Direct cellular reprogramming and transdifferentiation of fibroblasts on wound healing-Fantasy or reality?

Induced pluripotent stem cell (iPSC) technology is one of the de novo approaches in regeneration medicine and has led to new research applications for wound healing in recent years. Fibroblasts have attracted wide attention as the first cell line used for differentiation into iPSCs. Researchers have found that fibroblasts can be induced into different types of cells in variable mediums or microenvironments. This indicates the potential "stem" characteristics of fibroblasts in terms of direct cellular reprogramming compared with the iPSC detour. In this review, we described the morphology and biological function of fibroblasts. The stem cell characteristics and activities of fibroblasts, including transdifferentiation into myofibroblasts, osteogenic cells, chondrogenic cells, neurons, and vascular tissue, are discussed. The biological values of fibroblasts are then briefly reviewed. Finally, we discussed the potential applications of fibroblasts in clinical practice.

Silver nanoparticles promote osteogenic differentiation of mouse embryonic fibroblasts in vitro.

OBJECTIVE: This study investigated if silver nanoparticles (AgNps) could promote the proliferation and osteogenic differentiation of mouse embryonic fibroblasts. METHODS: Mouse embryonic fibroblasts were divided into two groups: Group 1 cells were cultured in DMEM/F12 medium and Group 2 cells were cultured in osteogenic medium. Both groups were then treated with 16, 32, or 100 µM AgNps. Fibroblast proliferation and viability were measured using BrdU and MTT methods at varying time points. Alizarin red staining and alkaline phosphatase (ALP) activity were measured to observe fibroblast differentiation into osteoblasts. Proteomics (cytokine array) was used to detect 111 different cytokines during differentiation. RESULTS: AgNps stimulated proliferation of mouse embryonic fibroblasts at a concentration of 16 µM. Marked enhancement of calcium mineralization was observed in cells cultured with AgNps compared with cells cultured without AgNps. Group 2 cells displayed nodules around the center where the cell density was high. ALP activity of mouse embryonic fibroblasts cultured in osteogenic medium increased during the whole culture period. Addition of AgNps at concentrations of 32 µM and 100 µM induced higher ALP activity at days 7 and 14. Proteomic array results show that low density lipoprotein receptor (LDL-R) and proprotein convertase subtilisin/kexin type 9 (PCSK-9) were significantly increased, while osteoprotegerin (OPG) was significantly reduced in medium containing 16 µM AgNPs. CONCLUSION: AgNps could promote differentiation of mouse embryonic fibroblasts into osteoblastic cells. LDL-R and PCSK-9, as well as OPG, may play a critical role in this process.

A Comparative Review of the Outcome Following MVD and PBC in Patients with Trigeminal Neuralgia.

BACKGROUND: This study aims to systematically review the treatment outcomes of percutaneous balloon compression (PBC) and microvascular decompression (MVD) in patients with trigeminal neuralgia. METHODS: A systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline was performed using PubMed, Embase, and Cochrane Central Registry of Controlled Trials databases. Only those articles with more than 5 years' follow-up length were included in this investigation. To uniformly assess the postoperative outcome, we defined pain relief as totally pain free, while the postoperative hospitalization and last follow-up period were defined as early and long term, respectively. The facial numbness was quantified with Barrow Neurological Institute Pain Intensity Score (BNI). RESULTS: After database searching and screening, 7,797 cases were finally included according to the criteria. The early pain relief rates were 94.1% (1,551/1,649) and 89.9% (4,962/5,482) following PBC and MVD (odds ratio [OR] = 0.603; p < 0.05), while the long-term rates were 58.1% (921/1,566) and 74.9% (4,549/6,074; OR = 2.089; p < 0.05), respectively. Although a significant higher facial numbness occurred in the PBC group in the early stage, it was mostly diminished 5 years later compared with the MVD group. At long-term follow-up, hypoacusis and facial palsy occurred more often in the MVD group (p < 0.05). CONCLUSIONS: Both MVD and PBC provide a satisfactory outcome for the patients in the long term. As a simple, safe, and reliable technique, PBC should be considered as a viable alternative.

Temporal and spatial development of intestinal smooth muscle layers of human embryos and fetuses.

