The formation of the enteric nervous system (ENS) primarily involves the migration of enteric neural crest-derived cells (ENCCs) and the subsequent maturation of enteric neurons. The developmental dysfunction of ENCCs and enteric neurons can result in ENS disorders, such as hypoganglionosis (HG). Although neurite outgrowth is fundamental to neuronal maturation, the mechanisms by which neurite outgrowth influences neuronal maturation remain poorly defined. Here, we identified EMB as a critical regulator of enteric neuronal maturation. In EMB mutant patients, the expression of EMB is reduced in the enteric neurons of the HG-affected colon. In mice, knockdown of Emb exhibited HG-like features and defects. In vitro experiments, along with analyses using Smart-seq2 and immunoprecipitation-mass spectrometry, demonstrated that EMB is essential for autophagic flux and physically interacts with ATG7, recruiting it to the autophagosomal membrane to facilitate autophagosome formation, and then EMB/ATG7-mediated autophagy promotes neurite outgrowth. Our findings elucidate EMB-mediated autophagy as a pivotal pathway in regulating neurite outgrowth and promoting the maturation of enteric neurons, which provides a mechanistic basis for understanding ENS disorders.

BACKGROUND:The enteric nervous system (ENS), which arises from enteric neural crest cells (ENCCs), plays important roles in many aspects of gastrointestinal tract function, including motility, secretions, blood flow and hormone release. Defects in ENS development could lead to a broad range of disorders, including Hirschsprung's disease (HSCR), which is characterized by missing nerve cells in the distal segment of the colon. Here, we identify EMB as an evolutionarily conserved regulator of ENS development. METHODS:We first examined EMB expression in human and mouse intestines using scRNA-seq data and immunofluorescence staining. To investigate its role in ENS development, we constructed Emb-knockout zebrafish and mouse models. To explore the underlying mechanisms, we focused on ENCCs and analyzed their proliferation and migration using migration assays in explant guts and organoid cultures. Finally, we assessed rare EMB variants in a cohort of HSCR patients. RESULTS:In zebrafish, loss of emb leads to a decrease number of enteric neurons and impaired intestinal transit ability. In mice, knockout of Emb causes HSCR-like phenotypes and defects. In vitro experiments, including explant mouse gut and organoid cultures, show that EMB is required for both the proliferation and migration of ENCCs. Mechanistically, EMB binds to and recruits the phosphatase complex PP2A to the cellular membrane to facilitate the activation of PI3K-AKT pathway, thereby promoting ENCCs development. Indeed, application of PI3K or AKT agonists partially restores the ENS developmental defects in zebrafish emb mutants. Furthermore, rare variants of EMB may potentially contribute to the pathology of HSCR in humans. CONCLUSIONS:EMB is required for ENS development by regulating the proliferation and migration of the ENCCs. Mechanistically, EMB recruits PP2A to the cell membrane, reducing cytoplasmic dephosphorylation activity and promoting the activation of the PI3K signaling pathway.

Hirschsprung's disease (HSCR) is a congenital enteric neuropathic disorder characterized by high heritability (>80%) and polygenic inheritance (>20 genes). The previous genome-wide association studies (GWAS) identified several common variants associated with HSCR and demonstrated increased predictive performance for HSCR risk in Europeans using a genetic risk score, there remains a notable gap in knowledge regarding Chinese populations. We conducted whole exome sequencing in a HSCR case cohort in Chinese. By using the common controls (505 controls from 1KG EAS and 10 588 controls from ChinaMAP), we conducted GWAS for the common variants in the exome and gene-based association for rare variants. We further validated the associated variants and genes in replicated samples and in vitro and vivo experiments. We identified one novel gene PLK5 by GWAS and suggested 45 novel putative genes based the gene-based test. By using genetic variant at RET and PLK5, we constructed a genetic risk score that could identify the individuals with very high genetic risk for HSCR. Compared with patients with zero or one risk allele from the three variants, the risk for HSCR was 36.61 times higher with six alleles. In addition, we delineated a HSCR risk gene landscape that encompasses 57 genes, which explains 88.5% and 54.5% of HSCR in Chinese and European, respectively. In summary, this study improved the understanding of genetic architecture of HSCR and provided a risk prediction approach for HSCR in the Chinese.

