Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital

Abstract Objectives To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

HEMATOLOGYCAL AND BLOOD BIOCHEMISTRY PARAMETERS OF CAPTIVE BIG-HEADED AMAZON RIVER TURTLES, Peltocephalus dumerilianus (TESTUDINES: PODOCNEMIDIDAE) / Parámetros hematológicos y bioquímica sanguínea de tortugas amazónicas cautivas de cabeza grande, Peltocephalus dumerilianus (Testudinas: Podocnemididae)

ABSTRACT The determination of hematological values is used to obtain knowledge about the health conditions of animal species. The big-headed Amazon River turtles, (Peltocephalus dumerilianus) are considered one of the least known testudine species concerning their biology and health status. Herein, we determined the hematological and plasma biochemical parameters of17 (eight males and nine females) adult P. dumerilianus to provide reference interval values for clinically healthy individuals. We collected the blood samples by puncturing the femoral vein using long heparinized hypodermic syringes. Sexual dimorphism for individuals was determined by external observation of the shape of the plastron. The average values obtained for the ten hematological and biochemical parameters analyzed were red blood cell count = 0.32 million μL-1; hematocrit = 20.6 %; hemoglobin = 8.5 g dL-1; mean corpuscular volume = 681.6 fL; mean corpuscular hemoglobin = 267.8 pg; mean corpuscular hemoglobin concentration = 41.9 g dL-1; glucose = 80.6 mg dL-1, total protein = 4.1 g dL-1, triglycerides = 388.9 mg dL-1, and total cholesterol = 79.3 mg dL-1. Despite the sexual dimorphism evidenced for the species, there was no significant statistical difference between males and females for both hematological and biochemical parameters analyzed herein. Based on these results, the population is considered healthy, with parameter values coinciding with previously reported reference ranges for testudines species in the region. The results obtained in this study can be used for assessing the health status of other Amazonian turtle populations, especially in actions aimed at cultivation strategies, management, and species conservation.

RESUMEN La determinación de valores hematológicos se ha utilizado para conocer las condiciones sanitarias de algunas especies animales. La tortuga cabezona del río Amazonas, Peltocephalus dumerilianus, se considera una de las especies de testudines menos conocidas en relación a su biología y estado de salud. Aquí, determinamos los parámetros bioquímicos hematológicos y plasmáticos de 17 adultos (ocho machos y nueve hembras) de P. dumerilianus con el fin de proporcionar valores de intervalo de referencia sobre los individuos clínicamente sanos. Recolectamos las muestras de sangre perforando la vena femoral con jeringas hipodérmicas largas heparinizadas. El dimorfismo sexual de los individuos se determinó mediante la observación externa de la forma del plastrón. Los valores medios obtenidos para los diez parámetros hematológicos y bioquímicos analizados fueron: recuento de glóbulos rojos = 0,32 millones μL-1; hematocrito = 20,6 %; hemoglobina = 8,5 g dL-1; volumen corpuscular medio = 681,6 fL; hemoglobina corpuscular media = 267,8 pg; concentración media de hemoglobina corpuscular = 41,9 g dL-1; glucosa = 80,6 mg dL-1, proteína total = 4,1 g dL-1, triglicéridos = 388,9 mg dL-1 y colesterol total = 79,3 mg dL-1. A pesar del dimorfismo sexual evidente para la especie, no hubo diferencia estadística significativa entre machos y hembras para los parámetros hematológicos y bioquímicos analizados aquí. Con base en estos resultados, la población se considera saludable y los valores de los parámetros coinciden con los rangos de referencia reportados previamente de las especies de testudines en la región. Los resultados obtenidos en este estudio pueden utilizarse en la evaluación del estado de salud de otras poblaciones de tortugas amazónicas, considerando especialmente aquellas acciones dirigidas al manejo, conservación y estrategias de cultivo de la especie.

