So many measures in ERAS protocol: Which matters most?

OBJECTIVES: Enhanced recovery after surgery (ERAS), which includes multiple measures, has gradually become the standard perioperative management in pediatric surgery. However, it is still unclear which of its many measures affects the outcomes more. METHODS: We retrospectively analyzed the medical records of children with congenital choledochal cysts who underwent surgical treatment in a specialized children's hospital from January 2019 to December 2022. Data including baseline factors, implementation of ERAS interventions, postoperative complications, and postoperative length of stay (PLOS) were collected. Univariate and multivariate analyses were performed to identify the association between PLOS and baseline factors or specific ERAS measures. RESULTS: The implementation rate of ERAS measures ranged from 5.02% to 100% in 219 cases who underwent 3 to 14 ERAS measures. Univariate analysis showed that body mass index-for-age z-scores, liver function indicators, and postoperative complications were the significant baseline factors for PLOS. At the same time, the measures with the greatest effect on PLOS were early postoperative feeding and early removal of tubes. Multivariate analysis with different models revealed that postoperative complications, early postoperative feeding, and early catheter removal influenced the PLOS the most. CONCLUSIONS: A prolonged PLOS was associated with poor preoperative nutritional status, elevated liver function indexes, and postoperative complications. Early postoperative feeding and removal of tubes appeared more likely with a reduced PLOS than other measures, requiring more attention when implementing the ERAS protocol.

A convenient nomogram for predicting early death or liver transplantation after the Kasai procedure in patients with biliary atresia.

PURPOSE: Many patients with biliary atresia (BA) after the Kasai procedure (KP) progress to death or require liver transplantation to achieve long-term survival; however, most cases of death/liver transplantation (D/LT) occur in the early period after KP (usually within 1 year). This study was designed to construct a convenient nomogram for predicting early D/LT in patients with BA after KP. METHODS: A BA cohort was established in May 2017, and up to May 2023, 112 patients with 1-5 years of follow-up were enrolled in the study and randomly (ratio, 3:1) divided into a training cohort for constructing a nomogram (n = 84) and a validation cohort (n = 28) for externally validating the discrimination and calibration. The training cohort was divided into two groups: the early D/LT group (patients who died or had undergone LT within 1 year after KP [n = 35]) and the control group (patients who survived through the native liver more than 1 year after KP [n = 49]). Multivariate logistic regression and stepwise regression were applied to detect variables with the best predictive ability for the construction of the nomogram. The discrimination and calibration of the nomogram were internally and externally validated. RESULTS: The Kaplan-Meier (K-M) curve showed an actual 1-year native liver transplantation (NLS) rate of 57.1% and an estimated 2-year NLS rate of 55.2%. By multivariate regression and stepwise regression, age at KP, jaundice clearance (JC) speed 1 month after KP, early-onset PC (initial time < 36.5 days) after KP, sex, aspartate aminotransferase-to-platelet ratio index (APRI), and weight at KP were identified as the independent variables with the best ability to predict early D/LT and were used to construct a nomogram. The developed nomogram based on these independent variables showed relatively good discrimination and calibration according to internal and external validation. CONCLUSION: Most D/LTs were early D/LTs that occurred within 1 year after KP. The established nomogram based on predictors, including sex, weight at the KP, the APRI, age at the KP, JC speed 1 month after the KP, and early PC, may be useful for predicting early D/LT and may be helpful for counseling BA patients about patient prognosis after KP. This study was retrospectively registered at ClinicalTrials.gov (NCT05909033) in June 2023.

Bioinformatics analysis identifies heparan sulfate proteoglycans acting as different progress subtypes of biliary atresia.

