Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

The Neglected Role of Education in Brazilian Research Regarding Nuclear Theme versus Scientific and Technological Knowledge, Perception and Risk Communication.

Studies of risk perception and risk communication concerning the nuclear area are quite common in scientific literature. However, though numerous studies on this topic point to the importance of scientific and technological knowledge in facilitating the reduction of perceived risks, there are few papers that effectively discuss the role of education in risk communication on the subject, particularly in Brazil. The objective of the present work was to reflect on this problem by analyzing the results obtained from two case studies on the implementation of the Brazilian Multipurpose Reactor, in the city of Iperó, in the state of São Paulo, Brazil. As was verified in the investigation, most participants of the study had high perception of risk with respect to the implementation of the reactor. Nevertheless, although important in the project of the reactor implementation, it was verified that the state, municipal and community schools of the city of Iperó had not participated, in the public hearings nor had they discussed the issue inside the school community, until the moment of the research.

Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain.

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins ßIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

Acceptability, Preferences, and Palatability of Diets Containing Summer and Winter Brassica Forage in Growing Pigs: A Pilot Study.

Brassica forage may be included in pigs' diet as a dietary fiber ingredient to reduce feeding costs, benefit gut health, immune system, reproductive traits, and welfare. However, they contain antinutritional factors which may affect feeding behavior. This study evaluated feeding behavior of growing pigs offered winter (kale and swede) and summer (turnip and forage rape) brassicas incorporated on their diets as dried ground meal. Two consecutive experiments with six growing castrated male pigs were conducted. Experiment 1 evaluated the inclusion of turnip bulbs and forage rape, while experiment 2 studied inclusion of kale and swede bulbs. Brassica meal was included at 15% of the diet by replacing wheat middlings (control diet). In each experiment, pigs were offered experimental diets over six consecutive days for 10 min to test their acceptability (day 1-3) and preferences (day 4-6). No differences were found between diets that included brassicas and control diet in pigs' acceptability or palatability (p > 0.05). However, during preference tests of winter brassicas, swede presented a higher consumption than control and kale (p < 0.05). This suggest that brassicas may be incorporated in growing pigs' diets without negative effects in animals' oral perception during short term feeding tests. Nevertheless, the long-term effects need to be explored.

Public space, socio-environmental responsibility and citizenship: a case study on a Brazilian borough.

Some researchers point out the decline in contemporary cities of the use of public spaces, whether free or restricted access. In this sense, the present essay proposes a reflection on a "public sphere of organized presence", according to Habermas classification, as a rescue of the importance of this space for the exercise of citizenship and social-environmental responsibility. The reflection was based on the results obtained from a study in the district of Riacho Grande, São Bernardo do Campo, São Paulo, Brazil, in which the environmental perception of the participants of the meetings about the region was known by documentary analysis, direct observation and interviews with questionnaires. It was possible to verify that this public space can facilitate a greater integration between the public administrators and citizens, helping even in the defense to the environment.

Liderazgo docente y la inclusión educativa en la educación superior / Teaching leadership and educational inclusion in higher education

El liderazgo docente se ha posicionado como uno de los temas emergentes de las últimas décadas en materia de educación y como factor que más influye en el aprendizaje de los estudiantes, considerando, que en la actualidad las instituciones educativas son más diversas que nunca. OBJETIVO: analizar la influencia que tiene el liderazgo docente en la promoción de la educación inclusiva. Metodología de tipo bibliográfica documental de revisión, descriptiva, exploratoria, transversal, critica y reflexiva. RESULTADO: se encuentran insuficiencias en la formación docente relacionada con el liderazgo y la inclusión, se requiere que las instituciones de educación cuenten con planta docente con liderazgo, preparación académica más integral y humana, que sean respetuosos de la diversidad, capaz de construir nuevos escenarios de aprendizaje que fortalezca el quehacer inclusivo. CONCLUSIONES: es necesario que los docentes se capaciten en procesos metodológicos inclusivos que los prepare para afrontar la heterogeneidad existente en los salones de clases, de esta forma contribuirán con el aprendizaje de los estudiantes para que estos aprendan al máximo, independientemente de las características e individualidades que presenten.