The sequential occurrence of three layers of smooth muscle layers (SML) in human embryos and fetus is not known. Here, we investigated the process of gut SML development in human embryos and fetuses and compared the morphology of SML in fetuses and neonates. The H&E, Masson trichrome staining, and Immunohistochemistry were conducted on 6-12 gestation week human embryos and fetuses and on normal neonatal intestine. We showed that no lumen was seen in 6-7th gestation week embryonic gut, neither gut wall nor SML was developed in this period. In 8-9th gestation week embryonic and fetal gut, primitive inner circular SML (IC-SML) was identified in a narrow and discontinuous gut lumen with some vacuoles. In 10th gestation week fetal gut, the outer longitudinal SML (OL-SML) in gut wall was clearly identifiable, both the inner and outer SML expressed α-SMA. In 11-12th gestation week fetal gut, in addition to the IC-SML and OL-SML, the muscularis mucosae started to develop as revealed by α-SMA immune-reactivity beneath the developing mucosal epithelial layer. Comparing with the gut of fetuses of 11-12th week of gestation, the muscularis mucosae, IC-SML, and OL-SML of neonatal intestine displayed different morphology, including branching into glands of lamina propria in mucosa and increased thickness. In conclusions, in the human developing gut between week-8 to week-12 of gestation, the IC-SML develops and forms at week-8, followed by the formation of OL-SML at week-10, and the muscularis mucosae develops and forms last at week-12.

The Allantois and Urachus: Histological Study Using Human Embryo and Fetuses.

Relatively little is known about allantois and urachal development in early humans.Serial sagittal histological sections from eight human embryos and fetuses were examined to determine allantois development.At gestational age 6-7 weeks, the primitive allantois consists of an enlarged tube located between the umbilical cord and abdominal cavity, whereas the urachus is not yet developed. At 8 weeks, the allantois gradually withdraws from the distal to the proximal end of the umbilical cord, and both the proximal allantois and the rectum (hindgut) start to develop into the cloaca. At 10 weeks, the allantois was located mostly in the abdominal cavity.The urachus forms from the distal end of the allantois and develops into a closed fibrous cord between the base of the urinary bladder and the umbilicus. The urogenital sinus forms from the proximal end of the allantois.

Gut lumen formation defect can cause intestinal atresia: evidence from histological studies of human embryos and intestinal atresia septum.

Intestinal atresia (IA), a common cause of neonatal intestinal obstruction, is a developmental defect, which disrupts the luminal continuity of the intestine. Here, we investigated (i) the process of lumen formation in human embryos; and (ii) how a defective lumen formation led to IA. We performed histological and histochemical study on 6-10 gestation week human embryos and on IA septal regions. To investigate the topology of embryonic intestine development, we conducted 3D reconstruction. We showed that a 6-7th gestation week embryonic gut has no lumen, but filled with mesenchyme cells and vacuoles of a monolayer of epithelial cells. A narrow gut lumen was formed by gestation week-9, the gut was filled with numerous vacuoles of different sizes, some vacuoles were merging with the developing embryonic gut wall. At gestation week-10, a prominent lumen was developed, only few vacuoles were present and were merging with the intestine wall. At IA septal regions, vacuoles were located in the submucous layer, covered by a single layer of epithelium without glandular structure, and surrounded with fibrous tissue. The mucosal epithelium was developed with lamina propria and basement membrane, but the submucosa and the longitudinal smooth muscle layers were not properly developed. Hence, the vacuoles in IA septum could represent a remnant of vacuoles of embryonic gut. In conclusion, the fusion of vacuoles with the developing intestine wall associates with the disappearance of vacuoles and gut lumen formation in human embryos, and perturbation of these developmental events could lead to IA.

Delayed development of vacuoles and recanalization in the duodenum: a study in human fetuses to understand susceptibility to duodenal atresia/stenosis.

ObjectiveWe compared the cross-sectional areas of the duodenum to the distal small intestine during early gestation to determine if there is a difference in age for recanalization.MethodsSerial sagittal sections of six fetuses of gestational age (GA) 8-10 weeks were examined morphologically to compare the degree of recanalization of the duodenum with to the more distal small intestine.ResultsAt GA 8-9 weeks, the duodenum had more epithelial plugs and vacuoles with no or narrower spaces compared to the distal small bowel. Quantitative assessment at GA 10 weeks showed that the cross-sectional area of the duodenal cavity was significantly less than the distal small bowel.ConclusionThe development and recanalization of vacuoles in the duodenum occurs later than the jejunum and ileum may be involved in the more frequent development of atresia/stenosis of the duodenum compared to more distal gastrointestinal tract.