Background: Biliary atresia (BA) is a life-threatening disorder, which is characterized by the obliteration of biliary tracts. Heparan sulfate proteoglycans (HSPGs) are important regulators in liver diseases. Whether HSPGs participate in the development of BA is poorly understood. Methods: RNA-seq dataset GSE122340, including 171 BA and 7 normal liver tissue, was integrated for bioinformatic analysis. R function "wilcox.test" was used to compare HSPGs expression levels, and "cor.test" was used to evaluate the correlation analysis. MCPcounter was used to assess the abundance of immunocytes. Molecular subtypes of BA were clustered via NMF clustering and LASSO regression was applied to screen hub HSPGs genes in BA clusters. RT-PCR analysis was used to assess the expression of hub HSPGs in BA liver. Immunohistochemical staining and immunofluorescence assay were used to evaluated the location and expression of hub HSPGs in BA liver tissue. Results: Majority of HSPGs was up-regulated in BA and correlated with liver fibrosis and ductular reaction markers. The abundance of immunocytes was higher in BA and associated with HSPGs. Based on the expression of HSPGs, BA patients were classified into 3 subtypes (C1, C2, and C3). Pathway enrichment analysis revealed C1 subtype had severe liver injury with SDC4 identified as the hub gene, while C3 subtype presented relatively normal liver condition with GPC3 identified as the hub gene. RT-PCR analysis demonstrated the expression levels of 2 hub genes in BA liver tissue with different jaundice clearance standards. Immunohistochemical staining and immunofluorescence assay showed that SDC4 was mostly expressed in ductular reaction area, while GPC3 was mostly expressed in hepatocytes. Conclusion: Majority of HSPGs are aberrant expressed in BA. The subtype hub gene SDC4 and GPC3 might be used as a potential indicator for different types of prognosis.

Initial surgical treatment of necrotizing enterocolitis: a meta-analysis of peritoneal drainage versus laparotomy.

Necrotizing enterocolitis (NEC) in premature infants is associated with high morbidity and mortality, and the optimal intervention remains uncertain. To compare the mortality of primary peritoneal drainage versus primary peritoneal laparotomy as initial surgical intervention for NEC. All data were extracted from PubMed, Embase, and the Cochrane Library. Studies published up to December 2021. Patients with NEC. Studies centered on primary peritoneal drainage and primary peritoneal laparotomy as the initial surgical treatment. Mortality outcomes were available for both interventions. Randomized controlled trials, retrospective cohort studies, and case series in peer-reviewed journals. Language limited to English. Odds ratio (OR) with 95% confidence intervals (CIs) was used to evaluate mortality outcome. Subgroup analyses and linear regression were performed to ascertain the association between mortality pre-specified factors. Data of 1062 patients received peritoneal drainage and 2185 patients received peritoneal laparotomy from five case series, five retrospective cohort studies, and three randomized controlled trials. Peritoneal drainage caused similar mortality (OR 1.49, 95% CI 0.99-2.26) compared with peritoneal laparotomy as initial surgical management for NEC infants. The subgroup analysis of study design, sample size, birth weight, and sex showed similar findings, but inconsistent results were found for country (USA: 1.47, 95% CI 0.90-2.41; Canada: 2.53, 95% CI 0.30-21.48; Australia: 10.29, 95% CI 1.03-102.75; Turkey: 0.09, 95% CI 0.01-0.63) and gestational age (age mean difference < 3: 1.23, 95% CI 0.72-2.11; age mean difference ≥ 3: 2.29, 95% CI 1.04-5.05). No statistically significance was found for the linear regression between mortality and sample size (P = 0.842), gestational age (P = 0.287), birth weight (P = 0.257), sex (P = 0.6). Small sample size, high heterogeneity, NEC, and spontaneous intestinal perforation (SIP) had to be analyzed together, lack of selection criteria for the future selection of an intervention, and no clear, standardized procedures.   Conclusion: There was no significant difference in mortality between peritoneal drainage and laparotomy as initial surgical intervention. The results suggest that either intervention could be used in selected patients. What is Known: • Necrotizing enterocolitis (NEC) in premature infants is associated with high morbidity and mortality, and the optimal intervention remains uncertain. What is New: • No significant difference of mortality between peritoneal drainage and laparotomy as initial surgical intervention.

Multi-Center Analysis of Predictive Factors of Enteral Autonomy and Risk Factors of Complications of Pediatric Intestinal Failure in China.

