Untargeted metabolomics of the intestinal tract of DEV-infected ducks.

INTRODUCTION: Duck enteritis virus (DEV) mainly causes infectious diseases characterized by intestinal haemorrhage, inflammation and parenchymal organ degeneration in ducks and other poultry. However, the mechanism by which it causes intestinal damage in ducks is not well understood. Metabolomics can provide an in-depth understanding of the full complexity of the disease. METHODS: In this study, 24 clinically healthy green-shell ducks (weight 1.5 kg ± 20 g) were randomly divided into 2 groups (experimental group, 18; control group, 6). The experimental group was intramuscularly injected with 0.2 mL of DEV virus in solution (TCID50 3.16 × 108 PFU/mL), and the control group was injected with 0.2 mL of sterile normal saline. Duck duodenum and ileum tissue samples were collected at 66 h, 90 h and 114 h post-injection (12 h of fasting before killing), and metabolomics analysis of duck duodenum and ileum tissues at the three time points (66, 90, 114 h) was performed by liquid chromatography-mass spectrometry (LC-MS) to screen for and analyse the potential differentiated metabolites and related signalling pathways. RESULTS: Screening was performed in the positive/negative mode (Pos: Positive ion mode; the ionization of substances at the ion source with positive ions such as H+, NH4+, Na+ and K+; Neg: Negative ion mode; the ionization of substances at the ion source with negative ions such as Cl-, OAc-), and compound abundance was compared to that in the control group. The total number of differentially abundant compounds in the duodenum at 66 h, 90 h and 114 h of DEV infection gradually increased, and metabolites such as cytidine, 2'-deoxyriboside and 4-guanidinobutyric acid were differentially abundant metabolites common to all three time periods. The metabolic pathways related to inflammatory response and immune response were tryptophan acid metabolism, cysteine-methionine metabolism, histidine metabolism and other amino acid metabolism and fat metabolism. Among them, the metabolic pathways with more differentially abundant metabolites were amino acid biosynthesis, cysteine and methionine metabolism, tryptophan metabolism, unsaturated fatty acid biosynthesis and purine metabolism, and the metabolic pathways with more enrichment factors were the IgA-related intestinal immune network pathway and lysosome pathway. Compared with the control group, there were 16 differentially abundant metabolites in the ileum tissue of DEV-infected ducks at 66 h of infection, 52 at 90 h of infection, and 40 at 14 h of infection with TD114. The metabolic pathways with more enriched differentially abundant metabolites were pyrimidine metabolism, tyrosine metabolism, phenylalanine metabolism and tryptophan biosynthesis. The metabolic pathways with the most enrichment factors were the mTOR signalling pathway, ferroptosis pathway, tryptophan metabolism pathway and caffeine metabolism pathway. CONCLUSION: Comparative analysis showed that the number of differentially abundant metabolites in the duodenum and ileum differed to some extent after DEV infection, with significantly more differentially abundant metabolites in duodenal tissues and fewer in ileal tissues; after DEV infection, the highest number of differentially abundant metabolites was obtained at 114 h of DEV infection, followed by the second highest at 90 h of infection and the lowest at 66 h of infection. The common differentially abundant metabolites in duodenal and ileal tissues were prostaglandins, arachidonic acid, and arachidonic ethanolamine. The main metabolic pathways in the duodenum were the IgA-associated intestinal immune network pathway and the lysosomal pathway, and the metabolic pathways with more enriched factors in the ileum were the mTOR signalling pathway, the ferroptosis pathway, and the tryptophan metabolism pathway.

Suprapatellar pouch effusion is associated with an increased risk of neglected osteochondral fractures in primary acute traumatic patellar dislocation: a consecutive series of 113 children.

