Effects of Retrograde Colonic Enema Based Fecal Microbiota Transplantation in the Treatment of Childhood Constipation: A Randomized, Double-blind Controlled Trial.

OBJECTIVE: Management of intractable childhood constipation is still challenging. The efficacy of retrograde colonic enema (RCE) with fecal microbiota transplantation (FMT) in intractable childhood constipation has not been established although both have demonstrated potential in gastrointestinal diseases. The aim of the current study was to determine the safety and efficacy of RCE-based FMT in the treatment of intractable constipation in children. METHODS: A randomized, double-blind, controlled trial with 110 children was conducted. The subjects were randomly assigned to the FMT with RCE group or the placebo with RCE group. All participants received a daily RCE followed by a 4-week FMT treatment (twice a week) and a 12-week follow-up period. Spontaneous bowel movements (SBMs) ≥ 3 per week were the main outcomes and the risk ratio (RR) with 95% CI was calculated. Changes in the intestinal bacterial profile were analyzed by BOX-PCR-based DNA fingerprinting and sequencing. The adverse effects were assessed based on symptoms. RESULTS: At the end of the follow-up period, 22 patients (40.0%) in the FMT with RCE group and 10 patients (18.2%) in the placebo with RCE group had ≥ 3 SBMs per week (net difference = 21.8%, 95% CI: 13.2%-30.4%; RR: 1.364, 95% CI: 1.063-1.749; P<0.05). Both RCE and FMT enriched the intestinal bacterial diversity of patients with constipation. The adverse events were all mild self-limiting gastrointestinal symptoms. CONCLUSIONS: FMT enhances the efficacy of RCE and the use of RCE-based FMT is a safe and effective method in the treatment of intractable constipation in children.

A novel genotype-phenotype between persistent-cloaca-related VACTERL and mutations of 8p23 and 12q23.1.

The mechanism underlying anorectal malformations (ARMs)-related VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, and renal and limb abnormalities) remains unclear. Copy number variation (CNV) contributed to VACTERL pathogenicity. Here, we report a novel CNV in 8p23 and 12q23.1 identified in a case of ARMs-related VACTERL association. This 12-year-old girl presented a cloaca (urethra, vagina, and rectum opening together and sharing a single tube length), an isolated kidney, and a perpetuation of the left superior vena cava at birth. Her intelligence, growth, and development were slightly lower than those of normal children of the same age. Array comparative genomic hybridization revealed a 9.6-Mb deletion in 8p23.1-23.3 and a 0.52-Mb duplication in 12q23.1 in her genome. Furthermore, we reviewed the cases involving CNVs in patients with VACTERL, 8p23 deletion, and 12q23.1 duplication, and our case was the first displaying ARMs-related VACTERL association with CNV in 8p23 and 12q23.1. These findings enriched our understanding between VACTERL association and the mutations of 8p23 deletion and 12q23.1 duplication. IMPACT: This is a novel case of a Chinese girl with anorectal malformations (ARMs)-related VACTERL with an 8p23.1-23.3 deletion and 12q23.1 duplication. Cloaca malformation is presented with novel copy number variation in 8p23.1-23.3 deletion and 12q23.1 duplication.

Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Mesenteric and omental lymphatic malformations in children: seven-year surgical experience from two centers in China.

PURPOSE: To compare the clinical characteristics, surgical management and prognosis of mesenteric lymphatic malformations (ML) and omental lymphatic malformations (OL) in children. METHODS: This retrospective study included 148 ML patients and 53 OL patients who underwent surgical treatment at two centers between January 2016 and December 2022. Details about the patients' clinical characteristics, cyst characteristics, preoperative complications, surgical methods, and prognosis were retrieved and compared. RESULTS: No significant differences in sex ratio, prenatal diagnosis, or age of diagnosis were noted between ML and OL patients. Vomiting was more common in ML patients than in OL patients (46.6% vs. 22.6%, P = 0.002), but OL patients were more likely to be misdiagnosed (35.8% vs. 18.9%, P = 0.012). The size of the cysts in OL patients was significantly larger than that in ML patients (14.0 [4.0-30.0] vs. 10.0 [2.0-50.0] cm, P<0.001), and cysts with turbid fluid were more common in OL patients (38.0% vs. 20.6%, P<0.001). More OL patients than ML patients had preoperative hemorrhage or infection of cysts (41.5% vs. 31.8%, P<0.016). Cyst excision was performed in 137 (92.6%) ML patients and 51 (96.2%) OL patients, and the incidence of postoperative complications was lower (12.6% vs. 4.2%, P = 0.165) among OL patients. The main postoperative complications included adhesive ileus and recurrence of cysts. Additionally, more OL patients than ML patients were treated with laparoscopic surgery (69.8% vs. 39.2%, P<0.001). CONCLUSIONS: There were differences in clinical characteristics, cyst characteristics and preoperative complications between ML and OL patients. Cyst excision was the most common surgical method that was used to treat both ML and OL patients, and laparoscopic surgery could be a feasible surgical approach for treating OL patients with a good prognosis. TRIAL REGISTRATION: Retrospectively registered.

