), and a maternal history of asthma (aOR=3.06; 95% CI: 1.89-4.98). The population attributable risk for exposure to indoor dogs was 18%. Notably, neither allergic rhinitis nor atopic dermatitis was found to be associated with dog or cat exposure (p> 0.05). CONCLUSION: Exposure to dogs in late adolescence is a factor associated with asthma, although its contribution to the development of asthma should be investigated in new studies.

Single strain versus multispecies probiotic on necrotizing enterocolitis and faecal IgA levels in very low birth weight preterm neonates: A randomized clinical trial.

BACKGROUND: According to the literature, probiotics are an attractive alternative to prevent necrotizing enterocolitis (NEC). However, due to differences in probiotic composition, randomized controlled trials are necessary to compare different probiotic mixtures. The objective of this study was to compare single strain (Lactobacillus acidophilus boucardii) versus multispecies probiotics on NEC incidence and faecal secretory Immunoglobulin A (sIgA) levels in very low preterm newborns. METHODS: We performed a double-blind randomized trial in 90 newborns. L. acidophilus boucardii strain or multispecies probiotics were randomly assigned to preterm newborns. As the primary outcome, we evaluated NEC incidence on the total length of neonatal intensive care unit (NICU) stay. As the secondary outcome, we measured the change in faecal sIgA levels from baseline to 3 weeks following the use of probiotics. RESULTS: NEC incidence was similar between groups (0% vs. 2.2% for the single strain and multispecies probiotic, respectively). Faecal sIgA levels increased significantly (p < 0.001) within groups (31% for single strain and 47% for multispecies probiotic), but this increase was not different between groups. Neonates with a faecal sIgA level increment >0.45 mg/dl showed higher gestational age, birth weight, and weight at the second and third weeks of follow up than neonates with a faecal sIgA level increment ≤0.45 mg/dl. No adverse effects were found after probiotics use. CONCLUSIONS: No difference between strains of probiotics used was found on NEC incidence or in the increase of faecal sIgA levels. Faecal sIgA levels were positively related to gestational age and body weight in very low preterm infants. ClinicalTrials.gov/NCT02245815.

Educación caligráfica durante la infancia y su efecto a largo plazo en la escritura en la vida adulta / Calligraphic education during childhood and its long-term effect on the adult handwriting

Antecedentes:La escritura es una parte esencial de la comunicación. Uno de los componentes más importantes es la prehensión fina, es decir el agarre de la pluma, de la cual existen cinco variantes. Reconociendo que la educación tiene un rol importante en la generación de buenos hábitos de escritura para un mejor producto: la legibilidad. Metodología:Se realizó un estudio transversal de correlación. Se usaron dos métodos de recolección de información: cuestionario electrónico y prueba caligráfica. Resultados:Se reclutaron 120 sujetos, con edades entre 18 y 30 años. No se encontró relación significativa entre la legibilidad del texto con la duración de la educación caligráfica, el sexo, la edad, la mano preferente, el promedio ni tipo de letra. Se encontró relación significativa entre la comparación de los agarres estandarizados con el agarre alterno respecto a su legibilidad (p=0.01). Discusión:El 77.5% de la muestra obtuvo un promedio alto en la prueba caligráfica; sin embargo, no se encontró relación estadísticamente significativa con la duración de la educación caligráfica en la infancia, la legibilidad según sexo y edad, mano preferente, instrumento, tipo de letra y promedio académico. La única asociación significativa (p=0.01) fue entre legibilidad en agarre estandarizado con el alterno. Conclusión:La legibilidad de la caligrafía es multifactorial, con peso principal en la educación caligráfica infantil. Sin embargo, se comprobó que la duración de esta educación no es significativa para la legibilidad. Igualmente, el uso de un agarre alterno a las cinco variantes se asocia con peores resultados en la prueba caligráfica.

Background:Handwriting is an essential part of human communication. Its successful development is decisive for its personal and academic future. One of the most important components is the grip of the pen, which there are 5 standard variants. Calligraphic education has a very important role in the generation of writing habits and a better legibility. Methods:In a cross-sectional study of correlation between variables, the aim was to correlate calligraphic education with many variables. The data was collected with an electronic questionnaire and a calligraphic test.Results:The sample consisted of 120 subjects between the ages of 18-30. No statistically significant correlation was found between legibility and years of calligraphic education, gender, age, dominant hand, GPA and preferred font. The only significant correlation was found between legibility and the use of one standard grips of the pen and an alternative (p=0.01). Discussion:77.5% of the subjects had a high average in the calligraphic test and no correlation was found between legibility and length of calligraphic education in childhood, gender, age, dominant hand, writing tool, font and GPA. However, a significant association between legibility and the use of the standard grips was found, in comparison with people that do not use one of those grips (p=0.01).Conclusions:Handwriting legibility is a multi-factor influenced skill, and calligraphic education during childhood is decisive to develop it properly. Nevertheless, the study showed no association between the length of the education and legibility, and a correlation was found between the use of an alternative pen grip and a worse result in the calligraphic test.