OBJECTIVES: The aim of this study was to identify predictors for enteral autonomy and intestinal failure (IF)-related complications and evaluate the outcomes of a multi-center pediatric cohort in China. METHODS: The medical records of pediatric patients with IF treated at four medical centers in China from January 1, 2012 to November 31, 2020 were retrospectively reviewed. Enteral autonomy was defined as sustained growth and cessation of parenteral nutrition for >90 days. Multivariate logistic regression analysis was used to identify factors predictive of enteral autonomy and the risk factors of complications, such as IF-associated liver disease (IFALD) and catheter-related bloodstream infection (CRBSI). RESULTS: The study cohort of 92 pediatric patients with IF included 71 (77%) who underwent surgery and 21 (23%) who received non-surgical treatment. Eventually, 63 (68.5%) patients achieved enteral autonomy by the end of the follow-up period. Multivariate logistic regression analysis indicated that longer duration of parenteral nutrition (PN), sepsis, and non-breastfeeding were risk factors for enteral autonomy. When considering the detailed intraoperative data, the presence of an ileocecal valve (ICV) and greater residual small bowel (RSB) length were reaffirmed as predictors of achieving enteral autonomy. Medium/long-chain (MCT/LCT) lipids or sepsis were identified as negative predictors for IFALD. Univariate analysis revealed that the use of MCT/LCT lipids was associated with a greater likelihood of CRBSI. CONCLUSION: In this cohort, enteral autonomy was achieved at a percentage of 68.5%, and the risk factors for not achieving enteral autonomy were a longer duration of PN, sepsis, and non-breastfeeding. The presence of an ICV and a greater RSB length were important predictors of achieving enteral autonomy.

Risk factors for the occurrence and recurrence of acute cerebellar ataxia: a retrospective observational study.

OBJECTIVE: There is little evidence to support a correlation between abdominal surgery and acute cerebellar ataxia (ACA). We reviewed the records of children with ACA treated at our institution to analyze risk factors for ACA. METHODS: Clinical data of 442 children with ACA treated at Children's Hospital of Nanjing Medical University between November 2015 and June 2019 were retrospectively analyzed. Univariate and multivariate analyses were performed to determine risk factors for the occurrence and recurrence of ACA. RESULTS: In total, 442 children with ACA were included in this study. Multivariate logistic regression analysis showed age (p = 0.009), infection (p < 0.001), vaccination (p < 0.001), head trauma (p < 0.001), intussusception surgery (IS) (p < 0.001), operation for indirect inguinal hernia (p < 0.001), and operation for congenital gastrointestinal malformation (p < 0.001) were independent risk factors for ACA occurrence. Univariate analysis showed that only IS (p < 0.001) was associated with ACA recurrence. CONCLUSIONS: Surgeons should be aware that age, infection, vaccination, head trauma, and history of abdominal surgery are associated with ACA, while IS is a risk factor for ACA recurrence.

Circular RNA CCDC66 targets DCX to regulate cell proliferation and migration by sponging miR-488-3p in Hirschsprung's disease.

It has been suggested that circular RNAs play critical roles in natural growth and disease development. Nevertheless, whether the circular RNAs were related in Hirschsprung's disease (HSCR) remains unknown. Thus, we discovered the cir-CCDC66 was downregulated in HSCR compared with the normal gut tissues. The cir-CCDC66 reduction might inhibit cells' proliferation and migration in vitro. Then, we found that DCX transcript was putative cir-CCDC66 competing endogenous RNA. Furthermore, the function of cir-CCDC66 as a sponge for miR-488-3p to regulate DCX RNA expression was demonstrated by immunoprecipitation and luciferase reporter assays. In conclusion, this is the first report revealing that cir-CCDC66 modulates DCX expression through sponging miR-488-3p and thus participates in the onset of HSCR.

Erratum: LncRNA AFAP1-AS Functions as a Competing Endogenous RNA to Regulate RAP1B Expression by sponging miR-181a in the HSCR: Erratum.

[This corrects the article DOI: 10.7150/ijms.18392.].