BACKGROUND: The aim of this study was to investigate the risk factors of neglected osteochondral fractures in primary acute traumatic patellar dislocation in the pediatric population. METHODS: A total of 113 patients with primary acute traumatic patellar dislocation for whom coincident osteochondral fractures could not be confirmed by X-ray examination at initial diagnosis between January 2010 and February 2022 were retrospectively analyzed. Medical history, physical examination, and radiographic images were recorded in detail. The greatest dimension of the suprapatellar pouch (SP) effusion on radiograph was measured. Computed tomography and magnetic resonance imaging were used to confirm the presence of neglected osteochondral fractures and measure the fragment size. Potential risk factors were calculated and correlated with reference to the neglected osteochondral fractures and fragment size using multivariate linear regression analysis. RESULTS: Weight, walking ability, effusion grade, and SP measurement had a significant correlation with neglected osteochondral fractures in primary acute traumatic patellar dislocation (p = 0.046; p < 0.001; p = 0.048; p < 0.001). The cutoff point was 53.5 kg for weight and 18.45 mm for SP measurement. In the neglected fractures group, SP measurement was statistically significant with larger fragment size (beta value = 0.457; p < 0.001), and the cutoff point was 26.2 mm. CONCLUSIONS: SP effusion is not only associated with an increased risk of neglected osteochondral fractures in primary acute traumatic patellar dislocation but also with larger fragment size. Knee radiograph, medical history, and physical examination can predict the need for further imaging examination and even surgery in primary acute traumatic patellar dislocation.

Application of 3D printing navigation system in pediatric epiphyseal complex lesion surgery.

To investigate 3D printing navigation system in pediatric epiphyseal complex lesion surgery. 10 children with epiphyseal complex lesions of the lower limb were recruited. After collecting imaging data such as CT and MRI in children with epiphyseal complex lesions of the lower limb, a three-dimensional model of bone was constructed using 3D printed computer modeling technologies for the localization of the lesion area. The extent of bone bridges was less than 30%, and all of them met the indications for bone bridge resection surgery. 3D printed navigation templates guided lesion resection. Epiphyseal block growth regulation with a figure-of-eight plate was also used in cases with preexisting abnormal alignment. During the operation, the average surgical incision was 4.0 cm, the bone bridge positioning was accurate, and the bone bridge tissue could be successfully and completely removed. As a result of follow-up, no cases had residual bone bridge tissue, no iatrogenic epiphyseal injury was found, and the epiphyseal plate was open in all children. 3D printing navigation system improved the accuracy of resection of lower limb epiphyseal complex lesions, significantly reduced the need for intraoperative fluoroscopy, avoided iatrogenic injury to the epiphyseal complex due to positioning errors, thereby reducing postoperative complications and considerably improving the prognosis of a series of lower limb epiphyseal complex lesion diseases in children.

Modified Smith-Petersen approach with rectus-sparing reduces severe avascular necrosis for developmental dysplasia of the hip at walking age: minimum 5-year follow-up.

BACKGROUND: Developmental dysplasia of the hip (DDH) is one of the most common orthopedic malformations in children. Open reduction for DDH at walking age remains a major concern. The goal of this study is to evaluate the mid-term effect of a modified Smith-Petersen approach which preserves the rectus femoris on DDH at walking age, in particular avascular necrosis (AVN). METHODS: A retrospective review of DDH patients aged between 12 and 24 months was carried out between January 2010 and June 2016. Open reduction through the Smith-Petersen approach (Group A) and modified Smith-Petersen approach, which preserves the rectus femoris (Group B), were both used. Measurement of hip geometry included acetabular index, the International Hip Dysplasia Institute classification, and AVN degree. Clinical records included operation time, bleeding volume, and abduction angle. RESULTS: There were 101 children (119 hips) with DDH who met the inclusion criteria. There were 66 hips in Group A and 53 in Group B. The mean surgical age at open reduction was 17.0 ± 2.4 months, with a mean 104.9 ± 19.5 months at last follow-up. There was no statistical difference in surgical age between the two groups (17.2 vs. 16.4 months). There was no significant difference in the incidence of all types of clinically significant AVN between group A and group B (27.3 vs. 18.9%), but the incidence of severe AVN was lower in group B (19.7 vs. 5.7%, P = 0.026). In addition, the lower the age at the time of open reduction, the lower the severity of AVN (P = 0.002). CONCLUSIONS: These mid-term data suggest that the modified Smith-Petersen approach with rectus-sparing could reduce severe AVN more than the classical Smith-Peterson approach in open reduction in DDH at walking age. In addition, early open reduction can reduce the postoperative degree of AVN.