Neuroblastoma is one of the highest incidence solid tumors of childhood. Therapy for neuroblastoma with MYCN proto-oncogene (MYCN) amplification is of great challenge, due to its aggressive invasion and chemoresistance. In our study, differentially expressed genes (DEGs) between MYCN-amplified and non-amplified neuroblastoma tissues were analyzed using "limma" R package. Then, DEGs were screened by silencing or enhancing expression of MYCN in neuroblastoma cells, and distal-less homeobox 5 (DLX5) was identified to be the downstream target gene. The positive correlation between DLX5 and MYCN expression was confirmed in neuroblastoma tissues and cell lines. Overall survival analysis suggested that high expression of DLX5 predicted poor clinical outcome.Then, DLX5 was demonstrated to promote proliferation and migration of neuroblastoma. Gene set enrichment analysis revealed that base excision repair and nucleotide excision repair processes were enriched in samples with DLX5 high expression. Thus, the half-maximal inhibitory concentration was measured and cisplatin treatment assay was carried out. The results revealed DLX5 promoted cisplatin chemoresistance of neuroblastoma. To explore the mechanism, mRNA sequencing was conducted and AKT signaling was suggested to be the mainly regulatory pathway, which was further verified in cells and tumor tissues. Then, we proved that heart and neural crest derivatives expressed 2, paired like homeobox 2B, GATA binding protein 3, and MYCN coregulated the expression of DLX5 at the transcriptional level, and identified the direct binding site of MYCN on DLX5 promoter. Finally, we demonstrated that DLX5 which was transcriptionally activated by MYCN, promoted growth, metastasis and chemoresistance of neuroblastoma through enhanced AKT phosphorylation. The findings in our study provided new insight for progression and chemoresistance of neuroblastoma.

N6-methyladenosine (m6A) is the most abundant modification controlling RNA metabolism and cellular functions, but its roles in placental development are still poorly understood. Here, we characterized the synchronization of m6A modifications and placental functions by mapping the m6A methylome in human placentas (n = 3, each trimester), revealing that the dynamic patterns of m6A were associated with gene expression homeostasis and different biological pathways in placental development. Then, we generated trophoblast-specific knockout mice of Wtap, a critical component of methyltransferase complex, and demonstrated that Wtap was essential for trophoblast proliferation, placentation and perinatal growth. Further in vitro experiments which includes cell viability assays and series molecular binding assays demonstrated that WTAP-m6A-IGF2BP3 axis regulated the RNA stability and translation of Anillin (ANLN) and VEGFA, promoting trophoblast proliferation and secretion. Dysregulation of this regulatory axis was observed in placentas from pregnancies with fetal growth restriction (FGR) or preeclampsia, revealing the pathogenic effects of imbalanced m6A modifications. Therefore, our findings provide novel insights into the functions and regulatory mechanisms of m6A modifications in placental development and placental-related gestational diseases.

In this editorial, we comment on the article by Chen et al . Metabolic dysfunction-associated fatty liver disease (MAFLD) is a global public health burden whose incidence has risen concurrently with overweight and obesity. Given its detrimental health impact, early identification of at-risk individuals is crucial. MAFLD diagnosis is based on evidence of hepatic steatosis indicated by liver biopsy, imaging, or blood biomarkers, and one of the following conditions: Overweight/obesity, type 2 diabetes mellitus, or metabolic dysregulation. However, in large-scale epidemiological studies, liver biopsies are not feasible. The application of techniques such as ultrasonography, computed tomography, magnetic resonance imaging, and magnetic resonance spectroscopy is restricted by their limited sensitivity, low effectiveness, high costs, and need for specialized software. Blood biomarkers offer several advantages, particularly in large-scale epidemiological studies or clinical scenarios where traditional imaging techniques are impractical. Analysis of cumulative effects of excess high-normal blood alanine aminotransferase (ALT) levels of blood ALT levels could facilitate identification of at-risk patients who might not be detected through conventional imaging methods. Accordingly, investigating the utility of blood biomarkers in MAFLD should enhance early detection and monitoring, enabling timely intervention and management and improving patient outcomes.