Automated prediction of early spontaneous miscarriage based on the analyzing ultrasonographic gestational sac imaging by the convolutional neural network: a case-control and cohort study.

BACKGROUND: It is challenging to predict the outcome of the pregnancy when fetal heart activity is detected in early pregnancy. However, an accurate prediction is of importance for obstetricians as it helps to provide appropriate consultancy and determine the frequency of ultrasound examinations. The purpose of this study was to investigate the role of the convolutional neural network (CNN) in the prediction of spontaneous miscarriage risk through the analysis of early ultrasound gestational sac images. METHODS: A total of 2196 ultrasound images from 1098 women with early singleton pregnancies of gestational age between 6 and 8 weeks were used for training a CNN for the prediction of the miscarriage in the retrospective study. The patients who had positive fetal cardiac activity on their first ultrasound but then experienced a miscarriage were enrolled. The control group was randomly selected in the same database from the fetuses confirmed to be normal during follow-up. Diagnostic performance of the algorithm was validated and tested in two separate test sets of 136 patients with 272 images, respectively. Performance in prediction of the miscarriage was compared between the CNN and the manual measurement of ultrasound characteristics in the prospective study. RESULTS: The accuracy of the predictive model was 80.32% and 78.1% in the retrospective and prospective study, respectively. The area under the receiver operating characteristic curve (AUC) for classification was 0.857 (95% confidence interval [CI], 0.793-0.922) in the retrospective study and 0.885 (95%CI, 0.846-0.925) in the prospective study, respectively. Correspondingly, the predictive power of the CNN was higher compared with manual ultrasound characteristics, for which the AUCs of the crown-rump length combined with fetal heart rate was 0.687 (95%CI, 0.587-0.775). CONCLUSIONS: The CNN model showed high accuracy for predicting miscarriage through the analysis of early pregnancy ultrasound images and achieved better performance than that of manual measurement.

Therapeutic potential of adenovirus-encoding brain-derived neurotrophic factor for spina bifida aperta by intra-amniotic delivery in a rat model.

Spina bifida aperta is a type of neural tube defect (NTD). Although prenatal fetal surgery has been an available and effective treatment for it, the neurological functional recovery is still need to be enhanced. Our previous results revealed that deficiencies of sensory, motor, and parasympathetic neurons were primary anomalies that occurred with the spinal malformation. Therefore, we emphasized that nerve regeneration is critical for NTD therapy. We delivered an adenoviral construct containing genes inserted for green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) into the amniotic fluid to investigate its prenatal therapeutic potential for rat fetuses with spina bifida aperta. Using immunofluorescence, TdT-mediated dUTP nick-end labeling staining, and real-time polymerase chain reaction analysis, we assessed cell apoptosis in the defective spinal cord and Brn3a positive neuron survival in the dorsal root ganglion (DRG); a protein array was used to investigate the microenvironmental changes of the amniotic fluid. We found that most of the overexpressed BDNF was present on the lesions of the spina bifida fetuses, the number of apoptosis cells in Ad-GFP-BDNF-transfected spinal cords were reduced, mRNA levels of Bcl2/Bax were upregulated and Casp3 were downregulated compared with the controls, the proportion of Brn3a positive neurons in DRG were increased by activating the BDNF/TrkB/Akt signaling pathway, and most of the significant changes in cytokines in the amniotic fluid were related to the biological processes of regulation of apoptotic process and generation of neurons. These results suggest that intra-amniotic Ad-GFP-BDNF gene delivery might have potential as a supplementary approach to treat congenital malformations of neural tubes.

BMP7 is Downregulated in Lumbosacral Spinal Cord of Rat Embryos With Anorectal Malformation.