Identification of two novel PCDHA9 mutations associated with Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a complex disorder with multiple pathogenic gene mutations. Protocadherin alpha 9 (PCDHA9) was identified as a potential candidate gene for HSCR by whole-exome sequencing in a Chinese family. Sanger sequencing in 298 HSCR cases revealed two sporadic Chinese patients with a novel missence PCDHΑ9 mutation (NM_031857; c.1280C > T[p.Ala427Val]) and one sporadic Chinese patient with another novel missence PCDHΑ9 mutation (c.1425C > G[p.Phe475Leu]).The silico predictions and 3D modeling suggest the deleterious effect of identified mutations on protein function. Immunohistochemistry analysis showed PCDHΑ9 was predominantly expressed in the myenteric plexus of human colon tissues. For mouse embryos, PCDHΑ9 was expressed in the stomach but rarely seen in the intestine during E10.5-12.5, then obviously expressed in the intestinal mucosa at E13.5 and extensively expressed in intestinal muscularis and mucosa at E14.5. Moreover, the down-regulation of PCDHΑ9 in the SH-SY5Y cell line promoted the proliferation and migration rate but inhibited the apoptotic rate. In summary, PCDHΑ9 is potentially related to HSCR and the clustered protocadherins (Pcdhs) may involve in the enteric nervous system (ENS) ontogeny.

Long non-coding RNA LOC100507600 functions as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p in Hirschsprung's disease.

Recently studies reported that long non-coding RNAs (lncRNAs) may take part in a lot of congenital diseases, meanwhile, Hirschsprung's disease (HSCR) is a major congenital digestive tract malformation. Nevertheless whether lncRNAs participate in the occurrence of HSCR and how it contributes to this disease are still unknown. LOC100507600 was selected from our gene expression microarray data obtained from bowel tissues from HSCR patients and negative controls. Subsequently, we used qRT-PCR to prove the result in 64 pairs of HSCR disease bowel stenosis tissues and negative controls. Transwell assay, CCK-8 assay and flow cytometry were employed to explore whether cellular functions change after knocking down the LOC100507600 in SH-SY5Y cell and human 293T cell. Dual-luciferase reporter assay was used to confirm the competitive relationship between BMI1 and LOC100507600 through their association with hsa-miR128-1-3p. Protein extraction and Western blotting were used to further confirm the relationship between LOC100507600 and BMI1. We found that LOC100507600 was obvious reduced in tissues from HSCR patients with noteworthy correlation with BMI1. Furthermore, Downregulation of LOC100507600 repressed cell migration and proliferation and didn't affect cell apoptosis or cycle. Dual-luciferase reporter assay, qRT-PCR and Western blotting assay verified that LOC100507600 serves as a competitive endogenous RNA of miR128-1-3p and down-regulates BMI1 expression by sponging miR128-1-3p in HSCR. In sum, our study researches the potential diagnostic value of LOC100507600 in HSCR and deduces that LOC100507600 can contributes to HSCR as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p.

LncRNA AFAP1-AS Functions as a Competing Endogenous RNA to Regulate RAP1B Expression by sponging miR-181a in the HSCR.

Background: Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in a broad spectrum of cellular processes including development and disease. Despite the known engagement of the AFAP1-AS in several human diseases, its biological function in Hirschsprung disease (HSCR) remains elusive. Methods: We used qRT-PCR to detect the relative expression of AFAP1-AS in 64 HSCR bowel tissues and matched normal intestinal tissues. The effects of AFAP1-AS on cell proliferation, migration, cell cycle, apoptosis and cytoskeletal organization were evaluated using CCK-8, transwell assay, flow cytometer analysis and immunofluorescence, in 293T and SH-SY5Y cell lines, respectively. Moreover, the competing endogenous RNA (ceRNA) activity of AFAP1-AS on miR-181a was investigated via luciferase reporter assay and immunoblot analysis. Results: Aberrant inhibition of AFAP1-AS was observed in HSCR tissues. Knockdown of AFAP1-AS in 293T and SH-SY5Y cells suppressed cell proliferation, migration, and induced the loss of cell stress filament integrity, possibly due to AFAP1-AS sequestering miR-181a in HSCR cells. Furthermore, AFAP1-AS could down-regulate RAP1B via its competing endogenous RNA (ceRNA) activity on miR-181a. Conclusions: These findings suggest that aberrant expression of lncRNA AFAP1-AS, a ceRNA of miR-181a, may involve in the onset and progression of HSCR by augmenting the miR-181a target gene, RAP1B.

