Quorum-quenching enzyme Est816 assisted antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in rats

IntroductionQuorum-quenching enzyme Est816 hydrolyzes the lactone rings of N-acyl homoserine lactones, effectively blocking the biofilm formation and development of Gram-negative bacteria. However, its applications in the oral field is limited. This study aimed to evaluate the efficacy of enzyme Est816 in combination with antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in vitro and in vivo.MethodsThe antimicrobial efficacy of enzyme Est816 in combination with minocycline, metronidazole, and amoxicillin was determined using the minimum inhibitory concentration test. The anti-biofilm effect of enzyme Est816 was assessed using scanning electron microscopy, live/dead bacterial staining, crystal violet staining, and real-time quantitative PCR. Biocompatibility of enzyme Est816 was assessed in human gingival fibroblasts (HGF) by staining. A rat model of periodontitis was established to evaluate the effect of enzyme Est816 combined with minocycline using micro-computed tomography and histological staining.ResultsCompared to minocycline, metronidazole, and amoxicillin treatment alone, simultaneous treatment with enzyme Est816 increased the sensitivity of biofilm bacteria to antibiotics. Enzyme Est816 with minocycline exhibited the highest rate of biofilm clearance and high biocompatibility. Moreover, the combination of enzyme Est816 with antibiotics improved the antibiofilm effects of the antibiotics synergistically, reducing the expression of the virulence factor leukotoxin gene (ltxA) and fimbria-associated gene (rcpA). Likewise, the combination of enzyme Est816 with minocycline exhibited a remarkable inhibitory effect on bone resorption and inflammation damage in a rat model of periodontitis.DiscussionThe combination of enzyme Est816 with antibiotics represents a prospective anti-biofilm strategy with the potential to treat periodontitis

Identification of Novel Biallelic TLE6 Variants in Female Infertility With Preimplantation Embryonic Lethality

Preimplantation embryonic lethality is a rare cause of primary female infertility. It has been reported that variants in the transducin-like enhancer of split 6 (TLE6) gene can lead to preimplantation embryonic lethality. However, the incidence of TLE6 variants in patients with preimplantation embryonic lethality is not fully understood. In this study, we identified four patients carrying novel biallelic TLE6 variants in a cohort of 28 patients with preimplantation embryonic lethality by whole-exome sequencing and bioinformatics analysis, accounting for 14.29% (4/28) of the cohort. Immunofluorescence showed that the TLE6 levels in oocytes from patients were much lower than in normal control oocytes, suggesting that the variants result in the lower expression of the TLE6 protein in oocytes. In addition, a retrospective analysis showed that the four patients underwent a total of nine failures of in vitro fertilization and intracytoplasmic sperm injection attempts, and one of them became pregnant on the first attempt using donated oocytes. Our study extends the genetic spectrum of female infertility caused by variants in TLE6 and further confirms previously reported findings that TLE6 plays an essential role in early embryonic development. In such case, oocyte donation may be the preferred treatment

