46,XY disorders of sex development: the use of NGS for prevalent variants.

46,XY disorders of sex development (DSD) present with diverse phenotypes and complicated genetic causes. Precise genetic diagnosis contributes to accurate management, and targeted next-generation sequencing (NGS) and whole-exome sequencing are powerful tools for investigating DSD. However, the prevalent variants resulting in 46,XY DSD remain unclear, especially those associated with mild forms, such as isolated hypospadias, inguinal cryptorchidism, and micropenis. From 2019 to 2021, 74 patients with 46,XY DSD (48 typical and 26 mild) from the First Affiliated Hospital of Sun Yat-sen University were enrolled in our cohort study for targeted NGS or whole-exome sequencing. Our targeted 46,XY DSD panel included 108 genes involved in disorders of gonadal development and differentiation, steroid hormone synthesis and activation, persistent Müllerian duct syndrome, idiopathic hypogonadotropic hypogonadism, syndromic disorder, and others. Variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, or benign following the American College of Medical Genetics guidelines. As a result, 28 of 74 (37.8%) patients with pathogenic and/or likely pathogenic variants acquired genetic diagnoses. The Mild DSD patients acquired a diagnosis rate of 30.7%. We detected 44 variants in 28 DSD genes from 31 patients, including 33 novel and 11 reported variants. Heterozygous (65%) and missense (70.5%) variants were the most common. Variants associated with steroid hormone synthesis and activation were the main genetic causes of 46,XY DSD. In conclusion, 46,XY DSD manifests as a series of complicated polygenetic diseases. NGS reveals prevalent variants and improves the genetic diagnoses of 46,XY DSD, regardless of severity.

GDNF family receptor alpha 2 promotes neuroblastoma cell proliferation by interacting with PTEN.

Neuroblastoma is a childhood tumor, and high-stage neuroblastoma has a poor prognosis. The regulatory mechanisms for neuroblastoma progression are poorly understood. In present study, we found that GDNF family receptor alpha 2 (GFRA2) was upregulated in neuroblastoma cells and tissues, and its overexpression promoted neuroblastoma cell proliferation, as revealed using colony formation, soft agar growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays Tumor suppressor phosphatase and tensin homolog (PTEN) is an inhibitor of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) pathway that interacts with GFRA2. A luciferase activity assay showed GFRA2 inhibits the transcriptional activity of the forkhead box O (FOXO) family proteins, which suggested that GFRA2 activated the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway in GFRA2 overexpressing cells decreased cell proliferation, confirming that GFRA2 promoted neuroblastoma cell proliferation by activating the PI3K/AKT pathway. In summary, cell proliferation via the GFRA2-PTEN-PI3K/AKT axis may represent new target to develop treatments for neuroblastoma.

A peptide tetramer Tk-tPN induces tolerance of cardiac allografting by conversion of type 1 to type 2 immune responses via the Toll-like receptor 2 signal-promoted activation of the MCP1 gene.

The plant protein trichosanthin (Tk) and its derived peptide tetramer Tk-tPN have been shown to stimulate the type 2 immune responses for treating autoimmune disease. This work explores the possibility of using Tk-tPN as a non-toxic immunosuppressant to induce transplantation tolerance using the mechanisms by which T-cell-mediated immune responses are transferred from type 1 to type 2 through innate immunity-related pathways. Immunocytes and cytokine secretions involved in the mouse cardiac allografting model with Tk-tPN treatment were characterized. Identification of critical genes and analysis of their functions through Toll-like receptor (TLR) -initiated signalling and the possible epigenetic changes were performed. Mean survival times of the cardiac allografts were delayed from 7.7 ± 0.3 days (control) to 22.7 ± 3.9 days (P < 0.01) or 79.1 ± 19.2 days (P < 0.0001) when Tk-tPN was introduced into the recipients alone or together with rapamycin, respectively. The grafting tolerance was donor-specific. The secretion pattern of the type 1 cytokine/transcription factor (IL-2(+) IFN-γ(+) T-bet(+)), which is responsible for the acute graft rejection, was shifted to the type 2 factor (IL-4(+) IL-10(+) Gata3+), together with a selective expansion of the IL-4/IL-10-producing CD8+ CD28- regulatory T-cell subset. A TLR2-initiated high expression of chemokine gene MCP1 was detectable simultaneously. Epigenetically Tk/Tk-tPN could also acetylate the histone H3K9 of MCP1 promoter to skew the immunity towards T helper type 2 responses. Tk/Tk-tPN is therefore capable of down-regulating the type 1 response-dominant rejection of cardiac allografts by evoking type 2 immunity through the activation of a TLR2-initiated signalling pathway and MCP1 gene to expand the IL-4/IL-10-secreting CD8+ CD28- regulatory T cells. Tk-tPN could be a promising novel immunosuppressant to induce tolerance in allotransplantation.