The Saccharomyces cerevisiae mitochondrial unselective channel behaves as a physiological uncoupling system regulated by Ca2+, Mg2+, phosphate and ATP.

It is proposed that the Saccharomyces cerevisiae the Mitochondrial Unselective Channel ((Sc)MUC) is tightly regulated constituting a physiological uncoupling system that prevents overproduction of reactive oxygen species (ROS). Mg(2+), Ca(2+) or phosphate (Pi) close (Sc)MUC, while ATP or a high rate of oxygen consumption open it. We assessed (Sc)MUC activity by measuring in isolated mitochondria the respiratory control, transmembrane potential (ΔΨ), swelling and production of ROS. At increasing [Pi], less [Ca(2+)] and/or [Mg(2+)] were needed to close (Sc)MUC or increase ATP synthesis. The Ca(2+)-mediated closure of (Sc)MUC was prevented by high [ATP] while the Mg(2+) or Pi effect was not. When Ca(2+) and Mg(2+) were alternatively added or chelated, (Sc)MUC opened and closed reversibly. Different effects of Ca(2+) vs Mg(2+) effects were probably due to mitochondrial Mg(2+) uptake. Our results suggest that (Sc)MUC activity is dynamically controlled by both the ATP/Pi ratio and divalent cation fluctuations. It is proposed that the reversible opening/closing of (Sc)MUC leads to physiological uncoupling and a consequent decrease in ROS production.

Retrospective analysis of patients undergoing bowel transit reconstruction in a tertiary referral hospital of São Paulo's east side / Análise retrospectiva dos pacientes submetidos à reconstrução de trânsito intestinal em hospital terciário de referência da zona leste de São Paulo

Introduction: The morbidity and mortality of patients undergoing bowel transit reconstruction reach significant values. Perhaps this and other factors could explain why 30-60% of patients end up with definitive ostomies, even those with initially temporary ostomies, due to the procedure risks. Objective: To analyze retrospectively the medical records of patients undergoing bowel transit reconstruction in one of the SUS referral hospitals in São Paulo from October 2008 to December 2011. Results: The mean age of our patients was 53.9 years and 54% of those 100 patients studied between October 2008 and December 2011 had significant comorbidity. The indication for creating an initial ostomy was malignancy in 43%, and the mean stoma duration 14.3 months. The mortality rate was 6%. Conclusion: Although the bowel transit reconstruction is a procedure quite desired by patients, its indication should be carefully evaluated, with an appropriate consent from the patient. .

Introdução: A morbi-mortalidade de pacientes submetidos à reconstrução de trânsito intestinal alcança valores significativos e, por esse e outros fatores, talvez se explique o fato que de 30 a 60% dos portadores de ostomia intestinal terminal passam a possuí-la de maneira definitiva, apesar de, na maior parte das vezes, ela ser realizada como procedimento provisório com o argumento de maior segurança do paciente. Objetivo: Analisar retrospectivamente os dados de prontuário de pacientes submetidos à reconstrução de trânsito intestinal em um dos hospitais de referência do SUS na cidade de São Paulo no período de outubro de 2008 a dezembro de 2011. Resultados: A média de idade dos pacientes foi de 53,9 anos e 54% dos 100 pacientes estudados no período de outubro de 2008 e dezembro de 2011 padeciam de alguma comorbidade. A indicação para confecção da ostomia inicial decorreu de doença maligna em 43% e o tempo médio de permanência com o estoma foi de 14,3 meses. A taxa de mortalidade foi de 6%. Conclusão: Embora a reconstrução do trânsito intestinal seja um procedimento bastante desejado pelos pacientes, sua indicação deve ser bastante criteriosa, com consentimento adequado por parte do paciente.