Teaching leadership has positioned itself as one of the emerging themes of education in recent decades and as a factor that most influences student learning, considering that educational institutions are currently more diverse than ever. OBJECTIVE: analyze the influence that teaching leadership has in promoting inclusive education. the documentary bibliographic type review, descriptive, exploratory, transversal, critical and reflective methodology. RESULT: there are insufficiencies in teacher training related to leadership and inclusion, it is required that educational institutions have a teaching staff with leadership, more comprehensive and humane academic preparation, that are respectful of diversity, capable of constructing new scenarios of learning that strengthens inclusive work. CONCLUSIONS: it is necessary for teachers to be trained in inclusive methodological processes that prepare them to face the heterogeneity existing in the classrooms, in this way they will contribute to the learning of students sothat they learn to the maximum, regardless of the characteristics and individualities that they present.

Histamine Modulates Midbrain Dopamine Neuron Differentiation Through the Regulation of Epigenetic Marks.

During midbrain development, dopamine neuron differentiation occurs before birth. Epigenetic processes such as DNA methylation and demethylation as well as post-translational modification of histones occur during neurogenesis. Here, we administered histamine (HA) into the brain of E12 embryos in vivo and observed significant lower immunoreactivity of Lmx1a+ and Tyrosine Hydroxylase (TH)+ cells, with parallel decreases in the expression of early (Lmx1a, Msx1) and late (Th) midbrain dopaminergic (mDA) genes. With MeDIP assays we found that HA decreases the percentage of 5-methylcytosine of Pitx3 and Th, without changes in 5-hydroxymethylcytosine. Additionally, HA treatment caused a significant increase in the repressive epigenetic modifications H3K9me3 in Pitx3 and Th, and also more H3K27me3 marks in Th. Furthermore, HA has a long-term effect on the formation of the nigrostriatal and mesolimbic/mesocortical pathways, since it causes a significant decrease in midbrain TH immunoreactivity, as well as alterations in dopaminergic neuronal fibers, and significant lower TH-positive area in the forebrain in whole-mount stainings. These findings suggest that HA diminishes dopaminergic gene transcription by altering several epigenetic components related to DNA and histone modifications, which affects mDA neuron progression during development.

CTCF-KDM4A complex correlates with histone modifications that negatively regulate CHD5 gene expression in cancer cell lines.

Histone demethylase KDM4A is involved in H3K9me3 and H3K36me3 demethylation, which are epigenetic modifications associated with gene silencing and RNA Polymerase II elongation, respectively. KDM4A is abnormally expressed in cancer, affecting the expression of multiple targets, such as the CHD5 gene. This enzyme localizes at the first intron of CHD5, and the dissociation of KDM4A increases gene expression. In vitro assays showed that KDM4A-mediated demethylation is enhanced in the presence of CTCF, suggesting that CTCF could increase its enzymatic activity in vivo, however the specific mechanism by which CTCF and KDM4A might be involved in the CHD5 gene repression is poorly understood. Here, we show that CTCF and KDM4A form a protein complex, which is recruited into the first intron of CHD5. This is related to a decrease in H3K36me3/2 histone marks and is associated with its transcriptional downregulation. Depletion of CTCF or KDM4A by siRNA, triggered the reactivation of CHD5 expression, suggesting that both proteins are involved in the negative regulation of this gene. Furthermore, the knockout of KDM4A restored the CHD5 expression and H3K36me3 and H3K36me2 histone marks. Such mechanism acts independently of CHD5 promoter DNA methylation. Our findings support a novel mechanism of epigenetic repression at the gene body that does not involve promoter silencing.

Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors.