BACKGROUND: Bone morphogenetic proteins (BMPs) comprise a highly conserved signaling protein family, which are involved in spinal cord formation, development and differentiation. Malformations of the lumbosacral spinal cord are associated with postoperation complications of anorectal malformation (ARM). However, the mechanism underlying the development of these malformations remains elusive. MATERIALS AND METHODS: Embryonic rat ARM model induced by ethylenethiourea (ETU) was introduced to investigate BMP7 expression in lumbosacral spinal cord. BMP7 expression was analyzed by immunohistochemical staining, qRT-PCR, and Western blot analysis on embryonic (E) days 16, 17, 19, and 21. The expression of the neuronal marker neurofilament (NF) and pSmad1/5 was determined by immunofluorescence double staining and Western blot analysis during peak BMP7 expression. RESULTS: BMP7 mRNA (E16, 1.041 ± 0.169 versus 0.758 ± 0.0423, P < 0.05; E17, 1.889 ± 0.444 versus 1.601 ± 0.263, P < 0.05; E19, 2.898 ± 0.434 versus 1.981 ± 0.068, P < 0.01; and E21, 2.652 ± 0.637 versus 1.957 ± 0.09, P < 0.05;) and protein (E16, 1.068 ± 0.065 versus 0.828 ± 0.066, P < 0.01; E17, 1.728 ± 0.153 versus1.4 ± 0.148, P < 0.05; E19, 2.313 ± 0.141 versus 1.696 ± 0.21, P < 0.01; and E21, 2.021 ± 0.13 versus 1.43 ± 0.128, P < 0.01) were downregulated, and their expressions were specifically low in interneurons (IN) located in the dorsal horn of the lumbosacral spinal cord in embryos with ARM. On E19, Western blot analysis revealed reduced P-Smad1/5(1.13 ± 0.08 versus 0.525 ± 0.06, P < 0.01). CONCLUSIONS: An implication of this study is the possibility that BMP7 downregulation contributes to maldevelopment of the lumbosacral spinal cord during embryogenesis in fetal rats with ARM, indicating that BMP7 may play an important role in ARM pathogenesis and the complications thereof.

Altered mRNA and lncRNA expression profiles in the striated muscle complex of anorectal malformation rats.

BACKGROUND: Striated muscle complex (SMC) dysplasia has been confirmed to contribute to postoperative defecation dysfunction of patients with anorectal malformations (ARMs). To date, the potential molecular mechanisms of SMC dysplasia underlying the development of ARMs have not been clearly explained. This study examined the expression profiles of mRNAs and lncRNAs in the malformed SMC of ARM rats using RNA sequencing (RNA-seq). METHODS: A rat model of ARMs was established by the intragastric administration of 1% ethylene thiourea (ETU) on an embryonic day 10 (E10). The rats were subjected to euthanasia and the SMC samples were collected on E19. The expression of mRNAs and lncRNAs was analyzed by RNA-seq on the Illumina HiSeq2500 platform. qRT-PCR was used to confirm the results of RNA-seq. RESULTS: Compared with the levels in control rats, 1408 mRNAs and 472 lncRNAs were differentially expressed in the SMC of E19 ARM rats. GO and KEGG pathway analyses showed that the top enriched GO terms were mainly related to muscle development and the enriched pathways were associated with muscle and synaptic development. Protein-protein interaction network analysis was also performed using the STRING database. The network map revealed the interaction between the WNT3 protein and NTRK1, NTF4, MUSK, and BMP5 proteins. Finally, the qRT-PCR results further confirmed the RNA-seq data. CONCLUSION: Our findings indicate the involvement of these dysregulated mRNAs and lncRNAs in the pathogenesis of SMC dysplasia in ARMs, providing a theoretical foundation for developing interventions to improve postoperative defecation function.

The Expression of Shh, Ptch1, and Gli1 in the Developing Caudal Spinal Cord of Fetal Rats With Anorectal Malformations.