Prevalência de hiperidrose entre estudantes de medicina / Prevalence of hyperhidrosis among medical students

OBJETIVO: Identificar a prevalência de hiperidrose entre os estudantes de Medicina de Manaus/AM. MÉTODOS: Estudo observacional, transversal, do tipo inquérito que analisou a prevalência de hiperidrose primária entre os alunos de Medicina da Universidade Federal do Amazonas e sua relação com o índice de massa corporal (IMC) e o stress. Os alunos foram pesados e entrevistados. Utilizou-se questionários com perguntas preconizadas pela International Hyperhidrosis Society, para relacionar a hiperidrose com as atividades diárias de cada pessoa. A análise dos resultados se deu com o cálculo da razão de prevalências e do intervalo de confiança. RESULTADOS: Entre os 293 estudantes analisados, verificou-se que um total de 16 (5,5 por cento) estudantes apresentavam sudorese excessiva dificilmente tolerável ou intolerável, interferindo em suas atividades diárias. Nenhum apresentava causas conhecidas de hiperidrose e 50 por cento possuíam história familiar. Em todos o acometimento foi bilateral, sendo os locais mais afetados: mãos (35,7 por cento), pés (21,4 por cento), axila (17,9), rosto (10,7 por cento), costas (7,1 por cento), tórax (3,6 por cento) e abdome (3,6 por cento). Não houve predomínio em relação ao sexo, idade ou IMC. Encontrou-se relação positiva com o IMC evidenciando sobrepeso e obesidade, sendo observado uma razão de prevalências de 2,48 superior em relação aos estudantes com peso normal ou abaixo do peso. CONCLUSÃO: A prevalência de hiperidrose primária entre os estudantes de Medicina de Manaus/AM foi de 5,5 por cento, existindo uma relação positiva não estatística com o sobrepeso e a obesidade. Foi constatada ainda uma relação observacional com o stress.

OBJECTIVE: To identify the prevalence of hyperhidrosis among medical students of Manaus, State of Amazonas, Brazil. METHODS: We conducted an observational, transversal, survey which examined the prevalence of primary hyperhidrosis among medical students of the Federal University of Amazonas and its relation to body mass index (BMI) and stress. Students were weighed and interviewed. We used questionnaires with questions recommended by the International Hyperhidrosis Society to relate hyperhidrosis to the daily activities of each person. Results were given by calculating the prevalence ratios and confidence intervals. RESULTS: Among the 293 students examined, it was found that a total of 16 (5.5 percent) students had barely tolerable or intolerable excessive sweating, interfering with daily activities. None had known causes of hyperhidrosis and 50 percent had family history. In all suffering from the condition the disease was bilateral, the mainly affected locations being: hands (35.7 percent), legs (21.4 percent), axilla (17.9), face (10 7 percent), back (7.1 percent), chest (3.6 percent) and abdomen (3.6 percent). There was no predominance regarding gender, age or BMI. We found a positive relationship with BMI and observed a prevalence ratio of 2.48 higher in overweight students than in normal weight or underweight ones. CONCLUSION: The prevalence of primary hyperhidrosis among medical students of Manaus was 5.5 percent. There is a positive non-statistical relationship with overweight and obesity. It was further noted an observational relationship with stress.

Prevalence of hyperhidrosis among medical students.

OBJECTIVE: To identify the prevalence of hyperhidrosis among medical students of Manaus, State of Amazonas, Brazil. METHODS: We conducted an observational, transversal, survey which examined the prevalence of primary hyperhidrosis among medical students of the Federal University of Amazonas and its relation to body mass index (BMI) and stress. Students were weighed and interviewed. We used questionnaires with questions recommended by the International Hyperhidrosis Society to relate hyperhidrosis to the daily activities of each person. Results were given by calculating the prevalence ratios and confidence intervals. RESULTS: Among the 293 students examined, it was found that a total of 16 (5.5%) students had barely tolerable or intolerable excessive sweating, interfering with daily activities. None had known causes of hyperhidrosis and 50% had family history. In all suffering from the condition the disease was bilateral, the mainly affected locations being: hands (35.7%), legs (21.4%), axilla (17.9), face (10 7%), back (7.1%), chest (3.6%) and abdomen (3.6%). There was no predominance regarding gender, age or BMI. We found a positive relationship with BMI and observed a prevalence ratio of 2.48 higher in overweight students than in normal weight or underweight ones. CONCLUSION: The prevalence of primary hyperhidrosis among medical students of Manaus was 5.5%. There is a positive non-statistical relationship with overweight and obesity. It was further noted an observational relationship with stress.