New evidences on the regulation of SF-1 expression by POD1/TCF21 in adrenocortical tumor cells

OBJECTIVES: Transcription Factor 21 represses steroidogenic factor 1, a nuclear receptor required for gonadal development, sex determination and the regulation of adrenogonadal steroidogenesis. The aim of this study was to investigate whether silencing or overexpression of the gene Transcription Factor 21 could modulate the gene and protein expression of steroidogenic factor 1 in adrenocortical tumors. METHODS: We analyzed the gene expression of steroidogenic factor 1 using qPCR after silencing endogenous Transcription Factor 21 in pediatric adrenal adenoma-T7 cells through small interfering RNA. In addition, using overexpression of Transcription Factor 21 in human adrenocortical carcinoma cells, we analyzed the protein expression of steroidogenic factor 1 using Western blotting. RESULTS: Transcription Factor 21 knockdown increased the mRNA expression of steroidogenic factor 1 by 5.97-fold in pediatric adrenal adenoma-T7 cells. Additionally, Transcription Factor 21 overexpression inhibited the protein expression of steroidogenic factor 1 by 0.41-fold and 0.64-fold in two different adult adrenocortical carcinoma cell cultures, H295R and T36, respectively. CONCLUSIONS: Transcription Factor 21 is downregulated in adrenocortical carcinoma cells. Taken together, these findings support the hypothesis that Transcription Factor 21 is a regulator of steroidogenic factor 1 and is a tumor suppressor gene in pediatric and adult adrenocortical tumors.

Promoção da saúde da população ribeirinha da região amazônica: relato de experiência / Health promotion on amazonic riverside population: experience report

Resumo:OBJETIVO:relatar as ações de promoção de saúde realizadas em comunidades ribeirinhas pelas equipes expedicionárias do projeto "FOB-USP em Rondônia".MÉTODOS:trata-se de um trabalho transversal observacional, que consiste em um relato de experiência de expedições interdisciplinares às comunidades de Tabajara-RO, Calama-RO, Demarcação-RO e Rio Preto-RO. A coleta de dados ocorreu por meio dos prontuários, anotação em diários de campo e observação participante. O enfoque adotado para guiar o desenvolvimento do presente trabalho está no trabalho da equipe, nos momentos de atendimento clínico e comunitário.RESULTADOS:foram atendidos 1042 ribeirinhos abrangendo ações assistenciais e educativas. As comunidades exibiram como principal fonte de renda a pesca e a agricultura de subsistência. O esgoto é coletado por fossas sépticas ou a céu aberto. A água provém dos rios ou poços artesianos. As casas em todas as comunidades, em sua maioria, são de madeira. Quanto aos procedimentos clínicos, foram realizados 974 fonoaudiológicos, sendo as principais queixas na área de audiologia, e 854 odontológicos com maior realização de exodontias e restaurações. Ações educativas foram realizadas em todas as comunidades.CONCLUSÃO:foram realizados 1828 procedimentos, dos quais 1100 foram clínicos e 728 educativos. As condições de acesso mostraram-se precárias e as alterações de saúde referentes às áreas fonoaudiológica e odontológica apresentaram-se como queixas e alterações auditivas e cárie dentária (principalmente) e doença periodontal. Verificou-se a necessidade de aprimorar o acesso das populações ribeirinhas a serviços essenciais, mantendo-se atendimentos regulares e contínuos, para a realização de atividades de educação e promoção de saúde com efetividade.