BACKGROUND: Postoperative incontinence and constipation still remain the major complications of anorectal malformations (ARMs), despite improvements in their treatment. One of the most important factors that affect postoperative anorectal function is malformations in the lumbosacral spinal cord. However, far too little attention has been paid to the underlying mechanism that produces these malformations. MATERIALS AND METHODS: The levels of sonic hedgehog (Shh), patched homolog 1 (Ptch1), and zinc finger-containing transcription factors 1 (Gli1) expression were investigated in the lumbosacral spinal cord in ethylenethiourea-exposed rat fetus with ARMs, and Shh, Ptch1, and Gli1 expression was confirmed with immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot analyses during lumbosacral spinal cord development both in the ARMs and normal rat embryos. RESULTS: Our results have shown that Shh, Ptch1, and Gli1 expression in the lumbosacral spinal cord of rat embryos with ARMs was decreased at both the messenger RNA and protein levels, when compared with their expression levels in normal tissues (P < 0.05). CONCLUSIONS: This study demonstrated that the expression of Shh, Ptch1, and Gli1 in lumbosacral spinal cord was remarkably reduced during late developmental stages in fetal rats with ARMs. These findings offered some important insights into the involvement of the Shh-Ptch1-Gli1 signaling pathway in the pathogenesis of lumbosacral spinal cord maldevelopment in rat fetus with ARMs, which leads to complications after procedures for ARMs.

Abnormal expression of TBX4 during anorectal development in rat embryos with ethylenethiourea-induced anorectal malformations.

BACKGROUND: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatio-temporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. RESULTS: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

Spatiotemporal Expression of Bcl-2/Bax and Neural Cell Apoptosis in the Developing Lumbosacral Spinal Cord of Rat Fetuses with Anorectal Malformations.

Fecal incontinence and constipation still remain the major complications after procedures for anorectal malformations (ARMs). Previous studies have demonstrated a decrease of neural cell in lumbosacral spinal cord of ARMs patients and rat models. However, the underlying mechanism remains elusive. In this study, the neural cell apoptosis and Bcl-2/Bax expression were explored during lumbosacral spinal cord development in normal and ARMs fetuses. ARMs rat fetuses were induced by treating pregnant rats with ethylenethiourea on embryonic day 10. TUNEL staining was performed to identify apoptosis, and the expression of Bcl-2/Bax was confirmed with immunohistochemical staining, RT-qPCR and Western blot analysis on E16, E17, E19 and E21. Apoptosis index (AI) in the ARMs group was significantly higher compared to normal group. Our results showed that TUNEL-positive cells were mainly localized in the ventral horn, which is the location of neural cells controlling defecation. And the expression of Bcl-2 decreased, whereas the level of Bax increased in the ARMs fetuses. In addition, there was a significantly negative correlation between protein expression of Bcl-2/Bax ratio and AI in the ARMs group. Abnormal apoptosis might be a fundamental pathogenesis for the number decrease of neural cells in lumbosacral spinal cord, which leads to complications after procedures for ARMs. The negative correlation between the ratio of Bcl-2/Bax and AI manifested that Bcl-2/Bax pathway might be the mechanism for neural cell apoptosis in ARMs.

Clinical and Ultrasonographic Features of Secondary Intussusception in Children.

OBJECTIVES: The aim of this study was to review the ultrasonographic features of secondary intussusception (SI) in children and assess the value of ultrasound in the diagnosis of pediatric SI. METHODS: The authors performed a retrospective analysis on the ultrasound findings of 1977 cases of primary intussusception (PI) and 37 cases of SI in children. The SI cases were diagnosed by ultrasonography and confirmed by laparotomy or histopathologic diagnosis. The clinical and ultrasonographic features were analyzed and compared between these two groups. RESULTS: The age, no flatus or defecation, position, diameter and length of intussusception, the presence of free intraperitoneal liquid, and intestinal dialation at the proximal end present, all contributed to the differentiation between PI and SI (all P < 0.05). Ultrasound was able to demonstrate the pathological lead point (PLP) shadows in all of the 37 SI cases, either in the cervical part or intussusceptum of the intussusception. Among the 37 SI patients, 21 cases (56.8 %) were accurately categorized with lesions, including intestinal polyps, cystic intestinal duplication, intestinal wall lymphoma, and a small part of Meckel's diverticulum. CONCLUSIONS: Ultrasound can be used as a feasible and effective method to discriminate PI from SI. Once the PLP is detected, a definite diagnosis can be made. KEY POINTS: • The clinical and ultrasonographic features were compared between SI and PI. • The age, location, diameter and length of intussusception, and intestinal dilation were distinguishing features. • The causes of SI were found to be polyps, intestinal duplication, lymphoma, and Meckel's diverticulum. • Ultrasound can be used as an important method to diagnose SI. • Demonstration and confirmation of PLP are vital to diagnosing SI.

Reduced Wnt3a expression correlates with poor development of the hindgut in rats with anorectal malformations.