BACKGROUND: Histamine (HA) regulates the sleep-wake cycle, synaptic plasticity and memory in adult mammals. Dopaminergic specification in the embryonic ventral midbrain (VM) coincides with increased HA brain levels. To study the effect of HA receptor stimulation on dopamine neuron generation, we administered HA to dopamine progenitors, both in vitro and in vivo. RESULTS: Cultured embryonic day 12 (E12) VM neural stem/progenitor cells expressed transcripts for HA receptors H1R, H2R and H3R. These undifferentiated progenitors increased intracellular calcium upon HA addition. In HA-treated cultures, dopamine neurons significantly decreased after activation of H1R. We performed intrauterine injections in the developing VM to investigate HA effects in vivo. HA administration to E12 rat embryos notably reduced VM Tyrosine Hydroxylase (TH) staining 2 days later, without affecting GABA neurons in the midbrain, or serotonin neurons in the mid-hindbrain boundary. qRT-PCR and Western blot analyses confirmed that several markers important for the generation and maintenance of dopaminergic lineage such as TH, Lmx1a and Lmx1b were significantly diminished. To identify the cell type susceptible to HA action, we injected embryos of different developmental stages, and found that neural progenitors (E10 and E12) were responsive, whereas differentiated dopaminergic neurons (E14 and E16) were not susceptible to HA actions. Proliferation was significantly diminished, whereas neuronal death was not increased in the VM after HA administration. We injected H1R or H2R antagonists to identify the receptor responsible for the detrimental effect of HA on dopaminergic lineage and found that activation of H1R was required. CONCLUSION: These results reveal a novel action of HA affecting dopaminergic lineage during VM development.

Hemangiopericytoma in a female dog with direct invasion of abdominal cavity and pulmonary metastasis / Hemangiopericitoma em uma cadela com invasão direta da cavidade abdominal e metástase pulmonar

An intact adult female Poodle dog was presented with a history of an increase in volume in the left and right mammary inguinal glands. The histopathological examination revealed a proliferation of spindle cells arranged in bundles with concentric arrangements surrounding blood vessels, occasionally collapsed. Three weeks after the surgery, the animal presented a recurrence of the tumor that extended from the left inguinal mammary gland to the vulva. Necropsy revealed direct invasion of the abdominal cavity by the tumor and pulmonary metastasis. The definitive diagnosis of hemangiopericytoma was made by histopathological and immunohistochemical examination. Pulmonary metastasis of CHP (canine hemangiopericytoma) is rare and there is no previous report of direct invasion to the abdominal cavity as observed in the present case.

Uma cadela adulta, intacta, da raça Poodle, apresentou histórico de aumento de volume na mama inguinal direita. O exame histopatológico revelou proliferação de células fusiformes dispostas em bandas e arranjos concêntricos ao redor de vasos, os quais se apresentavam ocasionalmente colapsados. Três semanas após a cirurgia, o animal apresentou recorrência do tumor que se estendia até a vulva. No exame necroscópico, observou-se que a massa invadia a cavidade abdominal e presença de metástase no pulmão. O diagnóstico definitivo de hemangiopericitoma foi realizado por meio dos exames histopatológico e imuno-histoquímico. A metástase pulmonar do hemangiopericitoma canino é rara e não há relatos prévios de invasão direta para cavidade abdominal, como observado no presente caso.

Assessment of neuromuscular blockade in children at the time of block reversal and the removal of the endotracheal tube.

BACKGROUND AND OBJECTIVES: Studies show the importance of monitoring neuromuscular function in preventing the residual block. However, most anesthesiologists in their daily practice base their evaluation of the recovery of neuromuscular function on clinical data. The aim of this study was to assess the degree of neuromuscular blockade in children undergoing general anesthesia at the time of block reversal and the removal of the endotracheal tube. METHOD: We evaluated children between 3 months and 12 years of age undergoing general anesthesia with the use of atracurium or rocuronium. Monitoring was initiated at the time of reversal of neuromuscular blockade and/or removal of the endotracheal tube. The anesthesiologist was not informed about the T4/T1 value; he/she was only alerted when the time was inadequate for the removal of the endotracheal tube. Since the start of the monitoring process, the degree of neuromuscular blockade was registered, as well as the interval of recovery of the T(4)/T1 ≥ 0.9 ratio, the doses of neostigmine and blocker used, the expired fraction of the inhalational agent, the duration of the anesthesia, and core and peripheral temperatures. RESULTS: Neuromuscular blockade was reversed in 80% of the children of the Rocuronium Group and in 64.5% of the Atracurium Group. The reversal was incorrect in 45.8% of the Rocuronium Group and in 25% of the Atracurium Group. The incidence of T4/T1 < 0.9 at the time of the removal of the endotracheal tube was 10% in both groups. CONCLUSIONS: When deciding to remove the endotracheal tube based on clinical criteria, 10% of children had T4/T1 < 0.9 regardless the blocker received. A considerable number of patients had the neuromuscular blockade incorrectly reversed when the blockade was still too deep or even already recovered.