Association Between Genetic Risk, Adherence to Healthy Lifestyle Behavior, and Thyroid Cancer Risk.

Importance: Genetic and lifestyle factors are related to thyroid cancer (TC). Whether a healthy lifestyle is associated with TC and could attenuate the influence of genetic variants in TC remains equivocal. Objectives: To examine the associations between genetics and healthy lifestyle with incident TC and whether adherence to a healthy lifestyle modifies the association between genetic variants and TC. Design, Setting, and Participants: A prospective cohort study using UK Biobank data recruited 502 505 participants aged 40 to 69 years between March 13, 2006, and October 1, 2010. A total of 307 803 participants of European descent were recruited at baseline, and 264 956 participants were available for the present study. Data analysis was conducted from November 1, 2021, to April 22, 2022. Exposures: Lifestyle behaviors were determined by diet index, physical activity, weight, smoking, and alcohol consumption. Lifestyle was categorized as unfavorable (scores 0-1), intermediate (score 2), and favorable (scores 3-5). The polygenic risk score (PRS) was derived from a meta-genome-wide association study using 3 cohorts and categorized as low, intermediate, and high. Main Outcomes and Measures: Thyroid cancer was defined using the International Classification of Diseases, Ninth Revision (code 193), International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (code C73), and self-report (code 1065). Results: Of 264 956 participants, 137 665 were women (52%). The median age was 57 (IQR, 49-62) years. During a median follow-up of 11.1 (IQR, 10.33-11.75) years (2 885 046 person-years), 423 incident TCs were ascertained (14.66 per 100 000 person-years). Higher PRSs were associated with TC (hazard ratio [HR], 2.25; 95% CI, 1.91-2.64; P = 8.65 × 10-23). An unfavorable lifestyle was also associated with a higher risk of TC (HR, 1.93; 95% CI, 1.50-2.49; P < .001). When stratified by PRS, unfavorable lifestyle was associated with TC in the higher PRS group (favorable vs unfavorable HR, 0.52; 95% CI, 0.37-0.73; P < .001). Furthermore, participants with both a high PRS and unfavorable lifestyle had the highest risk of TC (HR, 4.89; 95% CI, 3.03-7.91; P < .001). Conclusions and Relevance: In this prospective cohort study, genetic and lifestyle factors were independently associated with incident TC, which suggests that a healthier lifestyle may attenuate the deleterious influence of genetics on the risk of TC in individuals of European descent.

Identification of the genetic basis of sporadic polydactyly in China by targeted sequencing.

PURPOSE: Polydactyly is a highly heterogeneous group of skeletal deformities in clinical and genetic background. The variation spectrum in Chinese sporadic polydactyly has not been comprehensively analyzed. To elucidate genetic variation spectrum and genotype-phenotype correlations in Chinese patients with polydactyly, we conducted comprehensive genetic analysis of patients nationwide using targeted sequencing. METHODS: A total of 181 patients diagnosed with polydactylies were recruited. We designed a targeted capture panel for sequencing 721 genes that are associated with the pathogenesis of skeletal dysplasia. We performed rigorous variant- and gene-level filtrations to identify potentially damaging variants, followed by enrichment analysis and gene prioritization. RESULTS: A total of 568 deleterious variants of 293 genes were identified in 173 of 181 patients with a positive rate of 95.6% by targeted sequencing. For each sample, an average of 3.17 deleterious variants were identified. Especially, 14 pathogenic or likely pathogenic variants were identified in 10 genes in 14 patients out of the 181 patients, providing a positive molecular diagnostic rate of 7.7%. CONCLUSION: Targeted sequencing analysis provides a high efficiency approach for the genetic diagnosis of polydactyly. This is the largest next generation sequencing study performed to date in patients with polydactyly and represents the genetic basis of polydactyly typically encountered in genetics clinics.

A Novel Nonsense GLI3 Variant Is Associated With Polydactyly and Syndactyly in a Family by Blocking the Sonic Hedgehog Signaling Pathway.

