To compare the value of non-invasive liver fibrosis indicators, including serum matrix metalloproteinase-7 (MMP-7), APRI, collagen-related proteins [hyaluronic acid (HA), type IV collagen (IV-C), type III procollagen N-terminal peptide (PIIIP), laminin] and shear wave elastography (SWE), in assessing liver fibrosis for biliary atresia (BA). A retrospective cohort study was conducted in BA infants from July 2020 to December 2023. Liver fibrosis was classified according to the Batts-Ludwig criteria. A total of 471 BA infants were included. Serum MMP-7, HA, IV-C, PIIIP, laminin, and SWE all correlated with liver fibrosis, with correlation coefficients of 0.4, 0.28, 0.37, -0.15, 0.16, and 0.46 (all P < 0.01), respectively. For severe fibrosis, the AUROC was 0.675 (95

Spinal cord injury (SCI) predisposes patients to severe respiratory complications. Integrating clinical observations with a mouse model, we identified gut-lung axis perturbation as a key mechanism. SCI increased the similarity between gut and lung microbial communities, consistent with cross-compartment convergence driven by intestinal dysbiosis. Fecal microbiota transplantation from SCI donors reproduced this convergence in healthy recipients. Crucially, oral Bifidobacterium supplementation restored intestinal community structure, reduced gut-lung similarity, and attenuated pulmonary inflammation following an Escherichia coli challenge. These findings indicate that SCI disrupts gut-lung compartmentalization and that augmenting beneficial gut taxa mitigates downstream pulmonary consequences. Microbiota-targeted strategies therefore warrant evaluation as adjuncts to reduce post-SCI respiratory risk.

ObjectiveTo summarize the clinical features, diagnosis, treatment, and prognosis of congenital gluteal dermal sinus tracts in children, providing insights for better clinical management.MethodsA retrospective analysis was conducted on 20 patients diagnosed with congenital gluteal dermal sinus tracts at the Children's Hospital of Fudan University over 15 years. The analysis focused on age, gender, diagnostic delay, diagnostic methods, course and opening of the sinus tract, and postoperative follow-up.ResultsThe study included 6 males and 14 females, with symptom onset ranging from birth to 12 years (median age of 9.5 months). Initial symptoms included abnormal gluteal depression with recurrent infections (n = 8), unexplained recurrent infections (n = 6), and asymptomatic gluteal depression or small holes (n = 6). The time from symptom onset to diagnosis ranged from 1 to 102 months (median: 13.5 months). MRI was positive in 87.5%, identifying the tract in 81.25%. CT scans were positive in all cases, but only 50% identified the tract. Sinus tractography successfully identified the tract in 7 of 10 patients. The sinus tracts were classified into three types based on the opening location. All sinus tracts were excised during surgery, with an average length of 4.95 cm. Two patients experienced recurrence and underwent reoperation, while the remaining 15 had no recurrence.ConclusionCongenital gluteal dermal sinus tracts are more common in females and often misdiagnosed. CT is more sensitive but less specific than MRI. Sinus tractography helps determine the tract's path. Prognosis is generally good after complete excision, with recurrence being the main postoperative complication.

Objective Sacral dural arteriovenous fistula (SDAVF) is a rare spinal vascular malformation and often misdiagnosed or even mistreated. This study delved into the clinical characteristics, vascular architecture and treatment results of SDAVF, with the goal of enhancing upcoming diagnostic and therapeutic methodologies.Methods From March 2014 to March 2022, consecutive patients with SDAVF were retrospectively analysed. The data on demographics, symptom resolution, angioarchitectural features and postoperative course were studied. Spinal cord function was evaluated by modified Aminoff–Logue scale.Results A total of 36 patients with 36 SDAVFs were enrolled, 12 of whom were misdiagnosed on their initial visit. The SDAVFs were located at S1 in 24 (66.7%), S2 in 10 (27.8%) and S3 in 2 (5.6%) cases, respectively. The primary feeding arteries included lateral sacral artery (LSA) of internal iliac artery (31/36, 86.1%), the branches of external iliac artery (2/36, 5.6%) and median artery (3/36, 8.3%), most of which are straight. Venae terminalisis is the sole drainage vein, flowing back into perimedullary venous network. Endovascular embolisation is the main therapy method for 30 cases, while the other 6 cases were treated with microsurgical fistulectomy. MRI tests showed that the abnormal vascular signals around the medulla disappeared, and the spinal cord oedema was alleviated in the majority of cases (32/36, 88.9%). Six patients, who all were treated by endovascular embolisation at first time, had residual or recurrent and two of them were performed by microsurgical fistulectomy again. All patients by microsurgical fistulectomy had no residual or recurrent during follow-up. According to the spinal cord functional assessment, the Aminoff-Logue score was significantly decreased (Z=−3.449, p=0.001) postoperatively.Conclusion The misdiagnosis rate of SDAVF is very high. The most feeding artery of SDAVF came from the LSA, which was thicker and more straight, making it easier for microcatheters to reach the fistula site. So, endovascular embolism has become the first choice of treatment with minimal invasion, and safe and effective results.

