Therapeutic potential of proteasome inhibitors for dihydropyridine-induced gingival overgrowth.

OBJECTIVES: NF-κB plays a crucial role in collagen overproduction in dihydropyridine-induced gingival overgrowth (DIGO) fibroblasts. We aim to investigate the role of the kappa B (IκB) kinase (IKK)-NF-κB pathway and downstream collagen type I (Col I) synthesis in DIGO cells and to demonstrate the therapeutic strategy of interference of this pathway with proteasome inhibitors. METHODS: Gingival fibroblasts from DIGO (n = 5) and healthy (n = 5) patients were selected and stimulated with IL-1ß, nifedipine, or both. All experiments were run in triplicate and independently for each primary cell sample. RESULTS: The results demonstrated that both drugs additively mediated NF-κB activity by activating IKKα/ß phosphorylation. They also triggered nuclear translocation of NF-κB, Rela, and p50 (*p < .05) and increased Col I production in both healthy and DIGO cells. The addition of proteasome inhibitors, including bortezomib and MG132, promoted the accumulation of phosphorylated p-IκBα, prevented the subsequent cytosol-to-nuclear translocation of p50 and Rela (*p < .05), and abbreviated the biosynthesis of Col I in DIGO cells. CONCLUSIONS: We suggested that IKK-IκBα activation is mediated by proinflammatory cytokines and CCBs in DIGO cells and triggers downstream NF-κB-Col I synthesis. Proteasome inhibitors may strategically interfere with the IKK-IκBα-NF-κB-Col I pathway and inhibit the etiopathogenesis of DIGO.

Dynamic image clustering from projected coordinates of deep similarity learning.

Commonly used clustering algorithms typically require user parameters such as the number of clusters to be divided. Density-based algorithms do not have such requirements but are not suitable for high dimensional data. Recent studies have merged the cluster assignment task with deep similarity learning. In this paper, we propose a novel framework to perform dynamic image clustering without prior knowledge of the cluster count. A deep learning model first learns data similarity from scratch, followed by the use of a coordinate learning model to project high dimensional data onto a two-dimensional space. A new clustering algorithm, raster clustering, is proposed to evaluate and classify the projected data. This mechanism can be applied in high dimensional data clustering like image data, and it allows the prediction of unseen data in a consistent way without the need for consolidating with training data.

Research on the Impacts of Cognitive Style and Computational Thinking on College Students in a Visual Artificial Intelligence Course.

Visual programming language is a crucial part of learning programming. On this basis, it is essential to use visual programming to lower the learning threshold for students to learn about artificial intelligence (AI) to meet current demands in higher education. Therefore, a 3-h AI course with an RGB-to-HSL learning task was implemented; the results of which were used to analyze university students from two different disciplines. Valid data were collected for 65 students (55 men, 10 women) in the Science (Sci)-student group and 39 students (20 men, 19 women) in the Humanities (Hum)-student group. Independent sample t-tests were conducted to analyze the difference between cognitive styles and computational thinking. No significant differences in either cognitive style or computational thinking ability were found after the AI course, indicating that taking visual AI courses lowers the learning threshold for students and makes it possible for them to take more difficult AI courses, which in turn effectively helping them acquire AI knowledge, which is crucial for cultivating talent in the field of AI.

Macrophage inflammatory protein-3alpha is a novel serum marker for nasopharyngeal carcinoma detection and prediction of treatment outcomes.