A Trichosanthin-derived peptide suppresses type 1 immune responses by TLR2-dependent activation of CD8(+)CD28(-) Tregs.

A group of 15-aa-long Trichosanthin-derived peptides was synthesized and screened based on their differential abilities to induce low-responsiveness in mouse strains with high and low susceptibility. One of them was conjugated to form a homo-tetramer Tk-tPN. At concentrations of 0.1-50 µg/ml, Tk-tPN activated CD8(+)CD28(-) Tregs in vitro to induce immune suppression as effectively as the native Trichosanthin but did not exhibit cytotoxicity. In EAE mice which were pre-treated with Tk-tPN or Tk-tPN-activated CD8(+) T cells, a marked attenuation of clinical scores was recorded together with an expansion of the CD8(+)CD28(-) Treg from 2.2% to 36.1% in vivo. A pull-down assay and signal transduction analyses indicated that the ability of Tk-tPN to convert the CD8(+)CD28(-) Treg-related cytokine secretion pattern from type 1 to type 2 depends on the TLR2-initiated signaling in macrophages. The high production of IL-4/IL-10 by the Tk-tPN-activated CD8(+)CD28(-) Treg suggests the value of using Tk-tPN as a therapeutic reagent for Th1-dominant immunological diseases.

[Foley catheter versus urethral stent plus gastric tube for urine drainage following urethroplasty].

OBJECTIVE: To compare the advantages and disadvantages of the Foley catheter draining method versus the urethral stent plus gastric tube draining method for urine drainage following urethroplasty for hypospadias. METHODS: We retrospectively analyzed the clinical data of 361 cases of hypospadias treated by urethroplasty. After operation, 91 of the cases received urine drainage with the Foley catheter (group A) and 270 with a urethral stent plus a gastric tube (group B). We compared the incidence rates of bladder irritation, fistula, urethral stricture, and urethral diverticulum between the two groups of patients. RESULTS: No statistically significant differences were found between groups A and B in the incidences of bladder irritation (9.89% vs 10.70%, P > 0.05) and urethral diverticulum (1.09% vs 2.22%, P > 0.05). The incidence rate of fistula was markedly higher in group A than in B (20.80% vs 13.30%, P < 0.05), and so was that of urethral stricture (10.90% vs 5.55%, P < 0.05). CONCLUSION: The urethral stent plus gastric tube draining method is more effective than the Foley catheter draining method for urine drainage following urethroplasty.

Application of the modified Byars staged procedure for severe hypospadias repair.