PURPOSE:N6-methyladenosine (m6A) is involved in biliary atresia (BA), however the underlying mechanisms remain unknown. This study aims to explore the involvement of m6A modification and its role in epithelial-to-mesenchymal transition (EMT) in BA cholangiocytes. METHOD:A published single-cell RNA sequencing dataset (HRA003163) was analyzed, focusing on cholangiocytes for differential gene expression, pathway enrichment, and correlation analyses. Candidate genes were identified based on their high expression in BA cholangiocytes and association with EMT pathways. Selected markers were validated in liver tissues via immunofluorescence. Cholangiocytes with stable interference of METTL3 or CDH6 were established for functional assays, including CCK8, wound-healing, and Transwell experiments. RNA and protein stability were assessed, and RNA immunoprecipitation was conducted to confirm m6A reader protein. RNA sequencing (RNA-seq) was performed on CDH6-overexpressing cells to identify downstream pathways. RESULTS:Single-cell RNA sequencing data revealed significant enrichment of EMT and fibrosis-related pathways in BA cholangiocytes. Immunofluorescence confirmed upregulation of EMT and fibrosis markers in BA cholangiocytes. M6A regulatory genes were downregulated in BA cholangiocytes, with METTL3 identified as a key modulator. CDH6 was identified as a candidate gene regulated by m6A modification. Functional experiments revealed that METTL3-mediated hypomethylation stabilized CDH6 transcripts via YTHDF2. Downregulation of METTL3 enhanced cholangiocyte migration and EMT progression. RNA-seq and in vitro experiments demonstrated that CDH6 upregulation promoted proliferation and migration in cholangiocytes, contributing to EMT. CONCLUSION:This study highlights the role of m6A methylation in regulating CDH6 expression and its contribution to EMT progression in cholangiocytes, offering new evidence on potential mechanisms underlying BA.

Accurately predicting the prognosis of biliary atresia (BA) prior to Kasai portoenterostomy (KPE) remains a challenge. The identification of the specific BA population that may benefit from primary liver transplantation (pLT) instead of KPE remains elusive. A total of 196 BA patients and 31 age-matched non-BA cholestasis patients were recruited. BA patients were divided into training (February 2018–February 2019) and validation (March 2019–December 2021) cohorts. C-index was applied to evaluate the utility of indicators and models. Serum fibroblast growth factor 19 (FGF19) was elevated in BA patients [95.67 (58.97–140.6) vs. 58.73 (43.59–85.35) pg/ml, P = 0.0003]. Constructed with FGF19, GGT, DBIL, and ALB, nomogram A demonstrated optimal C-index in training (0.767 ± 0.039) and validation (0.721 ± 0.062) cohorts with ideal consistency in predicting 1-year NLS after KPE as well as potential clinically utility in BA patients with an age at KPE ≤ 60 days. Leveraging the risk score (RS) developed with nomogram A, our findings revealed a notable decrease in 2-year NLS after KPE among BA patients with a preoperative RS > 1.36, and the patients with a preoperative RS > 2.6 appear to be potential candidates for pLT [2-year NLS after KPE: 0