Mutations of MYH14 are associated to anorectal malformations with recto-perineal fistulas in a small subset of Chinese population.

BACKGROUND: Anorectal malformations (ARMs) are among the most commonly congenital abnormalities of distal hindgut development, ranging from anal stenosis to anal atresia with or without fistulas and persistent cloaca. The etiology remains elusive for most ARM cases and the majority of genetic studies on ARMs were based on a candidate gene approach. MATERIALS AND METHODS: In all eight family members of a non-consanguineous Chinese family, we performed whole-exome sequencing. Subsequently, exome sequencing of MYH14 in 72 unrelated probands with ARMs was performed. The accurate distribution of non-muscle myosin II heavy chain (NMHC II) was investigated by immunohistochemistry in serial sagittal sections of E11.5-13.5 mouse cloacal regions. RESULTS: A homozygous mutation in MYH14 was identified in the two siblings of family 1. Compound heterozygous MYH14 changes were identified in an unrelated individual. Immunohistochemical analysis suggest stronger NMHC IIC localization in the epithelium of the murine embryonic cloaca, urorectal septum and hindgut compared with another two NMHC II isoforms. CONCLUSION: This is the first identification of mutations in MYH14 as a cause of ARMs. The stronger localization of NMHC IIC in E11.5-13.5 mouse cloacal regions further supports the role of MYH14 in anorectal development.

Circular RNA ZNF609 functions as a competitive endogenous RNA to regulate AKT3 expression by sponging miR-150-5p in Hirschsprung's disease.

Research over the past decade suggested critical roles for circular RNAs in the natural growth and disease progression. However, it remains poorly defined whether the circular RNAs participate in Hirschsprung disease (HSCR). Here, we reported that the cir-ZNF609 was down-regulated in HSCR compared with normal bowel tissues. Furthermore, suppression of cir-ZNF609 inhibited the proliferation and migration of cells. We screened out several putative cir-ZNF609 ceRNAs of which the AKT3 transcript was selected. Finally, RNA immunoprecipitation and luciferase reporter assays demonstrated that cir-ZNF609 may act as a sponge for miR-150-5p to modulate the expression of AKT3. In conclusion, these findings illustrated that cir-ZNF609 took part in the onset of HSCR through the crosstalk with AKT3 by competing for shared miR-150-5p.

Downregulated Expression of Long Non-Coding RNA LOC101926975 Impairs both Cell Proliferation and Cell Cycle and Its Clinical Implication in Hirschsprung Disease Patients.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been reported to participate in various diseases. Hirschsprung disease (HSCR) is a common digestive disease in the new born. However, the relationship between lncRNAs and HSCR remains unclarified. METHODS: We used qRT-PCR to detect the relative expression of LOC101926975 in 80 pairs of HSCR bowel tissues and matched normal bowel tissues. CCK-8 assay, transwell assay and flow cytometry were then used to evaluate the function in vitro by knocking down the LOC101926975 in SK-N-BE(2) cells. Receiver operating characteristic (ROC) curve was used to evaluate the potential diagnostic value of LOC101926975. RESULTS: LOC101926975 was significantly downregulated in HSCR tissues with excellent correlation with FGF1. Dysregulation of LOC101926975 suppressed cell proliferation and induced G0/G1 arrest without impact on cell apoptosis or migration. Meanwhile, the AUC of LOC101926975 was 0.900 which presented great diagnostic value. CONCLUSIONS: Our study firstly investigates the potential function of LOC101926975 in HSCR and infers that LOC101926975 can distinguish HSCR from the normal ones.