PURPOSE: We herein examine whether macrophage inflammatory protein-3alpha (MIP-3alpha) is a biomarker for nasopharyngeal carcinoma (NPC) and whether it is involved in modulating NPC cell functions. EXPERIMENTAL DESIGN: The study population comprises 275 NPC patients and 250 controls. MIP-3alpha levels in tissues and sera were examined by immunohistochemistry and ELISA, respectively. EBV DNA load and EBV viral capsid antigen IgA were measured by quantitative real-time PCR and immunofluorescence assay, respectively. Effects of MIP-3alpha on NPC cell motility were investigated by Transwell migration/invasion assays and RNA interference. RESULTS: MIP-3alpha was overexpressed in NPC tumor cells. Serum MIP-3alpha levels were significantly higher in untreated patients, recurrent patients and patients with distant metastases versus non-NPC controls, patients with complete remission, and long-term disease-free patients. In the prospective cohort, serum MIP-3alpha levels were significantly higher in untreated NPC patients with advanced tumor-node-metastasis stage versus early stage and also correlated with EBV DNA load. Measurement of MIP-3alpha, EBV DNA, and viral capsid antigen IgA levels in serial serum/plasma samples from treated patients at 6-month intervals revealed a high association between MIP-3alpha level, EBV DNA load, and disease status. Among 155 consecutive NPC patients, subjects with pretreated MIP-3alpha serum levels over 65 pg/mL had worse prognoses for overall survival and distant metastasis-free survival in univariate and multivariate analysis. Additionally, cell functional assays showed that MIP-3alpha contributed to migration and invasion of NPC cells, which could be effectively inhibited by MIP-3alpha knockdown. CONCLUSIONS: MIP-3alpha may be a novel biomarker and prognosticator for NPC and is involved in migration and invasion of NPC cells.

Quantitative analysis of Miller mobility index for the diagnosis of moderate to severe periodontitis - A cross-sectional study.

BACKGROUND/PURPOSE: We assessed the mobility of single-root teeth by using Miller's mobility index (MMI) and to analyze the validity of MMI for the diagnosis of periodontitis. MATERIALS AND METHODS: A total of 30 patients were included and the Spearman correlation coefficient was used to assess the correlation between MMI, clinical attachment level (CAL), and probing depth (PD). The validity of MMI for the diagnosis of the severity of periodontitis was evaluated using the receiver operating characteristic (ROC) curve, area under curve (AUC) value, positive predictive value (PPV). RESULTS: Strong correlations were observed between MMI and CAL (r = 0.92) and between MMI and PD (r = 0.76). When the CAL = 3-4 mm and CAL ≥5 mm groups were pooled together, the AUC value was 0.81. The AUC was 0.86 for diagnosis with MMI in the CAL ≥5 mm group. A PPV of 100% was achieved for all grades when MMI >1. When the teeth with PD ≥ 5 to <7 mm and PD ≥ 7 mm groups were pooled together, the AUC value for MMI was 0.80. The PPV was 98.8%, 99%, and 100% for MMI Grade 1, Grade 2, and Grade 3, respectively. When PD ≥ 7 mm was defined as severe periodontitis, the AUC value for MMI was 0.72. CONCLUSION: MMI may provide valuable information for the diagnosis of moderate and severe periodontitis when CAL is not obtainable during routine practice.

Detection of Hunter syndrome (mucopolysaccharidosis type II) in Taiwanese: biochemical and linkage studies of the iduronate-2-sulfatase gene defects in MPS II patients and carriers.

BACKGROUND: Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. The result is impaired IDS enzyme function. METHODS: To characterize the biochemical and molecular defects in IDS-deficient patients and their families, we measured IDS enzyme activity by fluorimetric enzyme assay and identified the IDS gene mutations in 14 unrelated Taiwanese patients with varying clinical phenotypes. In addition, haplotype analysis was also performed. RESULTS: Three novel (IVS2+1G>C, 1055del12, and G489D) and 7 previously reported (N63K, P228L, K347E, R468Q, R468W, I485R, and 1241delAG) mutations were found. Together R468Q and R468W account for 42.8% mutations found in our patients. Haplotype analysis using IDS flanking markers DXS1113 and DXS1123 revealed that the unrelated R468Q alleles were independent in origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Once the mutation in a family was identified, restriction analysis was also performed for rapid diagnosis of female carriers in 8 families. Leukocyte IDS measurement revealed significantly wide range of IDS activity in normal controls and MPS II carriers (19.2 - 70.6 vs. 8.4 - 26.6 nmol/h/mg cell protein). The average leukocyte IDS activity of normal controls (n=43) was 43.9+/-13.3 nmol/h/mg protein, whereas patients with MPS II (n=14) had <5% of mean normal IDS activity (0.9+/-0.6 nmol/h/mg protein), and carriers (n=13) had a mean activity of 17.5 (+/-5.7) nmol/h/mg protein. The mean leukocyte IDS activity in female carriers was less than a half of the normal level. CONCLUSION: Due to a small overlapping range of normal and carriers, the level of enzyme activity cannot be used alone for carrier detection.