Polydactyly and syndactyly are congenital limb malformations that may occur either as non-syndromic or syndromic forms. In the present study, massively parallel sequencing was performed on a proband in a four-generation family with polydactyly and syndactyly to identify disease-causing variant(s). A pathogenic variant c.739C > T (p.Gln247∗) in the glioma-associated oncogene family zinc finger 3 (GLI3) gene was identified and co-segregated with the affected members of the family. Firstly, we examined GLI3 mRNA and GLI3 protein levels in peripheral blood mononuclear cells (PBMCs) of patients carrying this variant. The results showed that the truncated GLI3 p.Gln247∗ (c.739C > T) protein was detectable in patients and the GLI3 transcript and protein levels were not significantly altered in the PBMCs of patients compared with healthy controls. Furthermore, functional analysis showed that the truncated GLI3 p.Gln247∗ (c.739C > T) protein variant could lead to cytoplasmic accumulation of mutant protein and loss of ability to bind to the Suppressor of Fused protein. Alterations in protein expression levels of core components of the Sonic hedgehog signaling pathway were also observed. Our study shows that this novel GLI3 variant contributes to the malformations in this family and provides evidence for the mechanism by which GLI3 c.739C > T (p.Gln247∗) was implicated in the pathogenesis of polydactyly and syndactyly.

Methionine-Mediated Protein Phosphatase 2A Catalytic Subunit (PP2Ac) Methylation Ameliorates the Tauopathy Induced by Manganese in Cell and Animal Models.

The molecular mechanism of Alzheimer-like cognitive impairment induced by manganese (Mn) exposure has not yet been fully clarified, and there are currently no effective interventions to treat neurodegenerative lesions related to manganism. Protein phosphatase 2 A (PP2A) is a major tau phosphatase and was recently identified as a potential therapeutic target molecule for neurodegenerative diseases; its activity is directed by the methylation status of the catalytic C subunit. Methionine is an essential amino acid, and its downstream metabolite S-adenosylmethionine (SAM) participates in transmethylation pathways as a methyl donor. In this study, the neurotoxic mechanism of Mn and the protective effect of methionine were evaluated in Mn-exposed cell and rat models. We show that Mn-induced neurotoxicity is characterized by PP2Ac demethylation accompanied by abnormally decreased LCMT-1 and increased PME-1, which are associated with tau hyperphosphorylation and spatial learning and memory deficits, and that the poor availability of SAM in the hippocampus is likely to determine the loss of PP2Ac methylation. Importantly, maintenance of local SAM levels through continuous supplementation with exogenous methionine, or through specific inhibition of PP2Ac demethylation by ABL127 administration in vitro, can effectively prevent tau hyperphosphorylation to reduce cellular oxidative stress, apoptosis, damage to cell viability, and rat memory deficits in cell or animal Mn exposure models. In conclusion, our data suggest that SAM and PP2Ac methylation may be novel targets for the treatment of Mn poisoning and neurotoxic mechanism-related tauopathies.

Hip morphology in mucopolysaccharidosis type IVA through radiograph, magnetic resonance imaging and arthrogram assessment.

PURPOSE: This study examined the hip morphology of paediatric patients with mucopolysaccharidosis (MPS) type IVA (MPS IVA). METHODS: This was a retrospective chart review of 42 hips in 21 children with MPS IVA. Pelvic radiographs and magnetic resonance imaging (MRI) scans of 42 hips and arthrograms of 13 hips were analysed. The bony, cartilaginous and labral coverage of the acetabulum was determined by acetabular index (AI), centre edge angle (CEA) and femoral head coverage (FHC). RESULTS: The mean age at the time of radiography was 66.3 ± 21.7 months. The bony, cartilaginous and labral AI in the MRI assessment were 36.3 ± 5.3, 18.3 ± 4.7 and 12.1 ± 4.6 degrees, respectively. The inter-class correlation coefficients (ICCs) for the bony AI, CEA and FHC measurements on radiographs and MRI were 0.936, 0.879 and 0.810, respectively. In the MRI assessment, labrum in 12 of 42 hips appeared as a regular triangle, and it was flat on 30/42 hips. The average arthrographic AI (AAI) was 11.1 ± 2.7 degrees. The ICCs value of AAI versus cartilaginous and labral AI on MRI indicates good agreement but higher in labral AI. CONCLUSION: Hips in MPS IVA exhibited obvious cartilage and labrum compensation in response to abnormal ossification of bony acetabulum. Cartilage in MPS IVA hip increases the thickness in the longitudinal direction, while the labrum becomes flatten in the horizontal direction. The AAI may represent intraoperative labrum coverage. The femora-acetabular harmony is difficult to determine using radiography only, and pre-operative MRI and an intraoperative arthrogram are very important in a hip assessment in MPS IVA.