BACKGROUND AND OBJECTIVES: Intermittent reports of spinal cord arteriovenous shunts (SCAVSs)-induced paralysis during pregnancy and puerperium have raised significant concerns. This study aimed to assess whether women with SCAVSs are at an elevated clinical risk during this period. METHODS: Consecutive female patients with SCAVSs from 10 referral centers were included. Only clinical data within reproductive age were analyzed. The primary outcome was the occurrence of clinical deterioration associated with SCAVSs, categorized into acute and gradual patterns based on the modified Aminoff and Logue scale. RESULTS: A total of 480 patients were included. Before initial treatment, 262 patients experienced pregnancy. Both acute (17.88%/y vs 4.87%/y, P < .0001) and gradual (9.67%/y vs 3.27%/y, P < .0001) deterioration during pregnancy and puerperium were significantly elevated. In the matched cohort of 72 pregnant patients with untreated SCAVSs and their nonpregnant control, acute deterioration was significantly higher in the pregnant patients (26.09%/y vs 6.97%/y, P = .013). After partial SCAVSs obliteration, acute (33.74%/y vs 5.15%/y, P < .0001) and gradual (18.40%/y vs 2.61%/y, P = .0008) deterioration rates during this period were still significantly elevated. In the matched cohort of 23 pregnant patients with residual SCAVSs and their nonpregnant control, acute deterioration was still significantly higher in the pregnant patients (39.50%/y vs 6.25%/y, P = .043). In addition, no significant decrease in the clinical deterioration was observed after partial treatment compared with nontreated patients during this period. CONCLUSION: The risk of clinical deterioration associated with SCAVSs is significantly elevated during pregnancy and puerperium.

AIM:MMP7 has been identified as a potential biomarker for biliary atresia (BA) diagnosis. However, the mechanism of MMP7 and its downstream signaling pathway remain unknown in the pathogenesis of BA. Herein, this study was performed to figure out MMP7's downstream target. METHODS:Single-cell RNA sequencing (scRNA-seq) was performed to screen out MMP7's downstream molecule CXCL6. QRT-PCR, immunohistochemistry, western blot, and ELISA were used to determine the expressions of MMP7 and CXCL6 in the liver and serum of BA and controls. Immunofluorescence was conducted to validate the hepatic cellular expressions of MMP7 and CXCL6. In vitro, overexpression and knockdown of MMP7 in biliary epithelial cells (BECs) were established to verify the MMP7's regulation on CXCL6. RESULTS:ScRNA-seq demonstrated that MMP7 and CXCL6 were exclusively expressed on cholangiocytes and up-regulated in BA when compared with normal controls (NC). QRT-PCR, immunohistochemistry, western blot and ELISA validated the higher expressions of MMP7 and CXCL6 in the liver and serum of BA when compared with non-BA cholestasis (CS) and NC. Immunofluorescence further verified the biliary localization of MMP7 and CXCL6 in BA. Hepatic and serum CXCL6 expressions were positively correlated with MMP7 expressions, and both expressions were correlated with the stages of fibrosis in BA. Overexpression of MMP7 promoted CXCL6 expression, while knocking down MMP7 inhibited CXCL6 expression in BECs. CONCLUSION:CXCL6 is a downstream target of MMP7, and is identified as a novel biliary marker in BA. The production of CXCL6 by MMP7 may exert pathological roles in the liver fibrogenesis of BA.

BACKGROUND:Gastric adenomyoma (GA) in children is a relatively rare condition, and currently, there is limited knowledge regarding its optimal diagnostic methods and treatment strategies. To improve our understanding GA particularly in children, one case of a 4-month-old boy was reported and existing literature was comprehensively reviewed. CASE SUMMARY:A 4-month-old boy was admitted to the hospital with a 2-month history of recurrent vomiting of gastric contents. Ultrasound examination demonstrated that the pyloric canal muscle was 3.2 mm in thickness, 14.6 mm in diameter, and 17.6 mm in length. Pathological finding showed that a mass approximately 2.8 cm × 2 cm × 1.5 cm in size and grayish-white in color was detected. The cystic wall was composed of smooth muscles lined with the gastric mucosal epithelium and a small amount of intestinal epithelium. Scattered glandular epithelium and a small amount of pancreatic tissue were observed in smooth muscle. Immunohistochemistry revealed positivity for gastrin, insulin, and smooth muscle actin, confirming the diagnosis of GA. In addition, the Ki-67 proliferation index was low. CONCLUSION:We report a case of GA in children, successfully treated with laparoscopic surgery. Ultrasound and computed tomography may be helpful in differential diagnosis when detecting low-density septal lesions biased toward one side. We also review the existing literature data regarding GA in children.

