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.

Objective: This study aims to portray the characteristics of oxidative stress (OS) in cases of Necrotizing enterocolitis (NEC), identify the hub genes and associated mechanisms involved, and explore potential drugs for NEC. Methods: We performed a comprehensive analysis integrating bulk-RNA sequencing and single-cell RNA sequencing datasets, coupled with various techniques including differential analysis, gene set enrichment analysis, and immune infiltration analysis. We aimed to systematically elucidate the variations in functions related to OS among distinct cell populations within both NEC and non-NEC tissues. Additionally, we depicted the longitudinal changes in immune cells, with a particular focus on macrophages, throughout the progression of NEC. NEC mice model was established and RT-qPCR was performed to validate the expression of the hub genes. Results: In total, 465 OS related genes were found, and 53 of them were significantly differentially expressed. These genes were mainly involved in several signaling pathways, such as TNF signaling pathway, IL-17 signaling pathway, FOXO signaling pathway, inflammatory bowel disease. The top 10 hub genes were MMP2, IL1A, MMP3, HGF, HP, IL10, PPARGC1A, TLR4, MMP9 and HMOX1. Ten kinds of drug were discovered as the potential treatment for NEC. Four specific macrophages subtypes and relative function were identified in NEC. RT-qPCR and immunofluorescence staining confirmed the expression of the hub genes in NEC model. Conclusions: This investigation yielded innovative insights into the immune environment and therapeutic methodologies directed at oxidative stress in the pathogenesis of NEC.

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.

Electroceuticals based on peripheral nerve stimulation offer promising treatment for refractory inflammatory diseases such as inflammatory bowel diseases (IBDs). For pediatric IBD (PIBD) patients, wireless, biodegradable miniaturized bioelectronic devices are crucial to prevent neural damage and support neural development during and after therapy. Here we demonstrate a battery-free, miniaturized neurostimulator based on biodegradable materials and capacitive-coupling wireless power transfer. The biodegradable capacitive-coupling (BCC) neurostimulator consists of molybdenum (Mo) electronic components and self-healing biodegradable polyurethane elastomer (SBPUE) encapsulations. The self-healing property of SBPUE enables a stable neural interface. Capacitive coupling wirelessly transfers high-frequency electric fields through a single capacitor between wearable transmitters and implanted BCC neurostimulators. Programmed electrical stimulation of the vagus nerve alleviates PIBD symptoms by restoring CD4+ T cell balance, enhancing anti-inflammatory effects and suppressing pro-inflammatory effects in the intestines.

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 (NB), the most common extracranial solid tumor of childhood, originates from developing sympathetic nervous system. While cuproptosis has emerged as a critical regulator in oncobiology, its mechanistic involvement in NB remains poorly characterized. RNA-seq data of NB patients was analyzed using limma and ClusterProfiler. CRlncRNAs were identified through Pearson correlation between lncRNAs and CRGs, with co-expression networks visualized via ggalluvial. Prognostic signature was constructed through Lasso-penetrated Cox regression and validated via EFS analysis, C-index, and ROC curves. Somatic mutations and TMB were profiled using maftools. Immune infiltration landscapes were deciphered by CIBERSORT, while single-cell analysis integrated AddModuleScore, AUCell, and inferCNV to map CNV-driven transcriptional heterogeneity. Functional validation via siRNA knockdown confirmed the oncogenic role of CRGs. The risk model: Risk Score = (-1.637×DIRC3-AS1) + (0.6758×FOXN3-AS1) + (0.3032×LINC00682) + (-0.6812×RASSF8-AS1) was constructed. Stratification by median risk score revealed significantly prolonged EFS and OS. No significant TMB difference was observed between subgroups. scRNA-seq analysis highlighted malignant cell dominance with marked CNVs in high-risk patients. Functional validation confirmed CRlncRNAs’ roles in modulating proliferation and migration. Our findings establish a novel prognostic framework for NB that enhances risk stratification accuracy and provides actionable biomarkers to guide precision clinical management.