Embryogenesis is orchestrated by the wingless-type MMTV integration site family (WNT) signaling pathways, including Wnt3a. This study was performed to investigate the expression of Wnt3a in the terminal hindgut in ethylenethiourea (ETU)-exposed rat embryos with anorectal malformations (ARMs) and its potential association between Wnt3a and the maldevelopment of the terminal hindgut in ARMs. ARM rat embryos were induced by ethylenethiourea on embryonic day 10 (E10). The expression levels of protein and mRNA of Wnt3a were confirmed using immunohistochemistry staining, Western blotting analyses, and quantitative real-time PCR (qRT-PCR) in normal rat and ARM embryos. Immunostaining revealed a variation in the expression of Wnt3a in the developing terminal hindgut of ARM embryos. The expression of Wnt3a in the terminal hindgut of ARM rat embryos decreased at both the mRNA level and protein level (P<0.05) compared with normal tissues. This study demonstrated that the expression of Wnt3a in the ARMs of ETU-exposed rat embryos was remarkably reduced, which indicated its potential role in the pathogenesis of the terminal hindgut maldevelopment in ARMs.

Anti-miR-141-3p maintains homeostasis between autophagy and apoptosis by targeting Yy1 in the fetal lumbosacral defecation center of rats.

Anorectal malformations (ARMs) are congenital diseases that lead to postoperative fecal incontinence, constipation, and soiling, despite improvements in surgery; however, their pathological mechanisms remain unclear. Here, we report the role of microRNA-141-3p in maintaining homeostasis between apoptosis and autophagy in the lumbosacral defecation center of fetal rats with ARMs. Elevated microRNA-141-3p expression inhibited YIN-YANG-1 expression by binding its 3' UTR, and repressed autophagy and triggered apoptosis simultaneously. Then, adenylate cyclase 3 was screened to be the downstream target gene of YIN-YANG-1 by chromatin immunoprecipitation sequencing experiments, and Yin Yang 1 could positively activate the transcription of adenylate cyclase 3 by directly interacting with the motif GAGATGG and ATGG in its promoter. Intraamniotic microinjection of adeno-rno-microRNA-141-3p-sponge-GFP in fetal rats with ARMs on embryonic day 15 restored apoptosis-autophagy homeostasis. These findings reveal that microRNA-141-3p upregulation impaired homeostasis between apoptosis and autophagy by inhibiting the YIN-YANG-1/adenylate cyclase 3 axis, and that intraamniotic injection of anti-microRNA-141-3p helped maintain homeostasis in the lumbosacral defecation center of ARMs during embryogenesis.

Regulatory role of m6A epitranscriptomic modifications in normal development and congenital malformations during embryogenesis.

The discovery of N6-methyladenosine (m6A) methylation and its role in translation has led to the emergence of a new field of research. Despite accumulating evidence suggesting that m6A methylation is essential for the pathogenesis of cancers and aging diseases by influencing RNA stability, localization, transformation, and translation efficiency, its role in normal and abnormal embryonic development remains unclear. An increasing number of studies are addressing the development of the nervous and gonadal systems during embryonic development, but only few are assessing that of the immune, hematopoietic, urinary, and respiratory systems. Additionally, these studies are limited by the requirement for reliable embryonic animal models and the difficulty in collecting tissue samples of fetuses during development. Multiple studies on the function of m6A methylation have used suitable cell lines to mimic the complex biological processes of fetal development or the early postnatal phase; hence, the research is still in the primary stage. Herein, we discuss current advances in the extensive biological functions of m6A methylation in the development and maldevelopment of embryos/fetuses and conclude that m6A modification occurs extensively during fetal development. Aberrant expression of m6A regulators is probably correlated with single or multiple defects in organogenesis during the intrauterine life. This comprehensive review will enhance our understanding of the pivotal role of m6A modifications involved in fetal development and examine future research directions in embryogenesis.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Intra-amniotic mesenchymal stem cell therapy improves the amniotic fluid microenvironment in rat spina bifida aperta fetuses.

