MOLECULAR ASSESSMENT OF FECAL LACTOBACILLI POPULATIONS IN CHILDREN WITH FUNCTIONAL CONSTIPATION.

BACKGROUND: Investigation of the gut-specific bacterial strains including lactobacilli is essential for understanding the bacterial etiology of constipation. OBJECTIVE: This study aimed to compare the prevalence and quantity of intestinal lactobacilli in constipated children and healthy controls. METHODS: Forty children fulfilling Rome IV criteria for functional constipation and 40 healthy controls were recruited. Fecal samples were analyzed using species-specific polymerase chain reaction followed by random amplified polymorphic DNA-PCR and quantitative real-time PCR. RESULTS: Totally, seven different species of lactobacilli were detected. Out of 80 volunteers, 65 (81.3%) were culture and species-specific PCR positive from which 25 (38.46%) constipated children and 40 (61.54%) healthy subjects. The most prevalent species were L. paracasei 21 (32.3%) followed by L. plantarum 18 (27.7%) among both healthy and patient groups. Analysis of the RAPD dendrograms displayed that strains isolated from constipated and non-constipated children have similarity coefficients of more than 90%. The qPCR assays demonstrated constipated children had a lower amount of total lactobacilli population (per gram of feces) than healthy controls. CONCLUSION: Our findings showed that the mere existence of various species of Lactobacillus in the gut does not enough to prevent some gastrointestinal disorders such as functional constipation, and their quantity plays a more important role.

Avaliação molecular de populações fecais de lactobacilos em crianças com prisão de ventre funcional / Molecular assessment of fecal lactobacilli populations in children with functional constipation

ABSTRACT Background: Investigation of the gut-specific bacterial strains including lactobacilli is essential for understanding the bacterial etiology of constipation. Objective: This study aimed to compare the prevalence and quantity of intestinal lactobacilli in constipated children and healthy controls. Methods: Forty children fulfilling Rome IV criteria for functional constipation and 40 healthy controls were recruited. Fecal samples were analyzed using species-specific polymerase chain reaction followed by random amplified polymorphic DNA-PCR and quantitative real-time PCR. Results: Totally, seven different species of lactobacilli were detected. Out of 80 volunteers, 65 (81.3%) were culture and species-specific PCR positive from which 25 (38.46%) constipated children and 40 (61.54%) healthy subjects. The most prevalent species were L. paracasei 21 (32.3%) followed by L. plantarum 18 (27.7%) among both healthy and patient groups. Analysis of the RAPD dendrograms displayed that strains isolated from constipated and non-constipated children have similarity coefficients of more than 90%. The qPCR assays demonstrated constipated children had a lower amount of total lactobacilli population (per gram of feces) than healthy controls. Conclusion: Our findings showed that the mere existence of various species of Lactobacillus in the gut does not enough to prevent some gastrointestinal disorders such as functional constipation, and their quantity plays a more important role.

RESUMO Contexto: A investigação das cepas bacterianas específicas do intestino, incluindo lactobacilos, é essencial para a compreensão da etiologia bacteriana da prisão de ventre. Objetivo: Este estudo teve como objetivo comparar a prevalência e a quantidade de lactobacilos intestinais em crianças constipadas e controles saudáveis. Métodos: Foram recrutadas quarenta crianças que preenchem os critérios de Roma IV para prisão de ventre funcional e 40 controles saudáveis. As amostras fecais foram analisadas utilizando-se uma reação da cadeia de polimerase específica da espécie, seguida por DNA polimórfico amplificado aleatório e PCR quantitativo em tempo real. Resultados: Foram detectadas sete espécies diferentes de lactobacilos. Dos 80 voluntários, 65 (81,3%) eram cultura em PCR específico de espécies, dos quais 25 (38,46%) crianças constipadas e 40 (61,54%) indivíduos saudáveis. As espécies mais prevalentes foram L. paracasei 21 (32,3%) seguidas por L. plantarum 18 (27,7%) entre grupos saudáveis e de pacientes. A análise dos dendrogramas do RAPD mostrou que cepas isoladas de crianças constipadas e não constipadas têm coeficientes de similaridade superiores a 90%. Os ensaios qPCR demonstraram que as crianças constipadas apresentavam uma quantidade menor de população total de lactobacilos (por grama de fezes) do que os controles saudáveis. Conclusão: Nossos achados mostraram que a mera existência de várias espécies de Lactobacillus no intestino não é suficiente para prevenir alguns distúrbios gastrointestinais, como a prisão de ventre funcional, e sua quantidade desempenha um papel mais importante.

The Effect of Different Doses of Melatonin on in Vitro Maturation of Human Follicular Fluid-Derived Oocyte-Like Cells.

OBJECTIVE: Human follicular fluid (FF) contains different cell populations including mesenchymal stem cells. Studies tried to improve their differentiation to oocyte and use them in infertility treatments. Using an antioxidant may improve the quality of these cells. The present study investigated the effects of different doses of melatonin on FF-derived cells grown to oocyte-like cells (OLC). METHODS: Cell viability (MTT assay), flow cytometry, and ICC staining were utilized to evaluate CD105 and CD34 expression; colony forming unit assay (CFU-F) capability, qRT-PCR were used to investigate ZP1, ZP2, ZP3, GDF9, and SCP3 expression. AMH, Estradiol and Progesterone levels in the supernatant were measured. Morphological characteristics of fibroblast-like cells changing to a round shape were seen specifically in the group treated with melatonin 10-7M after 2 weeks. RESULTS: There was no difference between control and treatment groups for MTT and CFU assays. ICC staining was positive for CD105 marker and negative for CD34 hematopoietic stem cell marker. qRT-PCR results indicated that ZP1, ZP2, GDF9, and SCP3 expression increased in the group treated with melatonin 10-7M in Week 2, while ZP3 decreased in this group. Progesterone and AMH were detected in differentiation medium. CONCLUSIONS: Melatonin may improve in vitro formation of OLCs.