TGF-ßI Regulates Cell Migration through Pluripotent Transcription Factor OCT4 in Endometriosis.

Transforming growth factor (TGF-ß)/TGF-ß receptor signal is known to promote cell migration. Up-regulation of TGF-ß in serum/peritoneal fluid and increased levels of pluripotent transcription factor OCT4 in endometriotic tissues are frequently observed in patients with endometriosis. However, the mechanisms underlying how TGF-ß/TGF-ß receptor and OCT4 affect endometriotic cell migration still remain largely unknown. Therefore, endometriotic tissue with high cell migratory capacity were collected from patients with adenomyotic myometrium (n = 23) and chocolate cyst (n = 24); and endometrial tissue with low cell migratory capacity in normal endometrium or hyperplastic endometrium (n = 8) were collected as the controls. We found the mRNA levels of TGF-ß receptor I (TGF-ß RI) and OCT4 were significantly higher in the high-migratory ectopic endometriotic tissues than those of the low-migratory normal or hyperplastic endometrium. Positive correlations between TGF-ß RI and OCT4, and either TGF-ß RI or OCT4 with migration-related genes (SNAIL, SLUG and TWIST) regarding the mRNA levels were observed in human endometriotic tissues. TGF-ßI dose-dependently increased the gene and protein levels of OCT4, SNAIL and N-Cadherin (N-CAD) and silencing of endogenous OCT4 significantly suppressed the TGF-ßI-induced expressions of N-CAD and SNAIL in primary human endometriotic stromal cells and human endometrial carcinoma cell lines RL95-2 and HEC1A. Furthermore, TGF-ßI significantly increased the migration ability of endometriotic cells and silencing of OCT4 dramatically suppressed the TGF-ßI-induced cell migration activity evidenced by wound-closure assay, transwell assay, and confocal image of F-actin cellular distribution. In conclusion, the present findings demonstrate that the niche TGF-ß plays a critical role in initiating expressions of pluripotent transcription factor OCT4 which may contribute to the ectopic endometrial growth by stimulating endometrial cell migration. These findings would be useful for developing therapeutic strategies targeting TGF-ß-OCT4 signaling to prevent endometriosis in the future.

Mucopolysaccharidosis type I: Identification and characterization of mutations affecting alpha-L-iduronidase activity.

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). MPS I covers a broad spectrum of clinical severity ranging from severe Hurler syndrome through intermediate Hurler/Scheie syndrome to mild Scheie syndrome. Mutation screening was performed in two unrelated Taiwanese MPS I patients. A Hurler/Scheie patient had A79V (C to T transition in codon 79) in exon 2 and R619G (C to G transversion in codon 619) in exon 14. R619G has been shown to cause disease. Expression of A79V in COS-7 cells showed trace amounts of IDUA activity, demonstrating the deleterious nature of the mutation. A79V mutation did not cause a reduction in IDUA mRNA levels. The reduced level of IDUA protein suggests increased degradation of the mutant enzyme. A Hurler patient had 134del12 (in-frame deletion of codons 16-19 in signal peptide) in exon 1 and Q584X (C to T transition in codon 584) in exon 13. Transfection of COS-7 cells with Q584X did not yield active enzyme. Q584X mutation caused an apparent reduction in the IDUA mRNA level and no IDUA protein was detected. Conversely, 134del12 showed 124.6% of normal activity in transfected cells and a 77-kDa precursor protein was observed on Western blot, suggesting biologic activity of precursor IDUA without posttranslational cleavage. These findings provide further evidence of the molecular heterogeneity in mutations in MPS I.

Expression of the pluripotent transcription factor OCT4 promotes cell migration in endometriosis.