In hypertension-associated arteriolosclerosis cerebral small vessel disease (CSVD), various studies have shown that MRI-detected lesions—such as lacunes, white matter hyperintensities, enlarged perivascular spaces, and cerebral microbleeds—are more prevalent in deep brain regions (DBR) than in the cortex. However, the underlying mechanisms remain poorly understood. We propose that differential vascular remodeling between DBR small vessels and superficial cortical branches contributes to this heterogeneity. Using a stroke-prone renovascular hypertensive rat (RHRsp) model, we observed pronounced changes in vessel density, diameter, extracellular matrix deposition, and smooth muscle cell alterations in DBR small arteries compared to that of the cortex. These findings were further confirmed in human brain tissue of our study. Additionally, our mathematical modeling indicated greater hemodynamic alterations in DBR vessels, with increased shear and circumferential stress under hypertension conditions. Overall, our study highlights more severe vascular remodeling and hemodynamic changes in the deep brain regions, where CSVD-associated MRI lesions are frequently detected.

BACKGROUND:Ductular reaction (DR), characterized by the expansion of biliary epithelial cells in the portal area, is a typical hepatic pathology for biliary atresia (BA). The cellular source and function of DR remain poorly understood. Herein, we performed single-cell RNA sequencing (scRNA-seq) in BA to resolve the complexity of DR in BA. METHODS:A total of 4 BA and 3 normal control livers underwent scRNA-seq. The epithelial cells were extracted from all cells for further analysis. The cell types, functions, and differentiational trajectory of epithelial cells were determined. The biliary markers and transcription factors (TFs) were identified by combing public bulk and scRNA-seq data and validated by immunohistochemistry. RESULTS:ScRNA-seq identified the existence of biliary reprogramming in BA, and the reprogrammed cells expressed both hepatocyte and cholangiocyte markers. When compared with hepatocytes, genes of epithelial-mesenchymal transition, fibrosis, inflammation, and RNA metabolism were enriched in cholangiocytes and upregulated in BA. Pseudotime analysis depicted a differentiation trajectory from hepatocytes across reprogrammed cells to cholangiocytes in BA. Matrix metalloproteinase 7 (MMP7), VTCN1, and LAMC2 were identified as the biliary markers, and KLF5 and HNF1B were determined as the biliary TFs in BA. All the biliary markers and TFs were upregulated in BA when compared with controls. CONCLUSIONS:Dissecting the cellular source and function of cholangiocytes is essential to understand the pathological role of DR in BA. The identified specific biliary markers and TFs provide important insights into its potential diagnosis and mechanism exploration for BA in the future.

Introduction Immune factors are important antecedents in the pathophysiology of necrotizing enterocolitis (NEC). However, studies on the peripheral blood lymphocyte subsets changes in NEC patients among different Bell stages and in patients requiring surgery are scarce. Methods 34 infants with NEC and 33 age-matched controls were included. Peripheral blood was collected within 48 h after NEC diagnosis. Peripheral blood B and T lymphocytes subsets were detected by 12-color flow cytometry. Cell ratios were calculated, and their relationship to disease severity and their roles as indicators for surgery were assessed. Results NEC patients showed elevated percentages of unSwB cells (CD27+IgD+ unswitched memory/activated B cells)/B cells, SwB cells (CD27+IgD-switched memory B cells)/B cells, CD8+ T (CD3+CD8+ T cells)/T cells, Tem (CD45RA-CCR7-effector memory T cells)/CD4+ T cells, Tem/CD8+ T cells and decreased Bn (CD27-IgD+ naïve B cells)/B cells, CD4+T (CD3+CD4+ T cells)/T cells, CD45RA+ CCR7+ naïve T cells (CD45RA+CCR7+ naïve T cells)/CD8+T cells. Compared to NEC patients at BELL stage I + II, patients at BELL stage III showed increased percentages of SwB cells/B cells, antibody secreting cell (ASC, CD3-CD20-CD27high CD38high ASCs)/B cells and Tem/CD4+ T cells, and decreased percentages of CD45RA+CCR7+ naïve T cells/CD4+ T cells. The Receiver Operating Characteristic Curve analysis showed that the sensitivity of ASC/B cells ratio (0.52%) is 86.67% and the specificity of Tem/CD4+T ratio (5.22%) is 100%, indicating that NEC patients required surgery. Conclusions The severity of NEC exhibits codirectional changes with the maturation of B and T lymphocytes, especially CD4+ T cells. The increased ASC/B and Tem/CD4+ T cells could serve as potential indicators for NEC patients requiring surgery.