The effect and regulation mechanism of CircHIAT1 on polycystic ovary syndrome

Parte I: Expresión diferencial del circRNA en el síndrome de ovario poliquístico (SOP) Resumen Objetivo: Se ha encontrado que el ARN circular (ARNrc) desempeña un papel importante en muchas enfermedades, sin embargo, ha habido una investigación limitada centrada en el ARNcc en la enfermedad del SOP. Por lo tanto, intentamos detectar la expresión de circRNAs en el cumulus oophorus (CO) de pacientes con PCOS mediante secuenciación de alto rendimiento, para explorar el papel y el posible mecanismo de circRNAs en la patogénesis de PCOS. Métodos: Los CO de pacientes de FIV / ICSI se recolectaron al azar. Seleccionamos 3 pares de muestras para secuenciación de alto rendimiento. Se seleccionaron 14 circRNA expresados diferencialmente para la validación clínica en CO. Resultados: Se detectaron un total de 55,776 circRNAs expresados diferencialmente. En comparación con el grupo de control, 7,047 circRNAs fueron significativamente regulados por incremento y 48,729 fueron significativamente regulados por disminución en el grupo de SOP. El análisis GO mostró que los circRNA podrían ser activos en el metabolismo celular, la regulación biológica y la catálisis activa del SOP. El análisis de la ruta KEGG indicó que los circRNA podrían participar en la transducción de señales, el transporte de sustancias y el catabolismo, la endocitosis y la vía de proteólisis mediada por ubiquitina. En pacientes con SOP, la expresión de circ_0011693, circ_0042454, circ_0001365, circ_0006252 y circ_0003737 fue significativamente mayor y la expresión de circHIAT1 fue significativamente menor. Conclusiones: El estudio encontró los perfiles de expresión diferencial de circRNA en SOP por primera vez. El análisis bioinformático mostró que los circRNA expresados diferencialmente podrían participar en múltiples vías relacionadas con las enfermedades de SOP. Por lo tanto, el efecto y la regulación de circRNA en SOP merecen un estudio más a fondoParte II: Los efectos de CircHIAT1 sobre la esteroidogénesis en el síndrome de ovario poliquístico mediante esponja de miR-195-5p Objetivo: Nuestra hipótesis es que circHIAT1 podría regular la función de los CO esponjando miR-195-5p, participando así en la regulación del SOP. Métodos: El estudio detectó la co-localización de circHIAT1 y miR-195-5p en CO por RNA-FISH. Se utilizaron qRT-PCR y Western Blot para detectar los efectos de circHIAT1 en AR, CYP19A, GATA4 y VEGF en la línea celular KGN pretratada con DHT. Se utilizó FACS para detectar el ciclo celular. La relación de direccionamiento de circHIAT1 / miR-195-5p y miR-195-5p / CCNE1 se detectó mediante un ensayo de indicador de luciferasa dual. Se detectó la expresión de miR-195-5p y CCNE1 en muestras clínicas. Se verificaron los efectos de miR-195-5p y CCNE1 en AR, CYP19A, GATA4, VEGF y ciclo celular. Resultados: circHIAT1 y miR-195-5p se localizaron en el citoplasma de CO primarias y células KGN. El silenciamiento circHIAT1 podría aumentar significativamente los niveles de AR, CYP19A, GATA4 y VEGF, y conducir a la disminución de la viabilidad celular en las células KGN. circHIAT1 podría unirse a miR-195-5p. miR-195-5p se encontró altamente expresado en COs de pacientes con SOP. La sobreexpresión de miR-195-5p podría aumentar significativamente los niveles de AR, CYP19A, GATA4 y VEGF en células KGN, y dio como resultado una disminución significativa de la viabilidad celular. miR-195-5p podría unirse a CCNE1. El nivel de expresión de CCNE1 en el grupo de SOP fue significativamente menor. Conclusión: el silenciamiento de circHIAT1 podría aumentar significativamente los indicadores relacionados con la esteroidogénesis, inhibir el ciclo celular de las células KGN. Por lo tanto, circHIAT1 podría utilizarse como un nuevo objetivo para el diagnóstico y tratamiento del SOPPart I: Differential Expression of CircRNA in Polycystic Ovary Syndrome（PCOS） Abstract Objective: Circular RNA (circRNA) has been found to play an important role in many diseases, however, there has been limited research focusing on the circRNA in PCOS disease. Therefore, we intended to detect the expression of circRNAs in cumulus granulosa cells (CCs) of PCOS patients by high-throughput sequencing, to explore the role and possible mechanism of circRNAs in the pathogenesis of PCOS. Methods: The CCs from IVF/ICSI patients were collected, randomly. We selected 3 pairs of samples for high-throughput sequencing. 14 differentially expressed circRNAs were selected for clinical validation in CCs. Results: A total of 55,776 differentially expressed circRNAs was detected. Compared with the control group, 7,047 circRNAs were significantly up-regulated and 48,729 were significantly down-regulated in PCOS group. GO analysis showed circRNAs might be active in cell metabolism, biological regulation and active catalysis of PCOS. KEGG pathway analysis indicated that circRNAs could participate in signal transduction, substance transport and catabolism, endocytosis and ubiquitin mediated proteolysis pathway. The expression of circ_0011693, circ_0042454, circ_0001365, circ_0006252 and circ_0003737 were significantly higher in PCOS patients, while the expression of circHIAT1 was significantly lower in PCOS patients. Conclusions: The study found the differential expression profiles of circRNA in PCOS for the first time. Bioinformatics analysis showed that differentially expressed circRNAs could participate in multiple pathways related to PCOS diseases. Therefore, the effect and regulatory of circRNA on PCOS deserve further study. Part II: The Effects of CircHIAT1 on steroidogenesis in polycystic ovary syndrome by sponging miR-195-5p Abstract Objective: We hypothesised circHIAT1 could regulate the function of CCs by sponging miR-195-5p, thus participating in the regulation of PCOS. Methods: The study detected the co-localization of circHIAT1 and miR-195-5p in CCs by RNA-FISH. qRT-PCR and Western Blot were used to detect the effects of circHIAT1 on AR, CYP19A, GATA4, and VEGF in DHT pretreated KGN cell line. FACS was used to detect the cell cycle. The targeting relationship of circHIAT1/miR-195-5p, and miR-195-5p/CCNE1 were detected by dual-luciferase reporter assay. The expression of miR-195-5p and CCNE1 in clinical samples was detected. The effects of miR-195-5p and CCNE1 on AR, CYP19A, GATA4, VEGF, and cell cycle were verified. Results: circHIAT1 and miR-195-5p were both localized in the cytoplasm of primary CCs and KGN cells. circHIAT1 silencing could significantly increase the levels of AR, CYP19A, GATA4, and VEGF, and lead to the decrease of cell viability in KGN cells. circHIAT1 could bind to miR-195-5p. miR-195-5p was found highly expressed in CCs of PCOS patients. Overexpression of miR-195-5p could significantly increased the levels of AR, CYP19A, GATA4, and VEGF in KGN cells, and resulted in a significant decrease of cell viability. miR-195-5p could bind to CCNE1. The expression level of CCNE1 in PCOS group was significantly lower. Overexpression of CCNE1 could significantly decreased the levels of AR, CYP19A, GATA4 and VEGF, and increased the cell viability in KGN cells. miR-195-5p mimic could inhibit the regulation of circHIAT1 pEX-3 on AR, CYP19A, GATA4 and VEGF. Conclusion: Silencing circHIAT1 could significantly increase steroidogenesis related indicators, inhibit the cell cycle of KGN cells. Thus, circHIAT1 might be used as a new target for the diagnosis and treatment of PCOS