During enteric nervous system (ENS) development, pioneering wavefront enteric neural crest cells (ENCCs) initiate gut colonization. However, the molecular mechanisms guiding their specification and niche interaction are not fully understood. We used single-cell RNA sequencing and spatial transcriptomics to map the spatiotemporal dynamics and molecular landscape of wavefront ENCCs in mouse embryos. Our analysis shows a progressive decline in wavefront ENCC potency during migration and identifies transcription factors governing their specification and differentiation. We further delineate key signaling pathways (ephrin-Eph, Wnt-Frizzled, and Sema3a-Nrp1) utilized by wavefront ENCCs to interact with their surrounding cells. Disruptions in these pathways are observed in human Hirschsprung's disease gut tissue, linking them to ENS malformations. Additionally, we observed region-specific and cell-type-specific transcriptional changes in surrounding gut tissues upon wavefront ENCC arrival, suggesting their role in shaping the gut microenvironment. This work offers a roadmap of ENS development, with implications for understanding ENS disorders.

Abstract Background The prevalence of malnutrition is unknown in patients with Hirschsprung disease. Undernutrition is associated with poor clinical outcomes. This study aims to describe the nutrition status among patients with Hirschsprung disease at admission. Methods We retrospectively used data from children with Hirschsprung disease admitted to three pediatric surgery centers in China from January 2016 to December 2020. The weight‐for‐age z scores (WAZ), height‐for‐age z scores (HAZ), and body mass index‐for‐age z scores (BAZ) were calculated as the reference for nutrition risk according to the World Health Organization child growth standards. The nutrition status of enrolled children was described and nutrition risk in each clinical characteristic was compared. The association between nutrition status and clinical outcomes was analyzed using univariate and multivariate logistic regression. Results A total of 624 patients were included in this study. The mean WAZ, HAZ, and BAZ of all patients was −0.64 ± 1.40, −0.45 ± 1.78, and −0.43 ± 1.50, respectively. Moderate to severe overall undernutrition was 16.3% (102/624). We found that WAZ and BAZ were significantly reduced with the length of aganglionic segments (P = 0.001). Children who had a definitive surgery at 3 years of age or older had significantly lower HAZ (P = 0.001). A multivariate regression model assessing postoperative Hirschsprung‐associated enterocolitis showed that the WAZ was one of the independent risk factors (P = 0.001). Conclusion Undernutrition is prevalent among children with Hirschsprung disease. Nutrition assessment to identify individuals at risk of undernutrition for further intervention is necessary.

As a medium among pregnant women, environment and fetus, placenta owns powerful and delicate epigenetic processes to regulate gene expression and maintain cellular homeostasis. N6-methyladenosine (m6A) is the most prevalent modification that determines the fate of RNA, and its dynamic reversibility indicates that m6A may serve as a sensitive responder to environmental stimuli. Emerging evidence suggests that m6A modifications play an essential role in placental development and maternal-fetal crosstalk, and are closely related to gestational diseases. Herein, we summarized the latest techniques for m6A sequencing and highlighted current advances of m6A modifications in maternal-fetal crosstalk and the underlying mechanisms in gestational diseases. Therefore, proper m6A modifications are important in placental development, but its disturbance mainly caused by various environmental factors can lead to abnormal placentation and function with possible consequences of gestational diseases, fetal growth and disease susceptibility in adulthood.