This study aimed to introduce a modified Byars staged procedure and investigate its application value in patients with severe hypospadias. We retrospectively analyzed the clinical data of patients with severe hypospadias admitted to the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) between October 2012 and October 2022. In total, 31 patients underwent the conventional Byars procedure (conventional group), and 45 patients underwent the modified Byars staged procedure (modified group). Our modified strategy was built upon the standard Byars procedure by incorporating glansplasty during the first stage and employing a Y-shaped flap in conjunction with a glandular tunnel for urethroplasty during the second stage. Notably, there were no statistically significant differences in the preoperative baseline characteristics, duration of surgery, amount of blood loss, or occurrence of postoperative complications, including urethral fistula, stricture and diverticulum, or penile curvature, between the conventional and modified groups. However, there was a significantly lower incidence of coronal sulcus fistula (0 vs 16.1%, P = 0.02) and glans dehiscence (0 vs 12.9%, P = 0.02) in the surgical group than that in the conventional group. In addition, the modified group exhibited a notably greater rate of normotopic urethral opening (100.0% vs 83.9%, P = 0.01) and a higher mean score on the Hypospadias Objective Penile Evaluation (HOPE; mean ± standard error of mean: 8.6 ± 0.2 vs 7.9 ± 0.3, P = 0.02) than did the conventional group. In conclusion, the modified Byars staged procedure significantly reduced the risks of glans dehiscence and coronal sulcus fistula. Consequently, it offers a promising approach for achieving favorable penile esthetics, thereby providing a reliable therapeutic option for severe hypospadias.

A Nutrient-Deficient Microenvironment Facilitates Ferroptosis Resistance via the FAM60A-PPAR Axis in Pancreatic Ductal Adenocarcinoma.

Ferroptosis, a nonapoptotic form of cell death, is an emerging potential therapeutic target for various diseases, including cancer. However, the role of ferroptosis in pancreatic cancer remains poorly understood. Pancreatic ductal adenocarcinoma (PDAC) is characterized by a poor prognosis and chemotherapy resistance, attributed to its high Kirsten rats arcomaviral oncogene homolog mutation rate and severe nutritional deficits resulting from a dense stroma. Several studies have linked rat sarcoma (RAS) mutations to ferroptosis, suggesting that inducing ferroptosis may be an effective strategy against oncogenic RAS-bearing tumors. We investigated the role of Family With Sequence Similarity 60 Member A (FAM60A) in this study, a protein closely associated with a poor prognosis and highly expressed in PDAC and tumor tissue from KrasG12D/+;Trp53R172H/+; Pdx1-Cre mice, in regulating ferroptosis, tumor growth, and gemcitabine sensitivity in vitro and in vivo. Our results demonstrate that FAM60A regulates 3 essential metabolic enzymes, ACSL1/4 and GPX4, to protect PDAC cells from ferroptosis. Furthermore, we found that YY1 transcriptionally regulates FAM60A expression by promoting its transcription, and the Hippo-YY1 pathway is restricted in the low-amino-acid milieu in the context of nutrient deprivation, leading to downstream suppression of peroxisome proliferator-activated receptor and ACSL1/4 and activation of GPX4 pathways. Importantly, FAM60A knockdown sensitized PDAC cells to gemcitabine treatment. A new understanding of FAM60A transcriptional regulation pattern in PDAC and its dual function in ferroptosis reliever and chemotherapy resistance is provided by our study. Targeting FAM60A may therefore offer a promising therapeutic approach for PDAC by simultaneously addressing 2 major features of the disease (high RAS mutation rate and tumor microenvironment nutrient deficiency) and preventing tumor cell metabolic adaptation.

Mutated major histocompatibility complex class II transactivator up-regulates interleukin-33-dependent differentiation of Th2 subset through Nod2 binding for NLR (NOD-like receptor) signaling initiation.