Basilar invagination (BI) is a common disease in the craniocervical junction (CVJ) area. Posterior fossa decompression with/without fixation is a controversial surgical strategy for BI type B. This study aimed to evaluate the efficacy of simple posterior fossa decompression in treating BI type B. This study retrospectively enrolled BI type B patients who underwent simple posterior fossa decompression at Huashan Hospital, Fudan University between 2014.12 and 2021.12. Patient data and images were recorded pre- and postoperatively (at the last follow-up) to evaluate the surgical outcomes and craniocervical stability. A total of 18 BI type B patients (13 females), with a mean age of 44.2±7.9 years (range 37–62 years), were enrolled. The mean follow-up period was 47.7±20.6 months (range 10–81 months). All patients received simple posterior fossa decompression without any fixation. At the last follow-up, compared with preoperation, the JOA scores were significantly higher (14.2±1.5 vs. 9.9±2.0, p = 0.001); the CCA was improved (128.7±9.6° vs. 121.5±8.1° p = 0.001), and the DOCL was reduced (7.9±1.5 mm vs. 9.9±2.5 mm, p = 0.001). However, the follow-up and preoperative ADI, BAI, PR, and D/L ratio were similar. No patients had an unstable condition between the C1-2 facet joints that was observed in the follow-up CT and dynamic X-ray. In BI type B patients, simple posterior fossa decompression could improve neurological function and will not induce CVJ instability in BI type B patients. Simple posterior fossa decompression could be a satisfactory surgical strategy for BI type B patients, but preoperative CVJ stability assessment is crucial.

To develop a machine learning algorithm with prognosis data to identify different clinical phenotypes of biliary atresia (BA) and provide instructions for choosing treatment schemes. Six hundred thirty-nine cases of type III BA were retrospectively collected from the Children’s Hospital of Fudan University from Jan 1st, 2017 to Dec 1st, 2019 as a training dataset, and a survival-based forward clustering method, which can also be used to predict the subtype of a new patient was developed to identify BA subtypes. A total of 2 clusters were identified (cluster 1 = 324 and cluster 2 = 315), where cluster 2 had a lower 2 y native liver survival post-Kasai rate. The infant patients in cluster 2 have higher weight, liver, and spleen volume, wider portal vein width, and older operative age; worse coagulation and liver function results; higher grade of liver fibrosis and detection rate of hepatic portal fibrous mass, and higher recent infection detection rate of herpes simplex virus type I. With the proposed prognostic classification system, the authors predicted the subtypes of the 187 cases of type III BA in a testing dataset collected from the whole year of 2020. The p-value computed from the log-rank testing for the Kaplan–Meier survival curves of the predicted two testing groups was 0.0113. This classification system would be a convenient tool to choose appropriate treatment and accelerate the choice-making between clinicians and infant patients.

Introduction: Treatment of anterior cerebral artery (ACA) aneurysms is still not well established. The Leo stent with blood flow direction is a retrievable stent for intracranial aneurysms, whereas it needs to be studied clearly in patients with ACA aneurysms. Methods: Consecutive patients with ACA aneurysms were retrospectively enrolled in three neurosurgical centers between January 2016 and October 2021. The data on demographics, aneurysm characteristics, symptom resolution, and postoperative course were collected and analyzed. The aneurysm occlusion status was appraised by Raymond-Ray Occlusion Class (RROC). Results: A total of 57 patients with ACA aneurysms were included in our study. Immediate postprocedural angiograms showed that 20 aneurysms (35.1%) were in complete occlusion (RROC 1), 26 aneurysms (45.6%) were in near-complete occlusion (RROC 2), 11 aneurysms (19.3%) were in incomplete occlusion (RROC 3). The angiographic follow-up found that the rate of complete occlusion increased to 57.9%, and near-completion and incomplete occlusion dropped to 29.8% and 12.3%, respectively. The angiographic result of the last follow-up improved significantly (Z=- 2.805, P=0.005). Univariate analysis indicated that distal location of aneurysms (Z=4.538, P=0.033) and ruptured aneurysms (χ2=.6120, P=0.032) were potential risk factors for intra-parent artery narrowing. Furthermore, multivariate logistic regression analysis found that A3 aneurysms (95% CI 1.427~32.744, P=0.016) are the key risk factor for intra-parent artery narrowing. Conclusions: The Leo stent is safe and effective for aneurysms located in ACA circulations. The overall occlusion degree improved during follow-up. A distal, small artery was the risk factor for intra-parent artery narrowing.