PURPOSE:Congenital pulmonary airway malformation (CPAM) is a developmental lung malformation that seriously endangers children's health. The objective of this study was to investigate the etiology of CPAM by observing changes at the molecular and cellular levels. METHODS:Patient clinical data were collected and analyzed. Tissue samples were collected from CPAM lesions and marginal normal lung tissue during CPAM surgery. The samples were subjected to hematoxylin and eosin (H&E) staining for pathological analysis. Tissue RNA was isolated for RNA sequencing, and the differentially expressed genes (DEGs) were enriched for Gene Ontology (GO) analysis. The cytoskeletal and cell subtypes were subjected to immunofluorescence staining. RESULTS:RNA sequencing of 7 CPAM patients revealed 1252 DEGs, with 1041 upregulated and 211 downregulated. GO analysis revealed that biological processes related to cilium organization and movement were strongly enriched. Protein-protein interaction (PPI) network analysis highlighted genes such as CCDC65, DNAH5, DNAH11, DNAH12, CFAP43, CFAP70, PIH1D3, RSPH4A and DNAH6 as potentially important in CPAM. Immunofluorescence staining revealed abnormalities in cytoskeleton and cilia, and a reduction in the number of alveolar epithelial type II (AEC II) cells in CPAM lesions compared with normal lung tissue. CONCLUSION:Our study revealed a significant increase in cytoskeletal and cilia expression, along with a marked reduction in AEC II. These abnormalities provide potential insights into the etiology of CPAM and may guide the development of improved diagnostic and therapeutic strategies.

Background The myenteric plexus primarily consists of the myenteric ganglia, which include enteric neurons, synaptic neuropils, and glial cells. Abnormal myenteric plexus formation can result in gastrointestinal disorders. Comprehensive morphological classification studies of myenteric ganglia remain limited. Methods Whole-mount immunofluorescence staining was used to label myenteric ganglia in colon tissue of mice and children. The ganglionic area and the number of intraganglion neurons were quantified by the K-means clustering algorithm. The guts of embryonic day 11.5 (E11.5) mouse were cultured and immunostained to observe the characteristics of developing myenteric ganglia. Results Myenteric ganglia can be categorized into three groups in the colon tissues of mice and normal children. A similar classification was observed for Tuj1-positive neuronal cell clusters in the midgut of E11.5 mouse. Culture of the E11.5 mouse midgut revealed that the area of post-cultured clusters of developing neurons also fell into three distinct categories, with a noticeable increase compared to pre-culture. Conclusions The myenteric ganglia in mice and humans can be categorized into three groups based on both the ganglionic area and intraganglion neuron count, and distinct classes of myenteric ganglia may be present during early development.

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.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have become contaminants widely distributed in the environment due to improper disposal and discharge. Previous study has found several components might involve in impairing enteric nervous system (ENS) development of zebrafish, including NSAIDs cinchophen. Deficient ENS development in fetal could lead to Hirschsprung disease (HSCR), a congenital neurocristopathy characterized by absence of enteric neurons in hindgut. However, the intrinsic mechanism of neurotoxicity of cinchophen is unclear. We confirmed that cinchophen could impair ENS development of zebrafish and transcriptome sequencing revealed that disfunction of Replication protein A1 (RPA1), which is involved in DNA replication and repairment, might be relevant to the neurotoxicity effects induced by cinchophen. Based on previous data of single cell RNA sequencing (scRNA-seq) of zebrafish gut cells, we observed that rpa1 mainly expressed in proliferating, differentiating ENS cells and neural crest progenitors. Interestingly, cinchophen induced apoptosis and impaired proliferation. Furthermore, cinchophen caused DNA damage and abnormal activation of ataxia telangiectasia mutated/ Rad3 related (ATM/ATR) and checkpoint kinase 2 (CHK2). Finally, molecular docking indicated cinchophen could bind and antagonize RPA1 more effectively. Our study might provide a better understanding and draw more attention to the role of environmental factors in the pathogenesis of HSCR. And the mechanism of cinchophen neurotoxicity would give theoretical guidance for clinical pharmacy.

BackgroundMalnutrition has emerged as main side effects of inflammatory bowel disease (IBD) which might also affect the prognosis of IBD. However, whether these associations are causal remains unclear. We aimed to identify the causality of IBD on malnutrition and explore the causal relationship of malnutrition and nutrients intake on IBD by using Mendelian randomization (MR).MethodsSingle nucleotide polymorphisms associated with IBD, malnutrition and nutrients intake were obtained from previous researches of genome-wide association studies (GWAS) (p < 0.00000005). MR analysis was conducted to evaluate the causality with different methods based on OR and their 95% CIs. Meanwhile, heterogeneity, pleiotropy and MR-PRESSO were used for instrumental variables evaluation.ResultsThe results of MR analysis revealed that IBD, both Crohn disease (CD) and ulcerative colitis (UC), could directly impact the incidence of malnutrition (p-value <0.01). CD is directly related to nutrients such as sugar, fat, VA, VC, VD and zinc, while UC is correlated with carbohydrate, fat, VB12, VC, VD, VE, iron, zinc and magnesium. However, our results suggested that malnutrition could not affect the risk of IBD directly (p > 0.05). Further analysis showed similar results that nutrients intake had no direct effect on IBD, neither CD or UC.ConclusionOur results indicated that IBD increases the risk of malnutrition, however, malnutrition and nutrients intake might not directly affect the progression of IBD.