Dominant-negative mutants of class II transactivator (mCIITAs) with N-terminal depletion have been used to repress the transcription of class II genes in xenotransplantation. Here, we report that mCIITA overexpressing myeloid cell line Ana-1 (Ana-1-mCIITA) derived from a C57BL/6 mouse was able to down-regulate the MHC class II expression and reverse immune responses from Th1 (IL-2(+)IFN-γ(+)STAT4(+)) to Th2 (IL-4(+)IL-5(+)IL-10(+)IL-13(+)STAT6(+)) when cocultured with T cells. Mechanism analysis indicated that the mCIITA protein is able to initiate a NOD-like receptor-related signaling pathway via binding of the cytoplasmic Nod2 protein, which was followed by activating RIP2, caspase 1, and IKK-α/ß. This ensures the expression of the genes encoding the cytokines IL-33, IL-1ß, and TNF-α; however, only the highly expressed IL-33 is responsible for inducing the type 2 response, with a skewed Th2 cytokine secretion (IL-4(+)IL-5(+)IL-10(+)IL-13(+)IL-2(-)IFN-γ(-)), which was completely prevented by the deactivation of the Nod2 gene with siRNA or by the blockage of the IL-33-related signaling using the mAb ST2L against the IL-33 receptor. mCIITA-mediated Th2 conversion was also successfully induced in vivo in a mCIITA-transgenic C57BL/6 mouse model. These results indicate that the Th1/Th2 balance could be regulated by an N terminus-depleted CIITA molecule via NOD-like receptor-related signaling, a property valuable for disease control, especially for inducing transplantation tolerance via the repression of class II expression and the attenuation of a Th1-dominant response.

Ectopic scrotum and penoscrotal transposition: Case report and literature review.

Background: Ectopic scrotum (ES) is an extremely rare congenital scrotal malformation. Ectopic scrotum with VATER/VACTERL [vertebral defects (V), anal atresia or anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), cardiac defects, renal malformations (R), and limb defects (L)] association is even rarer. There are no uniform guidelines for diagnosis and treatment. Clinical case: We described a 2-year-5-month-old boy who has ectopic scrotum and penoscrotal transposition and reviewed relevant literature in this report. We performed laparoscopy exploration, rotation flap scrotoplasty, and orchiopexy and achieved a great result during the postoperative follow-up. Conclusions: Combined with the previous literature, we made a summary to come up with a plan for the diagnosis and treatment of ectopic scrotum. Rotation flap scrotoplasty and orchiopexy are worthy of considering operative methods in treating ES. For penoscrotal transposition or VATER/VACTERL association, we can treat the diseases individually.

Impact of Repeated Infantile Exposure to Surgery and Anesthesia on Gut Microbiota and Anxiety Behaviors at Age 6-9.

(1) Background: Preclinical as well as population studies have connected general anesthesia and surgery with a higher risk of abnormal cognitive development, including emotional development. Gut microbiota dysbiosis in neonatal rodents during the perioperative period has been reported, however, the relevance of this to human children who undergo multiple anesthesia for surgeries is unknown. Given the emerging role of altered gut microbes in propagating anxiety and depression, we sought to study whether repeated infantile exposures to surgery and anesthesia affect gut microbiota and anxiety behaviors later in life. (2) Methods: This is a retrospectively matched cohort study comparing 22 pediatric patients of less than 3 years of age with multiple exposures (≥3) to anesthesia for surgeries and 22 healthy controls with no history of exposure to anesthesia. The parent report version of the Spence Children's Anxiety Scale (SCAS-P) was applied to evaluate anxiety in children aged between 6 and 9 years old. Additionally, the gut microbiota profiles of the two groups were compared using 16S rRNA gene sequencing. (3) Results: In behavioral tests, the p-SCAS score of obsessive compulsive disorder and social phobia were significantly higher in children with repeated anesthesia exposure relative to the controls. There were no significant differences between the two groups with respect to panic attacks and agoraphobia, separation anxiety disorder, physical injury fears, generalized anxiety disorder, and the total SCAS-P scores. In the control group, 3 children out of 22 were found to have moderately elevated scores, but none of them had abnormally elevated scores. In the multiple-exposure group, 5 children out of 22 obtained moderately elevated scores, while 2 scored as abnormally elevated. However, no statistically significant differences were detected in the number of children with elevated and abnormally elevated scores. The data show that repeated anesthesia and surgical exposures in children led to long-lasting severe gut microbiota dysbiosis. (4) Conclusions: In this preliminary study, our findings demonstrated that early repeated exposures to anesthesia and surgical predisposes children to anxiety as well as long-term gut microbiota dysbiosis. We should confirm these findings in a larger data population size and with detailed analysis. However, the authors cannot confirm an association between the dysbiosis and anxiety.