Early open reduction of dislocated hips using a modified Smith-Petersen approach in arthrogyposis multiplex congenita.

BACKGROUND: Arthrogryposis multiplex congenita (AMC) is a rare syndrome with multiple joint contractures. Within the medical community, there is controversy surrounding AMC in terms of the ideal surgical approach and age for performing a reduction of dislocated hips. The purpose of this retrospective study was to evaluate the clinical outcomes of early open reduction of infant hip dislocation with arthrogryposis multiplex congenita following a modified Smith-Petersen approach that preserves the rectus femoris. METHODS: From 2010 to 2017, we performed this procedure on 28 dislocated hips in 20 infants under 12 months of age with AMC. The clinical and radiology data were reviewed retrospectively. The mean age at surgery was 6.9 ± 5.1 months, with a mean follow-up of 42.4 ± 41.1 months. RESULTS: After open reduction, the average hip acetabular index (AI), the international hip dysplasia institute classification (IHDI), and the hip range of motion significantly improved (all P < 0.001). After the surgery, 16 patients were community walkers, and four patients were home walkers. Three hips in two patients required secondary revision surgery for residual acetabular dysplasia with combined pelvic osteotomy and femoral osteotomy. Seven of the hips that had been operated on showed signs of avascular necrosis (AVN). Among them, four were degree II, two were degree III, and one was degree IV. Multiple linear regression analysis demonstrated that greater age (in months) heightened the risk for secondary revision surgery (P = 0.032). CONCLUSIONS: The modified Smith-Petersen approach preserving the rectus femoris is an encouraging and safe option for treating hip dislocation in young AMC patients (before 12 months). If surgery takes place at less than 12 months of age for patients with AMC, this earlier open reduction for hip dislocation may reduce the chances of secondary revision surgery. LEVEL OF EVIDENCE: IV, retrospective non-randomized study.

Systemic toxicity evaluation of novel tobacco products in Caenorhabditis elegans.

Under strictly Framework Convention on Tobacco Control, novel tobacco products are going to be promising alterations to consumers and manufactures. Even though the novel tobacco products have been considered less harmful than traditional tobaccos, there is a few knowledges about the subsequent substances during consume and their impacts to the consumers due to short introduction into the market. Thus, the present study aims to investigate the adverse effects of novel tobacco products on Caenorhabditis elegans(C. elegans) and to provide relevant references for novel tobacco products toxicity research and assessment. C. elegans individuals at L4 stage were exposed to different kinds of novel tobacco products, including electronic cigarettes liquid (e-liquid), the extract of e-cig aerosol (e-aerosol), mint and black tea flavor snus. After specific exposure time, the multiple toxic endpoints of C. elegans were measured, including acute toxicity, locomotion behavior, body length, and life-span. The oxidative stress was tested too. According to acute toxicity assays, the half lethal dose of four novel tobacco products calculated from theoretical nicotine concentration, ranked as follows e-liquid (0.29 mg/ml) > the extract of e-cig aerosol (0.43 mg/ml) > mint flavor snus (1.20 mg/ml) > black tea flavor snus (1.50 mg/ml). The equivalent lethal rate 5%~20% of four novel tobacco products were applied to following experiments. These novel tobacco products damaged nematode's locomotion including head thrashing and body bending, the damage was most evident in two flavors of snus. The similar trends were found in reproductive performance investigation. At tested concentrations, the retardation development of C. elegans was found throughout all stages with peak blockage at adulthood. Life-span tests showed that novel tobacco products at 5% lethal rate seemed no significant effect on affected the life-span of nematodes, with snus shortened the lifespan of C. elegans at 20% lethal rate. Imaging stress response indicted four types of tobacco productions causing stress response in C. elegans. Exposed to either 5% or 20% lethal levels (5% and 20%), the percentages of worms with DAF-16 redistribution among all groups varied, with higher frequencies in both snus. Summary, novel tobacco products caused multiple adverse impacts to C. elegans, including acute toxicity, locomotion behavior disruption, brood size reduction, development retardation, and life-span reduction. The toxicity was associated with both the feature and concentration of tobacco products, and oxidative stress was the main mechanism.