OBJECTIVE: To identify the impact of the pluripotent transcription factor OCT4 in endometrial cell migration and endometriosis. DESIGN: The OCT4 expression and cell migration study. SETTING: Research institution and reproductive medical clinic. PATIENT(S): Nine subjects with normal endometrium, 3 subjects with normal myometrium, 36 patients with hyperplastic endometrium, and 58 patients with endometriosis. INTERVENTION(S): The expression of OCT4 messenger RNA in normal endometrium, normal myometrium, hyperplastic endometrium, and ectopic endometriotic tissues was analyzed using reverse transcription and quantitative real-time polymerase chain reaction (PCR). The effect of OCT4 expression on the migration activity of the endometrial cells was examined. MAIN OUTCOME MEASURE(S): Reverse transcription and quantitative real-time PCR, Western blotting, and wound closure and transwell assays. RESULT(S): The expression of OCT4 and NANOG messenger RNA was significantly higher in ectopic endometriotic tissues, compared with that of the normal endometrium, the normal myometrium, and the hyperplastic endometrium. The level of OCT4 messenger RNA in endometriotic tissues was positively correlated with the expression of genes associated with cell migration. Overexpression of the OCT4 protein in primary human endometriotic stromal cells and human RL95-2 and HEC1A endometrial carcinoma cell lines resulted in decreased levels of E-CADHERIN, the increased expression of the VIMENTIN, TWIST, and SLUG proteins, and an increase in the migration activity of endometrial cells in transwell and wound closure assays. CONCLUSION(S): The transcription of the OCT4 gene is significantly up-regulated in human ectopic endometriotic tissues. The expression of OCT4 may contribute to the pathology of ectopic endometrial growth by stimulating the migration activity of endometrial cells.

Effects of rice bran oil on the blood lipids profiles and insulin resistance in type 2 diabetes patients.

The aim of this study was to investigate the influence of rice bran oil consumption on plasma lipids and insulin resistance in patients with type 2 diabetes. Thirty-five patients with type 2 diabetes were randomly assigned to a placebo group or a rice bran oil group. The placebo group consumed 250 mL soybean oil-modified milk (18 g soybean oil) daily for 5 weeks, and the rice bran oil group consumed 250 mL rice bran oil modified milk (18 g rice bran oil) daily for 5 weeks. At week 0 and week 5, anthropometric measurements, hematology tests, and an oral-glucose-tolerance test were conducted. The results showed that the homeostasis model assessment index of insulin resistance, the area under the curve for postprandial serum insulin, and serum low-density-lipoprotein cholesterol concentrations increased significantly in the placebo group. In the rice bran oil group, fasting and 2-h postprandial blood glucose concentrations and the area under the curve for postprandial plasma glucose increased significantly; however, total serum cholesterol and low-density-lipoprotein cholesterol concentrations decreased significantly. However, the homeostasis model assessment index of insulin resistance was not significantly different. Consumption of 18 g rice bran oil modified milk daily for 5 weeks significantly decreased total serum cholesterol concentrations and tended to decrease low-density-lipoprotein cholesterol concentrations in patients with type 2 diabetes. However, no significant influence on insulin resistance was observed.

Expression pattern and prognostic significance of claudins 1, 4, and 7 in nasopharyngeal carcinoma.

Claudins are major tight junction proteins that regulate the integrity and function of tight junctions. Aberrant expression of claudins has been shown in various carcinomas with diverse prognostic implications. However, their expression pattern and prognostic value have not been investigated in nasopharyngeal carcinoma. This is the first study to characterize the expression pattern of claudins 1, 4, and 7 in a cohort of 176 patients with nasopharyngeal carcinoma and a minimum follow-up of 9.4 years. Immunohistochemistry with semiquantitative assessment of expression of claudins 1, 4, and 7 was performed on 176 biopsy specimens. Results were correlated with sex, age, extent of tumor, lymph node status, the presence or absence of distant metastasis, and patient survival. High expression of claudins 1 and 4 (72.2% and 88.1%) and low expression of claudin 7 (82.4%) were frequently detected. Low claudin 4 expression (P = .020) and high claudin 7 expression (P < .001) were associated with distant metastasis. Elevated claudin 7 expression also correlated with high tumor stage (P = .001). Furthermore, decreased claudin 4 expression (P = .003) and increased claudin 7 expression (P < .001) independently predicted shorter distant metastasis-free survival by Cox regression analysis. Claudin 4 and claudin 7 may be a novel biomarker for the prediction of distant metastasis and unfavorable prognosis in nasopharyngeal carcinoma.