Effect of Hypertonic Saline Solution Combined with Furosemide on Acute Heart Failure: A Meta-Analysis.

Background: The efficacy of hypertonic saline solution (HSS) combined with furosemide in treating acute heart failure is controversial. This meta-analysis explores the efficacy of HSS combined with furosemide for the treatment of acute heart failure. Methods: Literature were searched from databases, including PubMed, Web of Knowledge, Embase, Central, CMKI, Wanfang, and VIP. The inclusion criteria were as follows: (1) subjects: patients with acute heart failure; (2) the experimental group and the control group were properly set up; (3) intervention measures: patients in the experimental group were treated with HSS + furosemide, and patients in the control group were treated with furosemide; (4) the outcomes included at least one of the following indicators: readmission rate, mortality, 24 h urine volume, weight loss, and serum creatinine; and (5) randomized controlled trial (RCT). The method recommended by Cochrane Collaboration Network was used to evaluate the risk bias. The heterogeneity among the studies was evaluated through the chi-square test, and the publication bias was assessed by the Egger test. The results were described using risk ratio (RR), mean difference (MD), and 95% confidence interval (CI). Results: The readmission rate in the HSS + furosemide group was lower than that in the furosemide group (RR = 0.53, 95% CI [0.46, 0.60], P < 0.00001), with no heterogeneity among the literature (P = 0.21, I 2 = 29%). Patients in the HSS + furosemide group had a lower mortality rate than that in the furosemide group (RR = 0.55, 95% CI [0.46, 0.65], P < 0.00001). The chi-square test result indicated no heterogeneity among the literature (P = 0.25, I 2 = 23%). Furthermore, the 24 h urine volume of patients in the HSS + furosemide group was higher than that in the furosemide group (MD = 497.29, 95% CI [457.61, 536.96], P < 0.00001). There was no heterogeneity among the literature (P = 0.58, I 2 = 0%). In contrast, patients in the HSS + furosemide group demonstrated a lower serum creatinine level than those in the furosemide group (MD = -0.45, 95% CI [-0.51, -0.39], P < 0.00001). However, heterogeneity was observed among the literature (P < 0.00001, I 2 = 81%). The weight loss in the HSS + furosemide group was higher than that in the furosemide group (MD = 1.83, 95% CI [1.51, 2.15], P < 0.00001). There was no heterogeneity among the literature (P = 0.42, I 2 = 2%). Egger test showed no publication bias among the literature (P > 0.05). Conclusion: Despite the heterogeneity and bias in our study, the combination of HSS with furosemide is promising in patients with acute heart failure. However, further research is still needed to confirm.

Application of the Mathieu combined tunnel technique for repairing glans dehiscence after failed hypospadias repair.

Repairing glans dehiscence after failed hypospadias repair is challenging for pediatric surgeons. Here, we introduced and evaluated a newly modified Mathieu technique, Mathieu combined tunnel (MCT), which involves multiple custom-designed flaps for the shortage of flap source material after repeated operations; we also constructed a tunnel to avoid the glans incision that may carry new risks of dehiscence. This retrospective study included 26 patients who were consecutively admitted to the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China) for glans dehiscence repair after failed hypospadias repair from October 2014 to October 2020; sixteen patients underwent surgery using the MCT (MCT group) and ten patients underwent surgery using the tubularized incised plate (TIP) technique (TIP group). The operative time, blood loss, postoperative complications, normal urethral meatus rate, success rate, and Hypospadias Objective Penile Evaluation (HOPE) score were compared between the two groups. The MCT group achieved an overall satisfactory penile appearance and voiding function, with a higher rate of normal urethral meatus (15/16, 93.8%) and a lower rate of glans dehiscence (1/16, 6.2%), compared with the TIP group (70.0% and 30.0%, respectively). However, these differences were not statistically significant, possibly because of the limited number of patients (all P > 0.05). Mean postoperative HOPE scores were similar in the MCT group (mean ± standard deviation: 8.83 ± 0. 89) and TIP group (8.94 ± 0.57) (P > 0.05). No significant differences were found between the two groups in terms of blood loss and success rate, nor in the rates of various complications (e.g., fistula, urethral stricture, and glans dehiscence). In conclusion, the MCT technique appears to be feasible and reliable for repairing glans dehiscence after failed hypospadias repair.