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability.

OBJECTIVES: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity. METHODS: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H2O2-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H2O2-treated vector control and LCMT1-overexpressing cells. RESULTS: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H2O2-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H2O2 treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton. DISCUSSION: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.

Hydrogen peroxide redistributes the localization of protein phosphatase methylesterase 1.

AIMS: Protein phosphatase methylesterase-1 (PME-1) is a serine hydrolase that catalyzes protein phosphatase 2A (PP2A) demethylation and negatively regulates its activity. PME-1 is compartmentalized within cells to precisely control the demethylation of PP2A. This study investigated the localization of PME-1 in human fibroblast cells (HDF) under oxidative stress. MAIN METHODS: Alkaline demethylation and peptide competition assays were applied to detect the methylation sensitivity of anti-PP2Ac. The localization of PME-1, leucine carboxyl methyltransferase 1 (LCMT1), demethylated-phosphorylated-PP2Ac (dem-p-PP2Ac) and total PP2Ac was determined by immunofluorescence analysis, and protein expression was measured by Western blot. A HEK293 cell line stably expressing constructed PME-1-EGFP was used to dynamically monitor the nuclear export of PME-1 under oxidative stress. KEY RESULTS: After hydrogen peroxide (H2O2) treatment, the protein expression of PME-1 remained unchanged, while PME-1 facilitated redistribution from the nucleus to the cytoplasm in HDF according to immunofluorescence analysis. In constructed HEK293 cells, the EGFP-tagged PME-1 was exported from the nucleus to the cytoplasm after H2O2 treatment, and nuclear export was eliminated after leptomycin B additions. Our observation of dem-p-PP2Ac species relocation from the nucleus to the cytoplasm under oxidative stress is consistent with the redistribution patterns of PME-1. Antioxidant N-acetyl cysteine can reverse the nuclear to cytoplasmic ratio of PME-1 proteins and dem-p-PP2Ac after H2O2 exposure. SIGNIFICANCE: We found that PME-1 is exported from the nucleus to the cytoplasm upon H2O2 treatment and redistributes dem-p-PP2Ac in subcellular compartments. These findings offer new insight into the regulation of PME-1 localization and PP2A demethylation under oxidative stress.

miR-214-3p promotes the proliferation, migration and invasion of osteosarcoma cells by targeting CADM1.

Although osteosarcoma (OS) is the most common type of primary bone tumor in adolescents and young adults, its mechanism remains unclear. A previous study by the authors demonstrated that miR-214-3p was upregulated in OS patients. Therefore, the present study aimed to investigate the effect and molecular mechanism of miR-214-3p in OS cells. OS cell lines, U2OS and MNNG/HOS Cl#5, were transiently transfected with miR-214-3p mimics, a control mimic, miR-214-3p inhibitors and a control inhibitor. Subsequent assays revealed that elevated miR-214-3p promoted the proliferative, migratory and invasive abilities of OS cells, while the opposite effects were observed in cells that were transfected with miR-214-3p inhibitors. The interaction between miR-214-3p and cell adhesion molecule 1 (CADM1) 3'untranslated region (UTR) was verified by a dual luciferase assay, which indicated that the relative luciferase activity was decreased in 293T cells that were co-transfected with miR-214-3p mimic and psiCHECK2-CADM1-3'UTR compared with cells that were co-transfected with psiCHECK2-CADM1-3'UTR and control mimic. The knockdown of CADM1 using small-interfering RNA enhanced the proliferative, migratory and invasive abilities of OS cells. Furthermore, downregulated CADM1 expression increased the expression of phosphorylated P44/42 mitogen activated kinase (MAPK). In conclusion, miR-214-3p was able to directly target CADM1 and decrease its expression. This resulted in the activation of the P44/42 MAPK signaling pathway, and thereby promoted the proliferation, migration and invasion of OS cells.