Depletion of Kruppel-like factor 15 sensitized gliomas to temozolomide cytotoxicity through O 6-methylguanine-DNA methyl-transferase.

Temozolomide (TMZ)-based chemotherapy is a standard strategy for gliomas, although chemoresistance remains a major therapeutic challenge. The chemical mechanism by which TMZ induces cell death is DNA methylation, leading to double-stranded breaks (DSBs) and thus to apoptosis. However, TMZ-induced N6-meG sites are efficiently repaired and mediated by the DNA repair protein O 6-methylguanine-DNA methyl-transferase (MGMT), leading to TMZ resistance. KLF15, a member of the Kruppel-like factors family, mainly functions as transcription factor and potential suppressor gene by inhibiting proliferation, migration, and inducing apoptosis. However, the roles and regulatory mechanisms of KLF15 in glioma tumorigenesis and chemoresistance are poorly understood. In this study, KLF15 expression was upregulated in glioma tissues and cell lines upon TMZ treatment. Knockdown of KLF15 amplified TMZ-induced repression of cell proliferation, while KLF15 overexpression reversed this process. Mechanistically, KLF15 functioned as a transcriptional activator of MGMT. Moreover, KLF15 knockdown sensitized tumors to TMZ treatment in vivo. Taken together, these results suggested that KLF15 up-regulated MGMT through direct binding to the promoter of MGMT, which plays an important role in glioma resistance to TMZ, and which may be a potential target for cancer diagnosis and treatment.

WITHDRAWN: Immunohistochemical study of inflammatory responses in septa arising from type I intestinal atresia.

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[An epidemiological survey of laryngopharyngeal refux disease in otorhinolaryngology head and neck surgery clinics in Chongqing area].

Objective:To investigate the prevalence and affecting factors of laryngopharyngeal reflux disease（LPRD） in otolaryngology head and neck surgery in Chongqing,and to provide a basis for the clinical diagnosis and therapy of LPRD. Methods:Multi-center cross-sectional survey method and systematic sampling method were used to select patients at fifteen hospitals in Chongqing from August to November in 2019. Then reflux symptom index（RSI） was investigated. At the same time, the information of the relevant dietary habits, including smoking and drinking, spicy diet, high-fat diet, and satiety was collected. Moreover, the factors related to LRPD（gender, age, symptoms, diet and lifestyle） were analyzed. Results:The composition ratio of LPRD was 11.90%（385/3234） in 3234 effective questionnaires and 385 positive ones. The composition ratio was 12.55%（173/1378） in men and 11.42%（212/1856） in women. The difference between the two groups was statistically significant（P<0.05）. The difference in composition ratio among different age groups was statistically significant（P<0.05）, with the highest composition ratio between 40 and 59 years old（170/1390）. Constant throat-clearing（symptom 2） and globus sensation（symptom 8） were most correlated with LPRD. Logistic regression analysis showed that spicy diet, over eating, and smoking were highly related to LPRD. Conclusion:Globus sensation and constant throat-clearing are the most common symptoms in LPRD patients. Spicy diet, over eating, and smoking are risk factors for LPRD.

Laparoscopic-assisted anorectoplasty for intermediate type rectovestibular fistula: a preliminary report.

PURPOSE: Laparoscopic-assisted anorectoplasty (LAARP) is considered to benefit the male patients with anorectal malformation (ARM). This study evaluates LAARP management for intermediate type rectovestibular fistula (IRVF) in the female patient with ARM. METHODS: Twelve patients with IRVF (aged 3-5 months) underwent LAARP from 2017 to 2019 in our institute. LAARP was performed for mobilization of the rectum, visualization and enlargement of the center of the sphincter muscle complex (SMC) from pelvic and perineal aspects, intra-fistula mucosectomy and rectal pull-through in the SMC with the fourchette and the perineal body unattached. RESULTS: LARRP was performed in all patients without conversion to open procedure. No patient suffered from wound infection, vaginal injury, recurrent fistula and anal stenosis. The parents were satisfied with the appearance of the wound. Rectal prolapse developed in one patient and needed surgical correction. The patients were followed up for a mean of 19.7 months (ranged from 12 to 35 months). CONCLUSION: Our preliminary experience shows that LAARP offers an alternative method of correction for the IRVF with good visualization of the SMC and may diminish the risks of wound dehiscence and vaginal injury.