Objective and design: Hirschsprung disease-associated enterocolitis (HAEC) is a common life-threatening complication of Hirschsprung disease (HSCR). We aimed to investigate the effectiveness, long-term safety and the underlying mechanisms of Mesenchymal stem cells (MSCs) based therapy for HAEC. Material or subjects: Specimens from HSCR and HAEC patients were used to assess the inflammatory condition. Ednrb knock-out mice was used as HAEC model. MSCs was intraperitoneally transplanted into HAEC mice. The therapy effects, long-term outcome, safety and toxicity and the mechanism of MSCs on the treatment of HAEC were explored in vivo and in vitro. Results: Intestinal M1 macrophages infiltration and severe inflammation condition were observed in HAEC. After the injection of MSCs, HAEC mice showed significant amelioration of the inflammatory injury and inhibition of M1 macrophages infiltration. The expression levels of pro-inflammatory cytokines (TNF- a and IFN- g ) were decreased and anti-inflammatory cytokines (IL-10 and TGF- b ) were increased. In addition, we found that effective MSCs homing to the inflamed colon tissue occurred without long-term toxicity response. However, COX-2 inhibitor could diminish the therapeutic effects of MSCs. Using MSCs and macrophages co-culture system, we identified that MSCs could alleviate HAEC by inhibiting M1 macrophages activation through COX-2-dependent MAPK/ERK signaling pathway. Conclusions: MSCs ameliorate HAEC by reducing M1 macrophages polarization via COX-2 mediated MAPK/ERK signaling pathway, thus providing novel insights and potentially promising strategy for the treatment or prevention of HAEC. (c) 2024 Elsevier Inc. All rights reserved.

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.

Background Hirschsprung’s disease (HSCR) is one of the most common malformations of the digestive tract. Patients with HSCR frequently manifest as having severe constipation and abdominal distension. The primary pathological feature of HSCR is the absence of ganglion cells in the distal bowel, and the arrangement of the circular and longitudinal muscles of the aganglionic segments is disorganized. To function properly, it requires an intact muscular layer as well as a neural network connection. Previous research has suggested that HSCR is a neurological disorder; however, HSCR may also be a muscular cell disorder of the intestinal smooth muscle. Methods To investigate the development rules of circular and longitudinal muscles and to research whether ENCCs affect smooth muscle function. αSMA immunohistochemistry was used to stain tissues of HSCR patients and HSCR model mice at different developmental stages. Results Under normal circumstances, the formation of circular muscles is later than that of longitudinal muscles, and the expression of αSMA in circular muscles is much lower than that in longitudinal muscles. The expression of αSMA in anganglionic segments of HSCR patients is much higher than that in the distal colon of normal control children. Conclusions Loss of ENCCs may influence the function of the circular muscles, αSMA is a biomarker for detecting the abnormal smooth muscle cell in Hirschsprung's disease patients' aganglionic segments.

BACKGROUND:Actin Alpha 2 (ACTA2) is expressed in intestinal smooth muscle cells (iSMCs) and is associated with contractility. Hirschsprung disease (HSCR), one of the most common digested tract malformations, shows peristaltic dysfunction and spasm smooth muscles. The arrangement of the circular and longitudinal smooth muscle (SM) of the aganglionic segments is disorganized. Does ACTA2, as a marker of iSMCs, exhibit abnormal expression in aganglionic segments? Does the ACTA2 expression level affect the contraction function of iSMCs? What are the spatiotemporal expression trends of ACTA2 during different developmental stages of the colon?METHODS:Immunohistochemical staining was used to detect the expression of ACTA2 in iSMCs of children with HSCR and Ednrb-/- mice, and the small interfering RNAs (siRNAs) knockdown technique was employed to investigate how Acta2 affected the systolic function of iSMCs. Additionally, Ednrb-/- mice were used to explore the changes in the expression level of iSMCs ACTA2 at different developmental stages.RESULTS:The expression of ACTA2 is higher in circular SM in the aganglionic segments of HSCR patients and Ednrb-/- mice than in normal control children and mice. Down regulation of Acta2 weakens the contraction ability of intestinal smooth muscle cells. Abnormally elevated expression of ACTA2 of circular smooth muscle occurs since embryonic day 15.5 (E15.5d) in aganglionic segments of Ednrb-/- mice.CONCLUSIONS:Abnormally elevated expression of ACTA2 in the circular SM leads to hyperactive contraction, which may cause the spasm of aganglionic segments in HSCR.