H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

Involvement of gap junctions in astrocyte impairment induced by manganese exposure.

Glutamate excitotoxicity, characterized as excessive glutamate stress, is considered to be involved in cerebral ischaemia, brain trauma, and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Glutamate homeostasis disruption was highlighted in Mn neurotoxicity caused by high levels of Mn. Astrocytes, accounting for approximately 50% of the neuronal cells in the central nervous system and maintain glutamate homeostasis, are sensitive to neurotoxicity induced by Mn exposure. Astrocytes are tightly coupled with gap junctions (GJ), which are comprised of connexins, mainly connexin43 (Cx43). The gap junctional intercellular communication (GJIC) pathway allows small signal molecules, such as glutamate, ATP (adenosine triphosphate, ATP) and tropic factors, etc., to transfer between adjacent cells. Evidence has shown that astrocytes execute the bystander effect during cell death through the GJIC pathway. However, the pathogenic mechanism of the gap junction underlying glutamate neurotoxicity induced by manganese exposure has not been elucidated yet. In the present study, primary astrocytes were cultured and then exposed to different levels of Mn (ranging from 0 to 1000 µM) for 4/16 h to investigate the function of the GJIC in apoptosis induced by Mn. The cellular toxicity was confirmed by cell viability and apoptotic percentage through MTT assay and flow cytometry (FC). The levels of intracellular/extracellular glutamate were measured by high-performance liquid chromatography (HPLC). The fluorescent dye, Lucifer Yellow (LY), was used to assess the status of gap junctions among astrocytes after Mn exposure. The protein/gene expression of major gap junctional forming protein, Cx43, was also investigated. Cell viability was distinctly reduced when exposed to 500 and 1000 µM MnCl2 compared with control cells at both time points. The percentage of apoptosis was significantly increased among all detected Mn levels (125, 500 and 1000 µM MnCl2) of exposure (p < 0.05) with a concentration-dependent manner at either time point. Mn administration for 4/16 h also caused a remarkable intracellular/extracellular glutamate increase in a concentration-dependent manner for extracellular glutamate levels (p < 0.01). Gap junctions were prominently inhibited by Mn with Cx43 protein shown as shortening of the LY dye transfer distance at both time points. In-cell western blot indicated that Mn caused a decrease in Cx43 protein/gene expression in a dose-dependent manner. These results suggested that the gap junction intercellular communication and its forming protein, Cx43, are likely involved in glutamate excitotoxicity induced by Mn exposure.

Characterization of overexpression of the alternatively spliced isoform of the protein phosphatase 2A catalytic subunit in cells.

PP2Acα2 is a recently discovered PP2Acα alternative splicing isoform that can be induced following serum withdrawal. It shows enhanced binding to immunoglobulin binding protein 1 and is overexpressed in chronic lymphocytic leukemia patients. Current knowledge concerning PP2Acα2 is limited. In this study, we induced and cloned PP2Acα2 from HL-60 cells and human lymphocytes, transfected them into human embryonic kidney 293 cells and constructed a stable overexpression cell line. We found that PP2Acα2 mRNA inhibits expression of its longer isoform PP2Acα mRNA but had no effect on the final protein expression and modification of this longer isoform. Moreover, PP2Acα2-overexpressed cells demonstrated increased expression of IGBP1, activated mTORC1 signaling to reduce basal autophagy and increased anchorage-independent growth. Our study provides new insights into the complex mechanisms of PP2A regulation.

Retrospective observational study comparing the international hip dysplasia institute classification with the Tonnis classification of developmental dysplasia of the hip.