Abstract:PURPOSE:to report the health promotion actions conducted on riverside communities by the expeditionary teams of "FOB-USP in Rondônia" project.METHODS:this is a cross-sectional, observational study, which consists in an experience report of interdisciplinary expeditions to the Tabajara-RO, Calama-RO, Demarcação-RO and Rio Preto-RO communities. Data collection has occurred by means of medical records, annotations on field diaries and participant observation. The adopted approach to guide the development of this paper lies on the work of the team during the clinical care and community attention moments.RESULTS:1042 riverside patients were assisted, being covered also educative and human aid actions the communities have shown as main income sources fishing and subsistence agriculture. Sewer is collected in septic tanks in the open Water comes from rivers or artesian wells. The houses in all of the communities are mostly made of wood. In regards to clinical procedures, 974 in the speech, language and hearing sciences area, being the most common complaint on the hearing area, and 854 dental procedures being mostly performed dental extractions and restorations. Educative actions were taken in all of the communities.CONCLUSION:1828 procedures were performed, of which 1100 were clinical and 728 were educative. Access conditions have proven to be precarious and health problems linked to the speech, language and hearing sciences and dental areas were presented as complaints, with dental caries and periodontal disease as main concerns. Need for improvement on access to essential services for the riverside populations has been demonstrated, maintaining regular and continuous care for the implementation of effective educative and health promotion activities.

Humoral immune response to antigens of Porphyromonas gingivalis ATCC 33277 in chronic periodontitis

Introduction: Periodontitis is a chronic disease that results from an interaction of a mixed bacterial challenge and the host response. Objective: The purposes of this study were to evaluate the IgG serum levels to Porphyromonas gingivalis antigens by ELISA in individuals with different periodontal conditions correlated with clinical parameters, and to analyze the immunoreactivity profiles by Western blotting. Methods: Serum IgG levels against the cell sonicate antigen from P. gingivalis ATCC 33277 of 28 patients with chronic periodontitis (CP), 10 patients with gingivitis (G) and 21 periodontally healthy individuals (H) were measured by ELISA and Western immunoblotting. Results: In the CP group, sera reactivity by ELISA was significantly higher than in the G and H groups (Kruskal-Wallis p<0.001; Dunnet t3 p= 0.001 and Dunnet t3 p= 0.0001). There was no statistically significant difference between G and HP reactivity (Dunnett t3 p=0.617). Among individuals with chronic periodontitis, the IgG-anti-P. gingivalis serum levels were positively correlated with percentage of clinical attachment level =5mm (r s = + 0.375, p<0.05) and a negative correlation was found between IgG-anti-P. gingivalis levels and percentage of probing pocket depth 0-3mm (r s = - 0. 411, p< 0.05). The analysis of sera immunoreactivity profiles to sonicate antigen by Western blotting showed differences between the sera of CP, G and H group individuals. The serum from CP frequently reacted with high molecular weight (103 kDa, 86 kDa, 72 kDa, 60 kDa, 58 kDa, 52 kDa) protein fractions. Conclusions: Serum levels of IgG anti-P. gingivalis distinguished individuals with chronic periodontitis, gingivitis and healthy periodontium. There was a correlation between clinical parameters and serum IgG levels against P. gingivalis. There was a difference in the recognition profile of protein fractions among the studied groups and some bands were more specific.

Ação moduladora da microbiota de portadores de síndrome de Down sobre bactérias cariogênicas / Modulating action of microbiota of carriers of Down’s syndrome on cariogenic microbiota

Os portadores da Síndrome de Down possuem baixa prevalência de cárie, apesar de apresentarem uma higiene oral deficiente. O estudo foi realizado para detectar cepas produtoras de bacteriocinas, com o intuito de avaliar a sua possível ação como moduladoras da microbiota cariogênica dos sindrômicos. Streptococcus mutans e Streptococcus sobrinus são os principais produtores de ácidos na cavidade oral. Em função disso, esses estreptococos foram testados antagonicamente com cepas isoladas dos indivíduos com Síndrome de Down, para que fosse avaliado se havia inibição das bactérias cariogênicas pelas cepas dos sindrômicos. Streptococcus mutans e Streptococcus sobrinus foram isolados utilizando o meio ágar sacarose bacitracina e identificados através de provas bioquímicas. Uma amostra de placa supragengival dos sindrômicos foi coletada e semeada por disseminação em Ágar Mitis-Salivarius, após o isolamento foram cultivados em caldo BHI. Dez culturas dos sindrômicos foram semeadas no Ágar Muller Hinton. Após o crescimento, as placas foram tratadas com clorofórmio. Sucessivamente, uma porção das culturas de bactérias cariogênicas foi transferida para meio semi-sólido BHI e vertidas nas placas com as culturas testes. Duas das dez cepas testadas apresentaram halo de inibição de crescimento contra cepas de Streptococcus mutans. A descoberta de cepas que produzem substâncias antimicrobianas pode ser responsável pela baixa prevalência de cárie em indivíduos com Síndrome de Down. Esse achado pode ajudar a entender a relação entre microbiota oral e patologia da cárie dentária.