Nuclear DLC1 exerts oncogenic function through association with FOXK1 for cooperative activation of MMP9 expression in melanoma.

A Rho GTPase-activating protein (RhoGAP), deleted in liver cancer 1 (DLC1), is known to function as a tumor suppressor in various cancer types; however, whether DLC1 is a tumor-suppressor gene or an oncogene in melanoma remains to be clarified. Here we revealed that high DLC1 expression was detected in most of the melanoma tissues where it was localized in both the nuclei and the cytoplasm. Functional studies unveiled that DLC1 was both required and sufficient for melanoma growth and metastasis. These tumorigenic events were mediated by nuclear-localized DLC1 in a RhoGAP-independent manner. Mechanistically, mass spectrometry analysis identified a DLC1-associated protein, FOXK1 transcription factor, which mediated oncogenic events in melanoma by translocating and retaining DLC1 into the nucleus. RNA-sequencing profiling studies further revealed MMP9 as a direct target of FOXK1 through DLC1-regulated promoter occupancy for cooperative activation of MMP9 expression to promote melanoma invasion and metastasis. Concerted action of DLC1-FOXK1 in MMP9 gene regulation was further supported by their highly correlated expression in melanoma patients' samples and cell lines. Together, our results not only unravel a mechanism by which nuclear DLC1 functions as an oncogene in melanoma but also suggest an unexpected role of RhoGAP protein in transcriptional regulation.

Circ_0058124 Upregulates MAPK1 Expression to Promote Proliferation, Metastasis and Metabolic Abilities in Thyroid Cancer Through Sponging miR-940.

BACKGROUND: Thyroid cancer (TC) is an endocrine disease, and its progression is regulated by many factors, including circular RNAs (circRNAs). However, as a new circRNA, the role of circ_0058124 in TC is worth further exploration. METHODS: The expression levels of circ_0058124, microRNA-940 (miR-940) and mitogen-activated protein kinase 1 (MAPK1) were assessed by quantitative polymerase chain reaction (q-PCR). The circular characteristic of circ_0058124 was identified by oligo (dT)18 primers, Ribonuclease R (RNase R) and Actinomycin D (ActD), and its localization was determined by nuclear-cytoplasmic separation assay. Also, cell proliferation was detected by colony formation assay, and cell migration and invasion were assessed by transwell assay. Further, Seahorse XF Extracellular Flux Analyzer was used to measure the oxygen consumption rate (OCR) of cells. Besides, dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were used to identify the mechanism of circ_0058124. Western blot (WB) analysis was used to test the MAPK1 protein level. In addition, mice xenograft models were constructed to test the effect of circ_0058124 on TC tumor growth in vivo. RESULTS: Circ_0058124 was highly expressed in TC and is a stable cyclic transcript, mainly located in the cytoplasm. Circ_0058124 knockdown suppressed proliferation, migration, invasion and metabolic abilities in TC cells. MiR-940 could be absorbed by circ_0058124, and the inhibition effect of its overexpression on TC progression could be reversed by overexpressed-circ_0058124. MAPK1 was a target of miR-940, and the suppression effect of its silencing on TC progression could be inverted by miR-940 inhibitor. Besides, MAPK1 expression was regulated by circ_0058124 and miR-940. Interference of circ_0058124 also reduced TC tumor growth in vivo. CONCLUSION: Circ_0058124 might play a carcinogenic role in TC progression by regulating the miR-940/MAPK1 axis, which might provide a new idea for the treatment of TC.

Exogenous Secretin Improves Parenteral Nutrition-associated Liver Disease in Rats.