METTL1 promotes neuroblastoma development through m7G tRNA modification and selective oncogenic gene translation.

BACKGROUND: Neuroblastoma (NBL) is the most common extra-cranial solid tumour in childhood, with prognosis ranging from spontaneous remission to high risk for rapid and fatal progression. Despite existing therapy approaches, the 5-year event-free survival (EFS) for patients with advanced NBL remains below 30%, emphasizing urgent necessary for novel therapeutic strategies. Studies have shown that epigenetic disorders play an essential role in the pathogenesis of NBL. However, the function and mechanism of N7-methylguanosine (m7G) methyltransferase in NBL remains unknown. METHODS: The expression levels of m7G tRNA methyltransferase Methyltransferase-like 1 (METTL1) were analyzed by querying the Gene Expression Omnibus (GEO) database and further confirmed by immunohistochemistry (IHC) assay. Kaplan-Meier, univariate and multivariate cox hazard analysis were performed to reveal the prognostic role of METTL1. Cell function assays were performed to evaluate how METTL1 works in proliferation, apoptosis and migration in cell lines and xenograft mouse models. The role of METTL1 on mRNA translation activity of NBL cells was measured using puromycin intake assay and polysome profiling assay. The m7G modified tRNAs were identified by tRNA reduction and cleavage sequencing (TRAC-seq). Ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq) was utilized to identify the variation of gene translation efficiency (TE). Analyzed the codon frequency decoded by m7G tRNA to clarify the translation regulation and mechanism of m7G modification in NBL. RESULTS: This study found that METTL1 were significantly up-regulated in advanced NBL, which acted as an independent risk factor and predicted poor prognosis. Further in NBL cell lines and BALB/c-nu female mice, we found METTL1 played a crucial role in promoting NBL progression. Furthermore, m7G profiling and translation analysis revealed downregulation of METTL1 would inhibit puromycin intake efficiency of NBL cells, indicating that METTL1 did count crucially in regulation of NBL cell translation. With all tRNAs with m7G modification identified in NBL cells, knockdown of METTL1 would significantly reduce the levels of both m7G modification and m7G tRNAs expressions. Result of RNC-seq shew there were 339 overlapped genes with impaired translation in NBL cells upon METTL1 knockdown. Further analysis revealed these genes contained higher frequency of codons decoded by m7G-modified tRNAs and were enriched in oncogenic pathways. CONCLUSION: This study revealed the critical role and mechanism of METTL1-mediated tRNA m7G modification in regulating NBL progression, providing new insights for developing therapeutic approaches for NBL patients.

Nicotine decreases beta-amyloid through regulating BACE1 transcription in SH-EP1-α4ß2 nAChR-APP695 cells.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects the elderly population. Deposition of beta-amyloid (Aß) in the brain is a hallmark of AD pathology. In our previous study, we have constructed a cell line expressing human APP695 (hAPP695) in SH-EP1 cells stably transfected with human nicotinic receptor (nAChR) α4 subunit and ß2 subunit gene. In present study, we found that activation of α4ß2 nAChR by nicotine and epibatidine decreased secreted Aß level in the cell line and hippocampal neurons, but had no effects on full-length APP695 and sAPP-α. Nicotine also decreases BACE1 and PSEN1 expression, as well as ERK1 and NFκB P65 subunit expression in the cell line. Furthermore, BACE1 promoter activity is, but PSEN1 not, decreased by nicotine in the cell line. All the results suggest that activation of α4ß2 nAChR decreases Aß through regulating BACE1 transcription by ERK1-NFκB pathway. Additionally, analysis of BACE1 promoter activity by dual-luciferase reporter assay may be useful for drug screening as a high throughput method.