Objective:To employ body weight as a reference index for nutritional risk screening to assess the nutritional status of hospitalized children with Hirschsprung disease (HSCR) and provide evidence-based rationales for clinical nutritional management.Methods:From January 2006 to December 2019, clinical data were reviewed for 472 hospitalized HSCR children. There were 386 boys and 86 girls with a median age of 10(0-120) months. Weight-for-age z score (WAZ) was calculated as an evaluation index of nutritional status using a standardized growth curve of height and weight of WHO 2006 as a reference. And WAZ < -2 was defined as moderate-to-severe malnutrition. To assess the overall nutritional status of HSCR children and compare the nutritional risk differences in children with different age of onset, different clinical types of HSCR and whether or not receiving conservative treatment.Results:The overall WAZ was (-0.81±1.56) and moderate-to-severe malnutrition accounted for 18.2%(86/472). According to diagnostic age, they were divided into 5 groups of ≤6 months, 6 months < age ≤12 months, 12 months < age≤24 months, 24 months < age ≤60 months and >60 months. The median body weight was 6.0, 8.0, 10.0, 13.7 and 21.7 kg. And WAZ of five groups were (-0.68±1.47), (-0.70±1.45), (-0.92±1.67), (-1.15±1.79) and (-1.29±1.51) respectively and there was no significant inter-group difference ( P=0.15). WAZ of children with different clinical types, namely short-segment, common, long-segment and pancolonic, were (-0.62±1.53), (-0.81±1.47), (-1.06±1.75), and (-2.20±1.79) respectively and the difference was statistically significant ( P=0.001). No significant differences existed in the incidence of WAZ or moderate-to-severe malnutrition between recipients and non-recipients of conservative treatment ( P>0.05). The results of correlation analysis hinted at a significant weak correlation between WAZ and whether or not postoperative enteritis occurring (r=0.21, P=0.001). Conclusions:Malnutrition is a prevalent condition among HSCR children. And clinical classification of HSCR is a influencing factor of malnutrition risk. Moderate-to-severe malnutrition is a significant influencing factor of occurrence of postoperative complications. Weight-for-age Z score may be employed as a simple and easy screening tool. Nutritional assessment should be performed and appropriate and timely nutritional intervention offered to children at a risk of malnutrition.

Background Preoperative evaluation of the dysganglionic bowel segment is critical for establishing the optimal resection strategy for Hirschsprung’s disease (HSCR), which facilitates patient outcomes. Objective We set out to determine the utility of the 24-h delayed film of barium retention in predicting the length of dysganglionic bowel segment in HSCR. Materials and methods A retrospective study of patients with clinically suspicious HSCR who underwent a preoperative 24-h delayed film of barium enema and were surgically treated from January 2015 to December 2019 was conducted. Results Two hundred and 58 patients were enrolled in this study. The sensitivity, specificity, positive and negative predictive values (NPVs) of the 24-h delayed film of barium enema to predict the neuropathological segment were 89.1, 91.5, 91.3, and 89.4%, respectively. The Youden index was 80.6%, with a kappa value of 0.806 (P < 0.001). The correlation rate between barium retention level and pathological results was 72.7% (16/22) when aganglionosis was restricted within the mid-distal rectum (short-segment type), increasing to 92.0% (46/50) and 93.5% (174/186) for patients that had aganglionosis extended beyond the mid-distal rectum (classical type) and sigmoid colon (long-segment type), respectively. Lastly, patients younger than 3 months showed a lower correlation rate (72.2%) compared to patients aged 3–12 months (91.0%) and > 12 months (92.6%). Conclusions Our investigation of the 24-h delayed film of barium enema performed for patients suspected of having HSCR indicated that the barium retention level remains crucial in predicting dysganglionic bowel segment, which contributes to the decision-making for surgical physicians.