OBJECTIVES: Intestinal failure-associated liver disease (IFALD) is a feared and life-threatening complication in neonates with intestinal failure (IF) receiving long-term total parenteral nutrition (TPN). This study aims to investigate the effect of exogenous secretin on liver pathology and hepatic function in a rat model of PN-associated liver disease (PNALD). METHODS: Male Sprague-Dawley rats underwent right jugular venous catheterization to receive 14-day continuous TPN therapy. All rats were allocated into 3 groups: the Control group (n = 8) did not have surgery or TPN and was fed standard rat chow ad libitum; the TPN group (n = 8) underwent catheter insertion and TPN treatment; and the TPN/S group (n = 8) also underwent catheter insertion, TPN treatment, and exogenous secretin treatment (2.5 nmol ·â€Škg ·â€Šday) daily. Fourteen days after initial surgery, we collected the animals' liver and blood samples for further test. RESULTS: The TPN/S group had diminished direct bilirubin (TPN, 2.1 ±â€Š0.7 µmol/L; TPN/S, 1.5 ±â€Š0.2 µmol/L) and liver total bile acid levels (TPN, 144.5 ±â€Š21.2 µmol/L; TPN/S, 123.4 ±â€Š10.4 µmol/L) and improved histological outcomes compared with those in the TPN group. Exogenous secretin also enhanced the canalicular transporter (BSEP, 0.5-fold, P = 0.011) and inhibited the basolateral transporter (OSTA, -0.48-fold, P = 0.002; OSTB, -0.6-fold, P = 0.013) of liver bile acid. CONCLUSIONS: In this animal model of PNALD, secretin may improve cholestasis by enhancing canalicular transport, inhibiting the basolateral export of liver bile acid, and eventually decreasing the total bile acid level in the liver. Exogenous secretin treatment may potentially prevent and treat IFALD in IF patients relying on long-term TPN therapy.

Laparoscopic surgical technique to enhance the management of anorectal malformations: 330 cases' experience in a single center.

PURPOSE: Laparoscopic-assisted anorectoplasty (LAARP) is considered to benefit the patients with vesico-prostatic fistula. The aim of this study is to present the details of our LAARP technique for improving the short- and long-term outcomes in the patients with high and intermediate types of anorectal malformations (ARMs). METHODS: 330 patients with high-type (174 cases) and intermediate-type (156 cases) anorectal malformation (aged 8 days to 15 years) underwent LAARP from 2001 to 2019. LAARP was performed for full mobilization and resection of the dilated rectum, intra-rectal closure of the fistula, visualization, and enlargement of the center of the longitudinal muscle tube (LMT) from pelvic and perineal aspects. RESULTS: LAARP was performed in all patients and no patient was converted to open procedure. The urethral diverticulum was found in three patients (1.02%, 3/294) according to postoperative protocol voiding cystourethrogram but was not associated with any symptoms such as urinary tract infection and dysuria. Rectal prolapse requiring surgical intervention developed in 25 (7.6%) of 330 patients. Anal stricture occurred in three patients and re-do anoplasty was performed 5 months after LAARP. Anal retraction occurred in two patients and re-pull-through was conducted at 5 and 6 days, respectively, after LAARP. 228 patients who were older than 3 years were followed up. The median follow-up period was 5.8 years (range 3-15 years). 217 patients (95.2%) had voluntary bowel movements; 202 patients (88.6%) were free from soiling or with grade 1 soiling; 30 patients (13.6%) and 25 patients (11.3%) suffered from grade 1 and grade 2 constipation, respectively, while no patient had grade 3 constipation. CONCLUSION: Our experience demonstrates that the LAARP has advantages on rectal mobilization and resection, intra-rectal fistula closure and accurate tunnel formation in the LMT with minimal trauma. The improvement of the short-term and long-term outcomes after LAARP has been shown not only for high-type ARM but also for intermediate-type ARM.

A narrative review of the challenges and countermeasures in hepatoblastoma management during COVID-19 epidemic.

An infectious disease named "coronavirus disease 2019" (COVID-19) currently has brought a threat to global health security and trends to be more and more severe in many countries. It also has introduced great challenges to the diagnosis and management of children with hepatoblastoma (HB). During the COVID-19 pandemic, pediatric surgeons should not only develop personalized treatment plans for HB therapy, but also emphasize the diagnosis, prevention, and treatment of this virus. Children with both HB and COVID-19 are recommended to undertake multidisciplinary assessment. Anti-SARS-CoV-2 therapy may be a preferred treatment for the infected without presenting a surgical emergency. However, emergent operation may be necessary for HB children with concurrent COVID-19 who developed a life-threatening surgical emergency condition. Otherwise, for children with negative virus examination results, treatment advice should be based on the impact of the epidemic and regional economic considerations. A "wait and see" strategy is recommended for children with resectable tumors after new adjuvant chemotherapy treatment (NACT). Assessment of liver transplantation is recommended for children with HB whose tumors cannot be resected after NACT. Children with HB with pulmonary metastasis may have abnormal findings on chest imaging due to COVID-19. Besides, the detailed therapeutic regimens may vary for children with HB with or without an emergency presentation. Based on previous consensus, current research, and the experiences of our hospital, we aim to offer available management plans for the above-mentioned concerns.