Polysomnographic characteristics in patients with mucopolysaccharidoses.

To evaluate the prevalence of obstructive sleep apnea (OSA) and to clarify sleep characteristics in patients with mucopolysaccharidoses (MPS), we performed overnight polysomnographic studies in 24 patients (22 males and 2 females; 3 with MPS I, 15 with MPS II, 1 with MPS III, 1 with MPS IV, and 4 with MPS VI; mean age, 10.8 ± 6.0 years; age range, 2.0-23.7 years; 2 patients ≥18 years of age). The nadir arterial oxygen saturation (SaO(2) ) was 74.5 ± 12.3%, and the average percentage of sleep time with an SaO(2) of <95% was 39.4%. The percentages of total sleep time spent in sleep stages N1, N2, N3, and R were 18.6 ± 10.8%, 50.3 ± 7.6%, 14.8 ± 8.1%, and 15.3 ± 4.6%, respectively. The respiratory disturbance index (RDI) was 21.8 ± 20.4/hr, and obstructive apnea-hypopnea index (OAHI) and central apnea index were 21.4 ± 19.9/hr and 0.4 ± 0.6/hr, respectively. The desaturation index was 17.6 ± 17.8/hr. All patients had some degree of OSA. For 22 children, the disorder was mild (OAHI 1.5-5) in 2, moderate (OAHI 5-10) in 7, and severe (OAHI > 10) in 13. Two patients with MPS II who received enzyme replacement therapy had reductions in RDI after treatment (38.9-10.8 and 3.5-2.0, respectively). The prevalence of moderate to severe OSA was 88% (21/24) in patients with MPS. The overnight polysomnography will help to determine the abnormalities of breathing during sleep more precisely and urge the clinicians to take necessary action for patients with severe manifestations.

beta-Carotene and canthaxanthin alter the pro-oxidation and antioxidation balance in rats fed a high-cholesterol and high-fat diet.

This study investigated the effects of beta-carotene and canthaxanthin on lipid peroxidation and antioxidative enzyme activities in rats fed a high-cholesterol, high-fat diet. Wistar rats were divided into six groups. Negative control group (group NC) received a high-fat (150 g/kg) diet; cholesterol control group (group CC) received a high-cholesterol (10 g/kg), high-fat diet. The other four groups were fed a high-cholesterol, high-fat diet supplemented with crystal beta-carotene (group BC), beta-carotene beadlet (group BB), canthaxanthin beadlet (group CX) or alpha-tocopherol (group AT). Blood and livers were collected for analysis after 6 weeks of feeding. Group BB had significantly lower hepatic thiobarbituric acid reactive substance (TBARS) and conjugated diene concentrations, whereas group CX had a significantly lower plasma TBARS concentration than did group CC. In erythrocytes, glutathione peroxidase activities were significantly greater in groups BC, BB and CX than in group CC. Moreover, compared with group CC, catalase activities were significantly greater in groups BB and CX, and superoxide dismutase (SOD) activity was significantly greater in group BB. In livers, SOD activities were significantly greater in groups BC, BB and CX, and glutathione reductase activities were significantly greater in groups BB and CX than in group CC. Compared with group CC, hepatic retinol and alpha-tocopherol concentrations were significantly greater in groups BC, BB and CX, whereas plasma and hepatic cholesterol concentrations were significantly lower in group BC. These findings suggest that beta-carotene and canthaxanthin altered the pro-oxidation and antioxidation balance and suppressed cholesterol-induced oxidative stress via modulation of antioxidant system and cholesterol metabolism.