BACKGROUND:Traumatic spinal cord injury (SCI) is still devastating. It was suggested that the inhibition of mTOR may alleviate neuronal inflammatory injury but its underlying mechanism remained to be elucidated. AIM2 (absent in melanoma 2) recruits ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 to form the AIM2 inflammasome, activate caspase-1, and elicit inflammatory responses. We designed this study to elucidate whether pre-treatments of rapamycin could suppress SCI induced neuronal inflammatory injury via AIM2 signaling pathway in vitro and in vivo.METHODS:We performed oxygen and glucose deprivation / re-oxygenation (OGD) treatment and rats clipping model to mimic neuronal injury after SCI in vitro and in vivo. Morphologic changes of injured spinal cord were detected by hematoxylin and eosin staining. The expression of mTOR, p-mTOR, AIM2, ASC, Caspase-1 and et al were analyzed by fluorescent staining, western blotting or qPCR. The polarization phenotype of microglia was identified by flow cytometry or fluorescent staining.RESULTS:We found BV-2 microglia without any pre-treatment cannot alleviate primary cultured neuronal OGD injury. However, pre-treated rapamycin in BV-2 cells could transform microglia to M2 phenotype and protects against neuronal OGD injury via AIM2 signaling pathway. Similarly, pre-treatment of rapamycin could improve the outcome of cervical SCI rats through AIM2 signaling pathway.CONCLUSIONS:It was suggested that resting state microglia pre-treated by rapamycin could protect against neuronal injury via AIM2 signaling pathway in vitro and in vivo. Pre-inhibition of mTOR pathway may improve neuronal protection after SCI.

Background Biliary atresia (BA) is characterized by a progressive fibroinflammatory cholangiopathy in early infants with unknown etiology. Although innate immune disorder is involved in its mechanism, role of NLRP3 inflammasome in BA remains largely undefined. Aim To explore the role of NLRP3 inflammasome in BA. Methods The expressions of NLRP3 inflammasome-related genes were determined in BA patients. Role of NLRP3 inflammasome was evaluated using MCC950 in experimental BA. Furthermore, gadolinium chloride, a macrophage scavenger, was applied to validate the inflammasome's cellular localization. Finally, the effects of NLRP3 inflammasome activation on liver fibrosis were explored in vivo and vitro in experimental BA. Results The components of NLRP3 inflammasome were up-regulated in BA patients. Inflammasome-related genes showed positively correlated with liver inflammation and fibrosis in BA patients. In experimental BA, inflammasome-related genes were up-regulated, and their expressions were inhibited by MCC950, which promoted mice growth, protected liver function, alleviated obstructive jaundice, inhibited liver inflammation, and reduced serum IL-1β level. NLRP3 inflammasome was expressed in macrophages, and macrophage elimination exerted the same protective roles as MCC950 did in BA. Additionally, NLRP3 inflammasome activation promoted liver fibrosis in experimental BA. Conclusions NLRP3 inflammasome activation in macrophages promoted liver inflammation and fibrosis in experimental BA.

Background: The pathogenesis of Hirschsprung's disease (HSCR) remains unclear but might involve genes participating in neural crest development. Gene methylation controls the expression of many genes and is involved in the development and migration of neural crest cells, but the involvement of demethylation in HSCR is unknown. This study aimed to investigate the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) (a demethylation protein) in patients with HSCR. Methods: This is a retrospective study of surgical specimens from paediatric patients with and without HSCR (e.g., intussusception and incarcerated hernia) obtained from 07/2015 to 08/2017. TETI expression was determined by qRT-PCR, western blotting, and immunohistochemistry. The levels of 5-hydroxymethylcytosine were determined by the dot blot assay. Results: The specimens of 35 patients with HSCR and 25 controls were collected. The median TET1 mRNA expression values were 1.028 [HSCR-stenotic (S)], 0.908 [HSCR-dilated (D)], and 0.467 (control) (HSCR-S vs. control: P = 0.002; HSCR-D vs. control: P = 0.008; HSCR-S vs. HSCR-D: P = 0.44). TET1 protein levels followed a similar pattern. The intensity of immunostaining identified higher expression of TET1 in HSCR colon tissues compared with control tissues. The 5-hmC levels in HSCR stenotic segment samples were significantly higher than those in controls. Conclusion: The expression of TET1 is higher in paediatric patients with HSCR than in controls. DNA demethylation initiated by TET1 may be related to HSCR, which demonstrates that TET1 may play a role in the development of HSCR. Copyright (C) 2022, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC.

Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide.1 Shifting the detection of CRC to earlier stages via massive screening has considerably reduced mortality.2 Metabolomics has shown great potential in the identification of noninvasive biomarkers of CRC. However, the huge number and inconsistent reports of putative markers make choosing the most appropriate biomarkers difficult. The aim of this study was to identify the metabolic markers of CRC by a multi-step strategy. We first identified and verified differential plasma metabolites of CRC by a two-stage case–control design. The tumour specificity of plasma markers was then confirmed by comparison with tumor-adjacent non-malignant paired tissue. Moreover, we conducted a systematic review of metabolomics studies of CRC to affirm the markers in multiple populations. Finally, the metabolites were quantitatively evaluated in an independent case–control population (Figure 1). A two-stage case–control study involving 170 cases and 197 controls was performed. All patients were diagnosed at the Third Affiliated Hospital of Harbin Medical University. Controls were recruited from patients in the orthopaedic and ophthalmology departments and volunteers from Xiangfang District of Harbin City during the same period. Fasting peripheral venous blood was obtained in the morning in the hospital or medical examination centre. Metabolic profiling analysis was conducted on a ultra performance liquid chromatography (UPLC)/Q–time-of-flight (TOF)–mass spectrometry (MS)/MS platform. Principal component analysis and orthogonal projections to latent structures discriminant analysis were performed to check the separation tendency. Student's t-test or Wilcoxon's rank-sum test with an adjusted P-value was applied to test the metabolites between cases and controls (details in Supporting Information, Section 1.1). No significant differences in age, sex or body mass index were observed between the cases and controls in either the first or second stage (Table S1). The datasets with 5394 and 7312 variables in electrospray ionization in negative and positive ion mode (ESI− and ESI+) in the first stage, and 6630 and 7760 variables in ESI− and ESI+ in the second stage, respectively, were imported for multivariate statistical analyses. By excluding ion fragments from the same parent ions and the duplicated variables in ESI− and ESI+, 388 variables were selected after univariate analysis. Finally, 147 metabolites were identified. Pathway-enrichment analysis was conducted using a hypergeometric test, and six pathways (including 62 of the 147 metabolites) were enriched (Supporting Information, Section 2.1.1). Eleven of the 147 metabolites were introduced into binary logistic regression because of an area under the curve (AUC) of > 0.85, and 4 showed statistical significance [eicosenoic acid, alpha-N-phenylacetyl-l-glutamine, 3a,7a-dihydroxycholanoic acid, and LysoPC(16:1(9Z))]. The combined AUCs [first-stage AUC: 1.000, 95% confidence interval (CI): 1.000–1.000; second-stage AUC: 0.989, 95% CI: 0.980–0.999] were higher than the AUC of any individual marker. However, all markers except LysoPC(16:1(9Z)) failed to be enriched in any biological pathway. Small-molecule metabolites perform biological functions in pathways. Our findings indicated that these three metabolites have unknown biological function or may not be functional. Thus, although these metabolites exhibited the greatest efficacy in distinguishing CRC cases, this lack of biological significance may result in false-positive diagnoses. Pavlova and Thompson3 suggested that there are some common features of cancer cell metabolism. Although few tumours display all of these hallmarks, most display several.4, 5 Thus, no single metabolic pathway can fully reflect tumorigenesis-associated metabolic alterations. One or a few metabolites from a single metabolic pathway also have limited diagnostic capacity. Choosing a group of metabolites encompassing a particular combination of hallmarks may ultimately improve diagnosis and tumor classification. Fifty-nine metabolites shared across the 141 metabolites with an AUC of > 0.65 and the 62 metabolites involved in the six biological pathways were analysed. Six of the 59 metabolites [2-aminobenzoic acid, 13-OxoODE, citric acid, 2′-deoxyinosine triphosphate, taurocholic acid, and LysoPC(16:1(9Z)] were focused on because they had the highest AUCs in each pathway. The combined AUCs were 0.982 (95% CI: 0.962–1.000) and 