Pediatric knowledge about acute viral hepatitis

Knowledge about hepatotropic viruses is crucial for pediatricians because of the high prevalence of viral hepatitis during childhood. The multiplicity of hepatotropic viruses, the spectrum of acute and chronic infections, and the sequels of viral hepatitis result in a need for physicians to better understand the clinical and epidemiological context of patients with viral hepatitis, as well as the importance of prevention measures for hepatitis. A descriptive cross-sectional study was made of pediatrician's knowledge about viral hepatitis, through questionnaires to 574 pediatricians, with no obligation of identification. The pediatricians were recruited among those who attended a national Congress of Pediatrics in Brasília, Brazil. Among these pediatricians, 50.1 percent frequently treated cases of hepatitis, and 74.7 percent indicated that they had knowledge of the existence of five hepatotropic viruses; 14.5 percent knew about at least four types of hepatitis complications, while only 7.7 percent and 4.3 percent were able to correctly diagnose viral hepatitis A and B, respectively. Many (28.4 percent) did not know how to treat the patients adequately. Only 37.5 percent had already recommended vaccination against hepatitis B. Only 50.2 percent of the pediatricians had been vaccinated against hepatitis B. We concluded that it is crucial to make pediatricians more knowledgeable about viral hepatitis, through continued education programs, especially emphasizing prevention procedures.

Microbiota estreptocócica associada com a formação inicial da placa dental / Streptococci microbiota associated with initial formation of dental plaque

A identificação e quantificação dos estreptococos que iniciam a colonização das superfícies dos dentes no biofilme complexo da placa dental, bem como a compreensão das relações funcionais entre eles e outros membros são fundamentais para avaliar e, possivelmente, controlar os processos que essa comunidade inicial desempenha. Placas bacterianas foram assepticamente removidas de dentes de pessoas sadias e experimentalmente inoculadas em unidades dentárias esterilizadas e previamente cobertas com fluido oral estéril. Cada unidade dentária (UD) inoculada foi incubada a 37°C em uma câmara úmida. Após a formação da placa, a UD foi colocada em uma solução desagregadora da placa (SD) constituída de 0,1 por cento de água peptonada, 0,1 por cento de tween 80 e 0,5 por cento de areia calcinada (p/v). A UD foi submetida sucessiva e progressivamente à agitação em cinco velocidades controladas. Imediatamente após cada agitação, alíquotas foram coletadas e diluídas em tampão fosfato estéril, pH 7,2. Após cada coleta das alíquotas e antes de nova agitação, a UD era lavada em solução PBS estéril e novamente imersa em nova SD. Experimentos in vivo foram também efetuados, em unidades dentárias recentemente extraídas, usando a mesma técnica. Alíquotas das diluições foram semeadas na superfície de meios apropriados por disseminação usando uma alça de Drigalsky. As placas foram incubadas a 37°C em anaerobiose por 48 horas, quando, então, efetuavam-se as contagens total e diferencial das colônias. Cada tipo colonial era subcultivado e submetido a testes padrões para identificação bacteriana. De um total de 86 culturas de Streptococcus estudadas, 40,7 por cento foram identificadas como S. sanguis, 37,2 por cento como S. oralis, 8,1 por cento como S. mutans, 7,0 por cento como S. gordonii e 7,0 por cento como outros estreptococos. Análises das culturas isoladas da última velocidade de agitação mostraram que, nos experimentos in vitro, somente S. oralis (66,7 por cento) e S. sanguis...