Background Hirschsprung’s disease (HSCR) is one of the most common congenital digestive tract malformations and can cause stubborn constipation or gastrointestinal obstruction after birth, causing great physical and mental pain to patients and their families. Studies have shown that more than 20 genes are involved in HSCR, and most cases of HSCR are sporadic. However, the overall rate of familial recurrence in 4331 cases of HSCR is about 7.6%. Furthermore, familial HSCR patients show incomplete dominance. We still do not know the penetrance and genetic characteristics of these known risk genes due to the rarity of HSCR families. Methods To find published references, we used the title/abstract terms “Hirschsprung” and “familial” in the PubMed database and the MeSH terms “Hirschsprung” and “familial” in Web of Science. Finally, we summarized 129 HSCR families over the last 40 years. Results The male-to-female ratio and the percentage of short segment-HSCR in familial HSCR are much lower than in sporadic HSCR. The primary gene factors in the syndromic families are ret proto-oncogene ( RET ) and endothelin B receptor gene ( EDNRB ). Most families show incomplete dominance and are relevant to RET , and the RET mutation has 56% penetrance in familial HSCR. When one of the parents is a RET mutation carrier in an HSCR family, the offspring’s recurrence risk is 28%, and the incidence of the offspring does not depend on whether the parent suffers from HSCR. Conclusion Our findings will help HSCR patients obtain better genetic counseling, calculate the risk of recurrence, and provide new insights for future pedigree studies.

Objective: This study aims to identify an immune-related signature to predict clinical outcomes of oral squamous cell carcinoma (OSCC) patients. Methods: Gene transcriptome data of both tumor and normal tissues from OSCC and the corresponding clinical information were downloaded from The Cancer Genome Atlas (TCGA). Tumor Immune Estimation Resource algorithm (ESTIMATE) was used to calculate the immune/stromal-related scores. The immune/stromal scores and associated clinical characteristics of OSCC patients were evaluated. Univariate Cox proportional hazards regression analyses, least absolute shrinkage, and selection operator (LASSO) and receiver operating characteristic (ROC) curve analyses were performed to assess the prognostic prediction capacity. Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) function annotation were used to analysis the functions of TME-related genes. Results: Eleven predictor genes were identified in the immune-related signature and overall survival (OS) in the high-risk group was significantly shorter than in the low-risk group. An ROC analysis showed the TME-related signature could predict the total OS of OSCC patients. Moreover, GSEA and GO function annotation proved that immunity and immune-related pathways were mainly enriched in the high-risk group. Conclusions: We identified an immune-related signature that was closely correlated with the prognosis and immune response of OSCC patients. This signature may have important implications for improving the clinical survival rate of OSCC patients and provide a potential strategy for cancer immunotherapy.

Objective:To explore the potential active components of intestinal adhesion prescription (IAP) and construct the " active components-targets-pathways" network of IAP and elucidate its potential mechanism.Methods:The active components and related targets of IAP were searched from the database of TCMSP and literature reports. These active components were filtrated by oral bioactivity (OB) and drug likeness (DL). Then potential targets of IAP related to intestinal adhesion (IA) , intestinal obstruction (IO) and necrotizing enterocolitis (NE) were searched from the database of GeneCards. The potential targets of IAP related to IA/IO/NE were obtained by aligning of two target lists mentioned above. GO enrichment and KEGG pathway of target genes were analyzed by the database of Matascape. Cytoscape_v3.7.2 software was utilized for constructing the "active components-targets-pathways" network of IAP related to these neonate intestinal diseases. Molecular docking hinted at the binding of active components and potential targets by using PDB , DiscoveryStudio, AutoDockTools and Pymol.Results:IAP contained 11 traditional Chinese medicines and 120 active components, including 246 gene targets containing 78 targets related to IA/IO/NE and affecting many signaling pathways (e. g. TNF). Molecular docking revealed the interactions between potential targets (VCAM1 & MAPK1) and selected active components (MOL000098 & MOL000791).Conclusion:Through suppressing the progression of inflammation through some signaling pathways, a large variety of active components of IAP may be used for treating neonatal intestinal adhesion, obstruction and necrotizing enterocolitis.

Background: Inflammatory myofibroblastic tumor (IMT) infrequently involves the sigmoid colon, and has not previously been described in an infant sigmoid colon. Case report: An inflammatory myofibroblastic tumor arose from the sigmoid colon of an 11-month-old boy, confirmed by anaplastic lymphoma kinase (ALK), smooth muscle actin (SMA) and desmin immunohistochemical staining. The patient recovered well after complete resection of the tumor. Discussion: Sigmoid IMT can occur in infancy. This eighth case is the youngest so far. The child did well after surgical resection.