Background: Previous studies have shown that oxidative stress contributes to hyperglycemia-induced erectile dysfunction. A preferential direct inhibitor of NOX1 and NOX4, GKT-137831, exhibited a strong anti-oxidative role via blockade of reactive oxygen species (ROS) generation in endothelial cells, but whether GKT-137831 could improve erectile function was not clear. Aim: Our study was designed to investigate the effect of NOX1/4 inhibition on improving diabetic erectile dysfunction (ED) in rats. Methods: We used streptozotocin to induce type 1 diabetes mellitus (DM) in 32 male Sprague Dawley (SD) rats (8 weeks old). Eight weeks later, type 1 diabetes mellitus-induced erectile dysfunction (DMED) in rats was confirmed using an apomorphine test. Our study consisted of 3 groups: (i) nondiabetic control group (n = 8), (ii) DMED + vehicle group (DMED group; n = 8), and (iii) DMED + GKT-137831 group (n = 9); GKT-137831 was given as a once-daily intraperitoneal injection for 4 weeks. Cavernous nerve electrostimulation was used to evaluate erectile function. Western blot, ELISA, immunohistochemistry, and immunofluorescence were used to measure expression of specific proteins, and DHE fluorescent probe was performed to detect ROS level. Outcomes: Intracavernous pressure (ICP), nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway, oxidative stress level, inflammatory response, corporal autophagy, and apoptosis were measured. Results: Erectile function in the DMED group was significantly impaired compared to the nondiabetic control group, whereas this impairment was improved with GKT-137831 treatment by 70%. Similarly, endothelial function and overactivated oxidative stress in the corpus cavernosum (CC) of the DMED + GKT-137831 group were improved. The DMED group showed serious inflammatory responses and excessive autophagy, which were inhibited by GKT-137831 treatment in the DMED + GKT-137831 group. Clinical Translation: Our study showed improvement in erectile function with GKT-137831 in a diabetic rat ED model. Strength and Limitations: This study suggested for the first time that GKT-137831, an NOX1/4 inhibitor undergoing clinical trials, is effective in improving erectile function in rats with type 1 DMED. However, we only investigated GKT-137831 treatment of streptozotocin-induced type 1 diabetic rats, and therapeutic evidence in other types of diabetes is lacking. Conclusion: GKT-137831 improves erectile function by 70% in type 1 DMED rats and constitutes a promising compound for the treatment of type 1 DMED, likely by inhibition of overactivated oxidative stress, down-regulation of proinflammatory factors, and amelioration of excessive autophagy and endothelial function. Copyright (C) 2021, International Society of Sexual Medicine. Published by Elsevier Inc. All rights reserved.

Background: Several studies report that sexuality is often affected by inflammatory bowel diseases (IBD). The aim of this meta-analysis was to investigate the association between IBD and sexual function. Methods: A literature search was conducted in PubMed, Web of Science, and EMBASE databases (up to September 1, 2020). Scores of sexual functions with a standard deviation and odds ratio (OR) or relative risk (RR) with a 95% CI were used to analysis the association between IBD and sexual function. Results: Eleven studies with 7,018 male IBD cases and 1,803 female IBD cases were included in the meta-analysis. In male individuals, the pooled results revealed that IBD was significantly associated with impaired erectile function and poor sexual satisfaction (RR for erectile function =1.50, 95% Cl: 1.22 to 1.84, P<0.0001; standard mean difference for sexual satisfaction =-0.24, 95% CI: -0.33 to -0.15, P<0.0001). And among female individuals, IBD had impact on most sub-domains of sexual function, except pains. Conclusions: IBD is associated with worse sexual function. It has significant impact on erectile function and satisfaction for male individuals and has impact on most sub-domains of sexual function for female individuals.

Background: Management of diabetes mellitus induced-erectile dysfunction (DMED) is challenging because of its poor responses to phosphodiesterase type 5 inhibitors. Increasingly important roles of 12-lipoxygenase (12-LOX) have been proven in diabetes mellitus. Aim: To investigate 12-LOX activity and therapeutic effect of its inhibitor, baicalein (BE), on DMED. Methods: Intraperitoneal streptozotocin injection was used to induce type I DM, and an apomorphine test was used to evaluate erectile function. In experiment A, we assessed 12-LOX expression alteration in the corpus cavernosum (CC) of rats with DMED of different levels of severity. In experiment B, rats with DMED were intraperitoneally injected with BE for 4 weeks, and control rats were injected with vehicles. The erectile function was tested by cavernous nerve stimulation before penile tissue was harvested. We performed Western blot, immunohistochemistry, immunofluorescence, Masson trichrome staining, and enzyme-linked immunosorbent assays to measure related proteins in CC. Main Outcome Measure: The main outcome measures included rectile response, histologic examination, and expression alteration of related proteins. Results: 12-LOX upregulation was associated with the progression of type I DMED. After 4 weeks treatment, compared with the DMED group, the DMED thorn BE group showed better erectile responses to cavernous nerve stimulation. In theDMEDthornBE group, significantly enhanced endothelial nitric oxide synthase/nitric oxide/cyclic guanosine monophosphate pathway, reduced 12-LOX expression, and inhibited p38 mitogen-activated protein kinase/arginase II/L-arginine pathway were showed in CC relative to the DMED group. In addition, overactivated oxidative stress and fibrosis in the DMED group were both partially ameliorated in the DMED thorn BE group. Clinical Implications: BE may be considered as an effective therapy for DMED, but needs to be verified in future human investigations. Strengths & Limitations: The role of 12-LOX and its inhibitor, BE, is firstly demonstrated in rats with type I DMED. However, the experimental data are derived from animal models with without evidences from cellularbased experiments. Conclusion: 12-LOX might serve as an important factor in the pathogenesis of type I DMED. BE alleviated erectile dysfunction in rats with type I DMED probably by inhibiting 12-LOX expression, ameliorating endothelial nitric oxide synthase dysfunction, as well as suppressing oxidative stress and fibrosis.