Ureteral triplication combined with right renal ectopia and ureteral cyst.

Ureteral triplication is one of the rarest malformations of the upper urinary tract. We report the case of a 12-year-old girl with right ureteral triplication combined with renal ectopia and ureteral cyst with stenosis at the junction of the ureteral cyst and distal ureter. The ureteral cyst was tailored and tubularized, and the tight junction was removed, as in Hynes-Anderson ureteropyeloplasty; on reevaluation almost 4 years later, kidney function was normal and computed tomography showed a normal kidney and ureter.

NLGN3 promotes neuroblastoma cell proliferation and growth through activating PI3K/AKT pathway.

Neuroblastoma is the most common extracranial solid tumor of childhood, previous studies show synaptic protein neuroligin-3 (NLGN3) promotes glioma proliferation and growth, However, no investigation about the role of NLGN3 in neuroblastoma was reported. Here, we found NGLGN3 was significantly upregulated in neuroblastoma cells and tissues, its overexpression significantly promoted neuroblastoma cell proliferation and growth determined by MTT analysis, colony formation assay, cell cycle progression analysis, BrdU incorporation assay and animal model, while its knockdown inhibited cell proliferation and growth. Then we found NLGN3 could increase the phosphorylation level of AKT and the transcription activity of FOXO family, suggesting NLGN3 activated PI3K/AKT pathway, inhibition of PI3K/AKT pathway in NLGN3 overexpressing cells inhibited cell proliferation, confirming NLGN3 promoted neuroblastoma proliferation through activating PI3K/AKT pathway. In summary, we found NLGN3 promoted neuroblastoma cell proliferation and growth through activating PI3K/AKT pathway and providing a new target for neuroblastoma therapy.

Construction of SH-EP1-alpha4beta2-hAPP695 cell line and effects of nicotinic agonists on beta-amyloid in the cells.

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer's disease. However, the most involved nicotinic receptor subtype in Alzheimer's disease is unclear. alpha4beta2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor alpha4 subunit and beta2 subunit gene, to observe effects of alpha4beta2 receptors activation on beta-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-alpha4beta2 cells. Function of the transfected alpha4beta2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective alpha4beta2 nicotinic receptor agonist) on beta-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-alpha4beta2 cells; Nicotine (1 muM) and epibatidine (0.1 muM) decreased intracellular and secreted beta-amyloid in the cells; and activation of alpha4beta2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor alpha4 subunit and beta2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer's disease. Alteration of Abeta level induced by activation of alpha4beta2 nAChR in our study might occur at a post-translational level.

The relationship between amniotic fluid miRNAs and congenital obstructive nephropathy.

Exosomes are small membrane vesicles with size of 30-100 nm, which were found in bodily fluids including amniotic fluid and saliva. The biological materials in exosomes, such as proteins and RNA, can be used as novel potential biomarkers for diagnostic assays. The purpose of this study was to assess whether exosomal microRNAs (miRNAs) could be used as biomarkers to prenatally diagnose congenital hydronephrosis and to evaluate fetal kidney function. Transmission electron microscopy (TEM), flow cytometry (FACS), and western-blot were applied to identify exosomes in the amniotic fluid from fetuses with congenital hydronephrosis and healthy controls. Exosomal miRNA was extracted according to the manufacturer's protocol and used for microarray. The differentially expressed miRNAs were selected for further study. The miRNA targets were analyzed to assess their possible function in the pathophysiology of obstructive nephropathy, and the miRNA array results were confirmed by qPCR. Amniotic fluid exosomes were identified based on CD24 and CD9 expression. The has-miR-942, has-miR-4289, has-miRPlus-A1073, and has-miR-195-3p were up-regulated in amniotic fluid exosomes from fetuses with congenital hydronephrosis comparing with those in healthy controls, and 35 had reduced expression levels. These results were confirmed by using qPCR. After integrating the miRNAs targets predicted via three databases and subjecting those target genes to KEGG pathway analysis, we found that the target genes of hsa-miR-300 and hsa-miR-299-5p were determined to be part of the Wnt signaling pathway. In addition, DVL2, PP2R5A, SRFP2, and SIAH1 predicted as target genes of has-miR-300 and has-miR-299-5p are informative for further exploration of congenital hydronephrosis pathologies. The reduced expression of hsa-miR-300 and hsa-miR-299-5p in the amniotic fluid of congenital hydronephrosis could be a biomarker for kidney fibrosis associated with congenital obstructive nephropathy.