SOX9 is a dose-dependent metastatic fate determinant in melanoma.

BACKGROUND: In this research, we aimed to resolve contradictory results whether SOX9 plays a positive or negative role in melanoma progression and determine whether SOX9 and its closely related member SOX10 share the same or distinct targets in mediating their functions in melanoma. METHODS: Immunofluorescence, TCGA database and qPCR were used to analyze the correlation between the expression patterns and levels of SOX9, SOX10 and NEDD9 in melanoma patient samples. AlamarBlue, transwell invasion and colony formation assays in melanoma cell lines were conducted to investigate the epistatic relationship between SOX10 and NEDD9, as well as the effects of graded SOX9 expression levels. Lung metastasis was determined by tail vein injection assay. Live cell imaging was conducted to monitor dynamics of melanoma migratory behavior. RHOA and RAC1 activation assays measured the activity of Rho GTPases. RESULTS: High SOX9 expression was predominantly detected in patients with distant melanoma metastases whereas SOX10 was present in the different stages of melanoma. Both SOX9 and SOX10 exhibited distinct but overlapping expression patterns with metastatic marker NEDD9. Accordingly, SOX10 was required for NEDD9 expression, which partly mediated its oncogenic functions in melanoma cells. Compensatory upregulation of SOX9 expression in SOX10-inhibited melanoma cells reduced growth and migratory capacity, partly due to elevated expression of cyclin-dependent kinase inhibitor p21 and lack of NEDD9 induction. Conversely, opposite phenomenon was observed when SOX9 expression was further elevated to a range of high SOX9 expression levels in metastatic melanoma specimens, and that high levels of SOX9 can restore melanoma progression in the absence of SOX10 both in vitro and in vivo. In addition, overexpression of SOX9 can also promote invasiveness of the parental melanoma cells by modulating the expression of various matrix metalloproteinases. SOX10 or high SOX9 expression regulates melanoma mesenchymal migration through the NEDD9-mediated focal adhesion dynamics and Rho GTPase signaling. CONCLUSIONS: These results unravel NEDD9 as a common target for SOX10 or high SOX9 to partly mediate their oncogenic events, and most importantly, reconcile previous discrepancies that suboptimal level of SOX9 expression is anti-metastatic whereas high level of SOX9 is metastatic in a heterogeneous population of melanoma.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

Single-port laparoscopic herniorrhaphy using a two-hooked cannula device with hydrodissection.

BACKGROUND: Laparoscopic percutaneous extraperitoneal closure (LPEC) with variable devices seems to be one of the most simple and reliable methods. We described our modifications of single-port laparoscopic herniorrhaphy using an inner two-hooked cannula device with preperitoneal hydrodissection. PATIENTS AND METHODS: 1568 children with 2114 inguinal hernias were treated by single-port LPEC. Under laparoscopic visualization, the two-hooked cannula device carrying a silk suture was inserted at the point of the internal ring and could be readily kept in an identical path. The hernia orifice was completely lassoed extraperitoneally by the suture with the assistance of hydrodissection. Any huge hernias of more than 1.5 cm were repaired with the incorporation of medial umbilical fold flap as reinforcement. RESULTS: All hernia repairs were successfully performed by LPEC. 1022 patients had unilateral inguinal hernia repair, and 546 patients underwent bilateral inguinal hernia repair. Of these, additional medial umbilical flap reinforcement was necessary in 68 cases, and an assisted grasping instrument was used in 19 cases owing to omental adhesion or sliding hernia. Mean operating times for unilateral and bilateral inguinal hernia repairs were 9.8 ±â€¯2.1 min and 13.6 ±â€¯2.2 min, respectively. There were no operative complications. Two recurrences and three hydroceles were observed during 6 to 30 months of follow-up. CONCLUSIONS: One-puncture LPEC using the two-hooked cannula device with preperitoneal hydrodissection has proved to be a safe and effective procedure with excellent cosmetic results. LEVEL OF EVIDENCE: IV.