0.974 (95% CI: 0.956–0.992) in the two-stage study (Figure 2B). Blood metabolites reflect the global metabolic perturbation throughout the body. However, the plasma metabolites replicated in targeted organ tissues represent the specific metabolic characteristics of tumour cells. Thus, tissue samples of the deepest infiltration of the tumour and the adjacent non-malignant mucosal tissues were analysed (Supporting information, Section 1.1.2). Ninety-three differential metabolites were identified (Supporting information, Section 2.1.2). Thirteen with an AUC of > 0.65 in plasma were overlapped in tissue (Dataset A). Five of the 13 [l-tryptophan, 13-OxoODE, IDP, glycochenodeoxycholate, and LysoPC(16:0)] were selected as CRC panel 1 because they had the highest AUCs in the pathways (Figure 2C). The combined AUCs were 0.982 (0.963–1.000) and 0.963 (0.943–0.984) in the two-stage study in plasma and 0.958 (0.924–0.991) in paired tissue. Metabolomics results may be affected by several factors, including the testing coverage, sensitivity of analytical instruments, sample pretreatment method, and data analysis process.6 Metabolites are sensitive to many endogenous pathologies and external stimuli,7, 8 such as dietary, environmental or gut microbial factors.9 A systematic review is a method to collect a complete summary of the current evidence relevant to a research question.10 Hence, it facilitates assessment of metabolic markers with reproducibility in diverse laboratories and populations. Sixty-two metabolomics studies of CRC were reviewed, and 635 differential metabolites were extracted (Supporting information, Sections 1.2 and 2.2). Twenty-seven of the 635 metabolites that had an AUC of > 0.65 and were enriched in six biological pathways were designated Dataset B. Six metabolites [l-phenylalanine (reported frequency = 20), linoleic acid (8), citric acid (9), inosine (4), glycocholic acid (3), and LysoPC(14:0) (3)] were selected when focusing on the reported frequency. The combined AUCs were 0.971 (0.949–0.994) and 0.948 (0.921–0.976) in plasma (CRC panel 2, Figure 3). Four metabolites [l-tryptophan, linoleic acid, glycocholic acid, and LysoPC(16:0)] were finally identified from the overlap of Datasets A and B (Dataset C) because they had multiple advantages of favourable AUCs, biological significance, tumor tissue reproducibility, and the highest reported frequency (Supporting information, Sections 1.3 and 2.3). The AUCs were 0.942 (0.901–0.983) and 0.937 (0.906–0.968) in plasma. In particular, the AUCs were > 0.93 in the diagnosis of stage I/II (Figures S14 and S15). Ultimately, we quantified five metabolites [l-tryptophan, linoleic acid, LysoPC(14:0), LysoPC(16:0), and LysoPC(18:0)] in the targeted analysis. A batch of 504 plasma samples (251 patients with CRC and 253 controls) was detected (Supporting information, Sections 1.4 and 2.4). The AUC of l-tryptophan, linoleic acid, LysoPC(14:0), and LysoPC(18:0) was 0.728 (0.681–0.775) in plasma (Figure 4). This study had some limitations. First, the fact that the biomarkers were not well validated might have been due to some other parameters that were either unknown or not tightly controlled. Second, change variation was not taken as a strict standard in the screening process because we did not want to exclude metabolites that may have better sensitivity in early cancer. The response of small molecular metabolites to nutritional or disease states is so sensitive that even subtle disturbances can be detected much earlier than genomic and proteomic variation. Third, UPLC/Q–TOF–MS/MS is not a good technique for quantitative analysis. Fourth, the model discrimination performance still requires validation internally and externally using targeted quantitative analyses. Finally, comparisons with the metabolites of other diseases are still required. In conclusion, we revealed the most promising metabolic biomarkers of CRC in plasma using the herein-described multi-step analysis strategy. Future application of multi-omics markers may achieve accurate noninvasive diagnosis. This work was supported by grants from the National Nature Science Foundation of China (81773503, 81573147, and 81473055), Scientific Research Foundation for the Returned Overseas Scholars of Heilongjiang Province (LC2018033), and Natural Science Foundation of Heilongjiang Province of China (for Youths, QC2018111). The authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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