Avaliação do bloqueio neuromuscular em crianças no momento da reversão do bloqueio e da retirada da cânula endotraqueal / Assessment of neuromuscular blockade in children at the time of block reversal and the removal of the endotracheal tube

JUSTIFICATIVA E OBJETIVOS: Estudos evidenciam a importância da monitoração da função neuromuscular na prevenção do bloqueio residual. No entanto, a maioria dos anestesistas em sua prática diária se abaliza em dados clínicos para avaliar a recuperação da função neuromuscular. O objetivo deste estudo foi avaliar o grau de bloqueio neuromuscular em crianças submetidas à anestesia geral no momento da reversão do bloqueio e da retirada da cânula endotraqueal. MÉTODO: Foram avaliadas crianças entre 3 meses e 12 anos, submetidas à anestesia geral com uso de atracúrio ou rocurônio. A monitoração foi iniciada no momento da reversão do bloqueio neuromuscular e/ou retirada da cânula endotraqueal. O anestesiologista não era informado sobre o valor de T4/T1; apenas era alertado quando o momento era inadequado para a retirada da cânula endotraqueal. Houve registro do grau de bloqueio neuromuscular desde o início da monitoração, bem como do intervalo para a recuperação da relação T4/T1 > 0,9, doses de bloqueador e neostigmina utilizadas, fração expirada do agente inalatório, duração da anestesia, temperatura central e periférica. RESULTADOS: O bloqueio neuromuscular foi revertido em 80 por cento das crianças no Grupo Rocurônio e 64,5 por cento no Grupo Atracúrio. A reversão foi incorreta em 45,8 por cento do Grupo Rocurônio e 25 por cento do Grupo Atracúrio. A incidência de T4/T1 < 0,9 no momento da retirada da cânula endotraqueal foi de 10 por cento em ambos os grupos. CONCLUSÕES: Na decisão de retirada da cânula endotraqueal, com base em critérios clínicos, 10 por cento das crianças apresentavam T4/T1 < 0,9, independentemente do bloqueador recebido. Um número considerável de pacientes teve o bloqueio neuromuscular revertido de maneira incorreta, quando o bloqueio ainda era profundo demais ou mesmo já estava recuperado.

BACKGROUND AND OBJECTIVES: Studies show the importance of monitoring neuromuscular function in preventing the residual block. However, most anesthesiologists in their daily practice base their evaluation of the recovery of neuromuscular function on clinical data. The aim of this study was to assess the degree of neuromuscular blockade in children undergoing general anesthesia at the time of block reversal and the removal of the endotracheal tube. METHOD: We evaluated children between 3 months and 12 years of age undergoing general anesthesia with the use of atracurium or rocuronium. Monitoring was initiated at the time of reversal of neuromuscular blockade and/or removal of the endotracheal tube. The anesthesiologist was not informed about the T4/T1 value; he/she was only alerted when the time was inadequate for the removal of the endotracheal tube. Since the start of the monitoring process, the degree of neuromuscular blockade was registered, as well as the interval of recovery of the T4/T1 > 0,9 ratio, the doses of neostigmine and blocker used, the expired fraction of the inhalational agent, the duration of the anesthesia, and core and peripheral temperatures. RESULTS: Neuromuscular blockade was reversed in 80 percent of the children of the Rocuronium Group and in 64.5 percent of the Atracurium Group. The reversal was incorrect in 45.8 percent of the Rocuronium Group and in 25 percent of the Atracurium Group. The incidence of T4/T1 < 0.9 at the time of the removal of the endotracheal tube was 10 percent in both groups. CONCLUSIONS: When deciding to remove the endotracheal tube based on clinical criteria, 10 percent of children had T4/T1 < 0.9 regardless the blocker received. A considerable number of patients had the neuromuscular blockade incorrectly reversed when the blockade was still too deep or even already recovered.