Objective To investigate the mechanism of sphingosine﹣1﹣phosphate receptor 3 (S1PR3) in the cavernous tissues of diabetes mellitus﹣induced erection dysfunction (ED ) rats. Methods Eighteen eight﹣week﹣old healthy male SD rats were randomly divided into 2 groups :Con﹣trol group (CO ,n＝6) ;Diabetes mellitus group (DM ,n＝12). Diabetes rats were constructed ,af﹣ter 8 weeks ,the maximum intracavernous pressure／mean arterial pressure were determined and the expression of S1PR3 were detected in the penis by IHC ,PCR and Western blot ,and the expression of RhoA ,ROCK1 and ROCK2 in rat penile cavernous tissue were detected by Western blot. Results T he erectile function significantly decreased in DM rats ,compared to that of age﹣matched control rats (P＜0.05) ; the expression level of S1PR3 ,RhoA ,ROCK1 and ROCK2 were higher in DM group than those in CO (P＜0.05). Conclusions Type Ⅰ DM can induce ED in rats ,w hich may be related to the up﹣regulation of S1PR3 expression and the activation of RhoA／Rho kinase pathway in rat penile cavernous tissue.

Objective:To compare the clinical effect of flexible ureteroscope incision and drainage and retroperitoneoscopic unroofing.Methods:We retrospectively analyzed the data of 45 patients with periplvic cyst adjacent to the renal collecting system from March 2015 to March 2016.Patients were divided into flexible ureteroscope group (incision and drainage by the flexible ureteroscope) and retroperitoneoscopic group (retroperitoneoscopic unroofing).The time of operation,hospital stay after operation,hospitalization expenture and postoperative follow-up outcomes were observed.Results:The operations were performed successfully in both two groups,there were no obvious complications after the operation,and the renal cysts disappeared or were significantly reduced after surgery.During a follow-up period of 6-12 months,the renal cyst did not recurred in the two groups.There were statistically significant differences (P＜0.05) between the two groups in the aspect of the time of operation,hospital stay after operation,and hospital expenture.Conclusions:The flexible ureteroscope incision and drainage method is minimally invasive,safe,effective,and worthy of clinical promotion.

Objective To explore the activation of inducible nitric oxide synthase(iNOS)by the recombinant adenovirus vec-tor of short hairpin RNA(shRNA)that targets rat inducible nitric oxide synthase(iNOS)in order to provide experimental evi-dence for gene therapy of erectile dysfunction.Methods Twenty-four-month-old male SD rats (n = 30)were divided into 3 groups at random:Ad5-iNOS-shRNA-EGFP group(AdU6/shiNOS group)(n=10),in which the recombinant adenovirus vector of shRNA Ad5-iNOS-shRNA-EGFP was injected;virus control group(AdU6/shControl group)(n=10);blank control group(n=10),in which PBS was given.One week after the injection,The erectile function was assessed by intracavernous pressure (ICP)/mean arterial pressure(MAP)in rats undergoing cavernous nerve electrical stimulation.Corpus cavernosum tissues were harvested,and the levels of iNOS mRNA and protein were examined by real-time quantitative PCR and Western blotting,re-spectively.Results At 2.5-,5.0-and 7.5-volt stimulation,the ICP/MAP was significantly higher in AdU6/shiNOS group than in both AdU6/shControl group and blank control group(P <0.05).The levels of mRNA and protein of iNOS were much higher in AdU6/shiNOS group than in AdU6/shControl group and blank control group(P <0.05).Conclusion Adenovirus-mediated RNAa technology can increase the iNOS gene expression,and improve the erectile function in senile rats.