The Tonnis radiographic classification of developmental dysplasia of the hip (DDH) has been widely used. The International Hip Dysplasia Institute (IHDI) classification, a new classification system recently developed by the IHDI, is beginning to be applied to evaluate DDH with the absence of an ossification center. This study aimed to validate its reliability in evaluating DDH with an ossification center and compared the 2 classifications in evaluating all DDH hips. In addition, the prediction values of the 2 classifications on clinical management selection were compared.In total, the pelvic radiographs of 212 DDH patients (318 hips) between the ages of 6 and 48 months admitted to Shanghai Children's Medical Center between 2007 and 2014 were assessed by 3 observers retrospectively using the 2 classifications. Intraobserver and interobserver agreements were evaluated using the kappa method. We also assessed the correlation of the 2 radiographic classifications in terms of treatment selection.In total, 216 hips received closed reduction, 61 hips received open reduction, and 41 hips received pelvic osteotomy. Both classifications showed excellent intraobserver and interobserver reliability. However, the IHDI demonstrated more interobserver reliability, especially for evaluating DDH without an ossification center. Both classifications were found to be relevant in detecting the DDH treatment type (P < 0.01). The Tonnis classification was also relevant, especially for evaluating DDH with an ossification center.The IHDI classification exhibited good practicability in classifying the radiographic severity of DDH compared to the Tonnis classification, particularly in hips without an ossification center. Like the Tonnis classification, the IHDI classification can predict treatment plans. Therefore, the IHDI classification seems to be the upgraded version of the Tonnis classification.

Mutational screening of GLI3, SHH, preZRS, and ZRS in 102 Chinese children with nonsyndromic polydactyly.

BACKGROUND: Polydactyly is a group of congenital limb malformations that show high degree of phenotypic variability and genetic heterogeneity. RESULTS: In the present study, four genomic regions (exons of GLI3, SHH, and noncoding sequences of preZRS and ZRS) involved in hedgehog (Hh) signaling pathway were sequenced for 102 unrelated Chinese children with nonsyndromic polydactyly. Two GLI3 variants (c.2844 G > G/A; c.1486C > C/T) and four preZRS variants (chr7:156585336 A>G; chr7:156585421 C>A; chr7: 156585247 G>C; chr7:156585420 A > C) were observed in 2(2.0%) and 6(5.9%) patients, respectively. These variants are not over-represented in the Chinese healthy population. All the 8 cases showed preaxial polydactyly in hands. Additionally, no specific patterns of malformation predicted mutations in other candidate genes or sequences. CONCLUSIONS: This is the first report of the assessment of the frequency of GLI3/SHH/preZRS/ZRS in Chinese patients to show any higher possibility of mutations or variants for the 4 genes or sequences in China. Developmental Dynamics 246:392-402, 2017. © 2017 Wiley Periodicals, Inc.

Open reduction combined with CORA-based osteotomy of the ulna in the treatment of missed Bado type I Monteggia injury: A retrospective study of 5 cases.

Open reduction combined with ulnar osteotomy is the most common approach to treating missed Monteggia injuries. The osteotomy is usually performed at the proximal ulna to ensure better healing and fewer complications. The purpose of this study is to present a center of rotation angulation (CORA)-based osteotomy of the ulna for treating Bado type I Monteggia injuries.We retrospectively reviewed the cases of patients who were treated with open reduction combined with a CORA-based ulnar osteotomy between February 2014 and December 2016. Each patient provided his or her internal control, and paired data of the involved and uninvolved sides were analyzed to evaluate forearm rotation function.Five patients (3 male, 2 female) with median age 5.7 years (range, 3.4-6.8 years) were operated on by the senior author in our hospital. The median interval between the original injury and the corrective surgery was 3 months (range, 1-4 months). In a median follow-up of 10 months (range, 6-17 months), all patients obtained stable reduction of the radial head and uneventful healing of the ulnar osteotomy. All patients had pain-free elbows with no neurological or vascular complications and no implant breakage. Patients showed excellent outcomes evaluated using the Broberg and Morrey index.Open reduction with a CORA-based osteotomy of the ulna for the treatment of missed Bado type I Monteggia injury with an obvious ulnar bowing deformity resulted in stable reduction of the radial head and excellent forearm function.