JUSTIFICATIVA Y OBJETIVOS: Algunos estudios demuestran la importancia de la monitorización de la función neuromuscular en la prevención del bloqueo residual. Sin embargo, la mayoría de los anestesiólogos en su práctica diaria, se basa en datos clínicos para evaluar la recuperación de la función neuromuscular. El objetivo de este estudio fue evaluar el grado de bloqueo neuromuscular en niños sometidos a la anestesia general, en el momento de la reversión del bloqueo y de la retirada de la cánula endotraqueal. MÉTODO: Se evaluaron niños entre 3 meses y 12 años, sometidos a la anestesia general con el uso de atracurio o rocuronio. La monitorización se inició al momento de la reversión del bloqueo neuromuscular y/o retirada de la cánula endotraqueal. El anestesiólogo no conocía el valor de T4/T1; apenas se le avisaba cuando el momento no era el adecuado para la retirada de la cánula endotraqueal. Se registró el grado de bloqueo neuromuscular desde el inicio de la monitorización, como también el intervalo para la recuperación de la relación T4/T1 < 0,9, dosis de bloqueante y neostigmina utilizadas, fracción expirada del agente inhalatorio, duración de la anestesia, temperatura central y periférica. RESULTADOS: El bloqueo neuromuscular fue revertido en un 80 por ciento de los niños en el Grupo Rocuronio y en un 64,5 por ciento en el Grupo Atracurio. La reversión fue incorrecta en un 45,8 por ciento del Grupo Rocuronio y en un 25 por ciento del Grupo Atracurio. La incidencia de T4/T1 < 0,9 al momento de la retirada de la cánula endotraqueal fue de un 10 por ciento en los dos grupos. CONCLUSIONES: En la toma de decisión de la retirada de la cánula endotraqueal, en base a los criterios clínicos, el 10 por ciento de los niños presentaron T4/T1 < 0,9, independientemente del bloqueante recibido. Un número considerable de pacientes tuvo el bloqueo neuromuscular revertido de manera incorrecta, cuando el bloqueo todavía era muy profundo...

Triclabendazole resistance involving Fasciola hepatica in sheep and goats during an outbreak in Almirante Tamandare, Paraná, Brazil.

Fasciolosis is a disease of extreme importance, occurring throughout Brazil, with great economic losses to the animal industry. This study aims to determine the effectiveness of treatment against Fasciola hepatica on a sheep and goat farm during an outbreak of fasciolosis, in which a high proportion of deaths occurred. The farm harbored 33 sheep and 60 goats of mixed breeds with varying weights and ages. Parasite control was based on suppressive, monthly treatments with moxidectin. Over the course of the study, assessments with Famacha method, body condition score, faecal exam (EPG), coproculture and the egg sedimentation technique were made on seven occasions. At the time of the first visit 81% of the goats and 100% of the sheep were positive for F. hepatica. Salvage treatment with triclabendazole failed to relieve the heavy infection due to resistance, with an efficacy of only 66.3% being obtained against the F. hepatica population consensual in sheep and 57.3% in goats. There was no record of triclabendazole having been administered previously on the farm. A low level of correlation was found between the Famacha values and the incidence of anemia due to clinical fasciolosis (C<0.5). There is an urgent need to investigate the correct use of available drugs against F. hepatica and the implementation of alternative control strategies in endemic areas in order to ensure optimum sustainability of the efficacy of available fasciolicides.