miR-1303 promotes the proliferation of neuroblastoma cell SH-SY5Y by targeting GSK3ß and SFRP1.

Neuroblastoma (NB) is one of the most common solid tumors in children, many microRNAs regulate progression and development of NB. Here, we found miR-1303 was upregulated in NB cells and tissues, miR-1303 overexpression promoted the proliferation of SH-SY5Y NB cell investigated by MTT assay, colony formation assay and anchorage-independent growth ability assay, while miR-1303 knockdown reduced this effect. mechanism analysis suggested glycogen synthase kinase 3 beta (GSK3ß) and secreted frizzled-related protein 1 (SFRP1) were the target of miR-1303, luciferase assay revealed miR-1303 directly bound to the 3'UTR of GSK3ß and SFRP1. miR-1303 increased expression of MYC and CyclinD1, and decreased the expression of p21 and p27, and further demonstrated miR-1303 promotes NB proliferation. Moreover, there was a negative correlation between miR-1303 expression and GSK3ß and SFRP1 expression in NB tissues, confirming GSK3ß and SFRP1 were the targets of miR-1303 in NB tissues. Collectively, our findings suggested miR-1303 promotes NB proliferation by targeting GSK3ß and SFRP1, and might be a target for NB therapy.

Protein kinase A-dependent phosphorylation of Dock180 at serine residue 1250 is important for glioma growth and invasion stimulated by platelet derived-growth factor receptor α.

BACKGROUND: Dedicator of cytokinesis 1 (Dock1 or Dock180), a bipartite guanine nucleotide exchange factor for Rac1, plays critical roles in receptor tyrosine kinase-stimulated cancer growth and invasion. Dock180 activity is required in cell migration cancer tumorigenesis promoted by platelet derived growth factor receptor (PDGFR) and epidermal growth factor receptor. METHODS: To demonstrate whether PDGFRα promotes tumor malignant behavior through protein kinase A (PKA)-dependent serine phosphorylation of Dock180, we performed cell proliferation, viability, migration, immunoprecipitation, immunoblotting, colony formation, and in vivo tumorigenesis assays using established and short-term explant cultures of glioblastoma cell lines. RESULTS: Stimulation of PDGFRα results in phosphorylation of Dock180 at serine residue 1250 (S1250), whereas PKA inhibitors H-89 and KT5720 oppose this phosphorylation. S1250 locates within the Rac1-binding Dock homology region 2 domain of Dock180, and its phosphorylation activates Rac1, p-Akt, and phosphorylated extracellular signal-regulated kinase 1/2, while promoting cell migration, in vitro. By expressing RNA interference (RNAi)-resistant wild-type Dock180, but not mutant Dock180 S1250L, we were able to rescue PDGFRα-associated signaling and biological activities in cultured glioblastoma multiforme (GBM) cells that had been treated with RNAi for suppression of endogenous Dock180. In addition, expression of the same RNAi-resistant Dock180 rescued an invasive phenotype of GBM cells following intracranial engraftment in immunocompromised mice. CONCLUSION: These data describe an important mechanism by which PDGFRα promotes glioma malignant phenotypes through PKA-dependent serine phosphorylation of Dock180, and the data thereby support targeting the PDGFRα-PKA-Dock180-Rac1 axis for treating GBM with molecular profiles indicating PDGFRα signaling dependency.