OBJECTIVES: Spina bifida aperta (SBA) is one of the most common neural tube defects. Neural injury in SBA occurs in two stages involving failed neural tube closure and progressive degeneration through contact with the amniotic fluid. We previously suggested that intra-amniotic bone marrow-derived mesenchymal stem cell (BMSC) therapy for fetal rat SBA could achieve beneficial functional recovery through lesion-specific differentiation. The aim of this study is to examine whether the amniotic fluid microenvironment can be improved by intra-amniotic BMSC transplantation. METHODS: The intra-amniotic BMSC injection was performed using in vivo rat fetal SBA models. The various cytokine expressions in rat amniotic fluid were screened by protein microassays. Intervention experiments were used to study the function of differentially expressed cytokines. RESULTS: A total of 32 cytokines showed significant upregulated expression in the BMSC-injected amniotic fluid. We focused on Activin A, NGF, BDNF, CNTF, and CXCR4. Intervention experiments showed that the upregulated Activin A, NGF, BDNF, and CNTF could inhibit apoptosis and promote synaptic development in fetal spinal cords. Inhibiting the activity of these factors weakened the anti-apoptotic and pro-differentiation effects of transplanted BMSCs. Inhibition of CXCR4 activity reduced the engraftment rate of BMSCs in SBA fetuses. CONCLUSION: BMSC transplantation can improve the amniotic fluid environment, and this is beneficial for SBA repair.

The expression analysis of Notch-1 and Jagged-2 during the development of the hindgut in rat embryos with ethylenethiourea induced anorectal malformations.

BACKGROUND: This study was designed to investigate the expression of Notch-1 and Jagged-2 in the terminal hindgut in ethylenethiourea (ETU)-exposed rat embryos with anorectal malformations (ARMs) and its potential association with the maldevelopment of the terminal hindgut in ARMs. MATERIAL AND METHODS: ETU-exposed ARMs model was introduced to investigate the expression pattern of Notch-1 and Jagged-2 during the hindgut development using immunohistochemical staining, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: Immunostaining revealed that the expression of Notch-1 and Jagged-2 showed changes in the developing terminal hindgut of ARMs. The expression of Notch-1 and Jagged-2 in the terminal hindgut of ARMs rat embryos decreased at both mRNA level and protein level (P < 0.05) compared with normal tissues. CONCLUSION: This study demonstrated that the expression of Notch-1 and Jagged-2 in ARMs of ETU-exposed rat embryos was remarkably reduced, which implied its potential role in the pathogenesis of the terminal hindgut maldevelopment in ARMs.

Diagnosis and management of fecal incontinence in children and adolescents.

Fecal incontinence (FI) is a commonly occurring disease of high concern. It is characterized by voluntary and involuntary defecation in children and adolescents. It is not only a physical disease but also a psychological and behavioral disorder. FI poses a serious burden on individuals and their families and therefore has become a social problem. Unfortunately, the management of FI among children is still a challenge because the etiology varies widely. Constipation has been found to be the most common cause, while sphincter dysfunction and neurogenic abnormalities may also play a role. Currently, no consensus guidelines exist, and the criteria for selecting optional methods remain unclear. It is therefore necessary to improve the efficacy of diagnosis and management strategies of FI in children. This review focused on the classification and etiology, discussed the diagnosis and management methods of FI in children and adolescents, and aimed to guide future studies.

Transcriptome analysis reveals the spinal expression profiles of non-coding RNAs involved in anorectal malformations in rat fetuses.

BACKGROUND: Despite improvements in anorectal malformation (ARM) therapy, patients might still experience post-operative problems such as fecal incontinence, constipation, and soiling. In particular, the dysplasia of the lumbosacral spinal cord in ARM patients is a major disorder that affects fecal function post-operation. However, the pathological mechanisms involved are still unclear. METHODS: The non-coding RNAs (ncRNAs) in the lumbosacral spinal cord of fetal rats with ethylenethiourea-induced ARM were identified using RNA sequencing (RNA-seq) and examined to determine their potential function. The lumbosacral spinal cord was isolated on embryonic day 17 for subsequent RNA extraction and RNA-seq. The transcriptome data was analyzed using bioinformatics analysis, followed by validation using quantitative reverse transcription PCR. RESULTS: Compared to the control group, 26 differentially expressed microRNAs (DEMs; 22 upregulated, 4 downregulated) and 112 differentially expressed long non-coding RNAs (63 upregulated, 49 downregulated) were identified in the ARM group. Several DEMs related to development, namely miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-200a-5p, and miR-429, were selected for further analysis. Notably, compared to the control, the relative expression of miR-200 family members was highly upregulated in ARM fetal rats. Furthermore, GO and KEGG enrichment and miRNA-transcription factor-lncRNA/mRNA network analysis was explored to show molecular mechanism underlying DEMs. CONCLUSIONS: Our findings suggest the involvement of ncRNAs, especially the miR-200 family members, in the pathogenesis of lumbosacral spinal cord dysplasia in ARM fetal rats.