Mutation identification and characterization of a Taiwanese patient with fucosidosis.

Fucosidosis is a rare lysosomal storage disease caused by a defect of the alpha-L: -fucosidase (FUCA1) gene. Worldwide 26 mutations underlying the disease have been reported. By direct DNA sequencing of exons and flanking introns, homozygous Y126X mutation and Q281R polymorphism were found in a Taiwanese patient with fucosidosis. Upon expressing in COS-7 cells, 97.4% of alpha-L: -fucosidase activity compared with that of the wild-type construct was observed in the cDNA containing Q281R polymorphism. Western blot analysis revealed a 58-kDa precursor and 56-kDa mature forms for cells transfected with wild-type and Q281R enzymes. Using the fluorogenic substrate, the Michaelis constants and maximal velocities of both enzymes were very similar. While no appreciable enzyme activity (0.0%) was observed with Y126X mutation, no apparent decrease in FUCA1 mRNA level was seen with Y126X mutation. The expressed truncated Y126X protein was unstable and largely degraded. The delineation of the molecular defect could serve to complement future prenatal diagnosis for this family when necessary.

Expression studies of mutations underlying Taiwanese Hunter syndrome (mucopolysaccharidosis type II).

Nearly 300 different mutations underlying mucopolysaccharidosis type II (MPS II) have been identified worldwide. To investigate the molecular lesions underlying Taiwanese MPS II, probands and families were identified and screened for iduronate-2-sulfatase (IDS) mutation by single-strand conformation polymorphism and DNA sequencing. Five novel and five previously reported mutations were found. Together with those previously reported, a total of 17 identified missense, small deletion, and nonsense mutations were further characterized by transient expression studies. Transfection of COS-7 cells by the mutated cDNA did not yield active enzyme, demonstrating the deleterious nature of the mutations. A 57% decrease in IDS mRNA level was seen with the 231del6 mutation. Among the 11 missense mutations examined, K347E substitution showed apparent normal maturation and targeting on immunoblot and confocal fluorescence microscopy examination. The other 10 missense mutations showed apparent normal precursor with little or reduced mature forms, indicating normal maturation but incorrect targeting of the mutant enzymes. Among the six deletion and nonsense mutations examined, 1055del12 and E521X showed abnormal maturation. The staining pattern of the truncated W267X and 1184delG proteins suggested retention within early vacuolar compartments. The mutated 231del6 and 1421delAG proteins were unstable and largely degraded. Molecular analysis of the IDS gene will clearly identify the cause of the disease within patients and allow antenatal and family studies. The further characterization of gene mutations may delineate their functional consequences on IDS activity and processing and may enable future studies of genotype-phenotype correlation to estimate a prognosis and to lead to possible therapeutic interventions.

Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese.

DNA screening for LDL receptor mutations was performed in 170 unrelated hyperlipidemic Chinese patients and two clinically diagnosed familial hypercholesterolemia patients. Two deletions (Del e3-5 and Del e6-8), eight point mutations (W-18X, D69N, R94H, E207K, C308Y, I402T, A410T, and A696G), and two polymorphisms (A370T and I602V) were identified. Of these mutations, C308Y and Del e6-8 were found in homozygosity, and D69N and C308Y were seen in unrelated patients. The effects of mutations on LDL receptor function were characterized in COS-7 cells. The LDL receptor level and activity were close to those of wild type in A696G transfected cells. A novel intermediate protein and reduction of LDL receptor activity were seen in D69N transfected cells. For R94H, E207K, C308Y, I402T, and A410T mutations, only approximately 20-64% of normal receptor activities were seen. Conversely, Del e3-5 and Del e6-8 lead to defective proteins with approximately 0-13% activity. Most of the mutant receptors were localized intracellularly, with a staining pattern resembling that of the endoplasmic reticulum and Golgi apparatus (D69N, R94H, E207K, C308Y, and I402T) or endosome/lysosome (A410T and Del e6-8). Molecular analysis of the LDL receptor gene will clearly identify the cause of the patient's hyperlipidemia and allow appropriate early treatment as well as antenatal and family studies.