Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation.

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.

Epithelial-specific isoforms of protein 4.1R promote adherens junction assembly in maturing epithelia.

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states. The membrane-cytoskeleton interface plays a crucial role in junctional reorganization. Protein 4.1R (4.1R), expressed as a diverse array of spliceoforms, has been implicated in linking the AJ and TJ complex to the cytoskeleton. However, which specific 4.1 isoform(s) participate and the mechanisms involved in junctional stability or remodeling remain unclear. We now describe a role for epithelial-specific isoforms containing exon 17b and excluding exon 16 4.1R (4.1R+17b) in AJs. 4.1R+17b is exclusively co-localized with the AJs. 4.1R+17b binds to the armadillo repeats 1-2 of ß-catenin via its membrane-binding domain. This complex is linked to the actin cytoskeleton via a bispecific interaction with an exon 17b-encoded peptide. Exon 17b peptides also promote fodrin-actin complex formation. Expression of 4.1R+17b forms does not disrupt the junctional cytoskeleton and AJs during the steady-state or calcium-dependent AJ reassembly. Overexpression of 4.1R-17b forms, which displace the endogenous 4.1R+17b forms at the AJs, as well as depletion of the 4.1R+17b forms both decrease junctional actin and attenuate the recruitment of spectrin to the AJs and also reduce E-cadherin during the initial junctional formation of the AJ reassembly process. Expressing 4.1R+17b forms in depleted cells rescues junctional localization of actin, spectrin, and E-cadherin assembly at the AJs. Together, our results identify a critical role for 4.1R+17b forms in AJ assembly and offer additional insights into the spectrin-actin-4.1R-based membrane skeleton as an emerging regulator of epithelial integrity and remodeling.

Protein 4.1R Influences Myogenin Protein Stability and Skeletal Muscle Differentiation.

Protein 4.1R (4.1R) isoforms are expressed in both cardiac and skeletal muscle. 4.1R is a component of the contractile apparatus. It is also associated with dystrophin at the sarcolemma in skeletal myofibers. However, the expression and function of 4.1R during myogenesis have not been characterized. We now report that 4.1R expression increases during C2C12 myoblast differentiation into myotubes. Depletion of 4.1R impairs skeletal muscle differentiation and is accompanied by a decrease in the levels of myosin heavy and light chains and caveolin-3. Furthermore, the expression of myogenin at the protein, but not mRNA, level is drastically decreased in 4.1R knockdown myocytes. Similar results were obtained using MyoD-induced differentiation of 4.1R-/- mouse embryonic fibroblast cells. von Hippel-Lindau (VHL) protein is known to destabilize myogenin via the ubiquitin-proteasome pathway. We show that 4.1R associates with VHL and, when overexpressed, reverses myogenin ubiquitination and stability. This suggests that 4.1R may influence myogenesis by preventing VHL-mediated myogenin degradation. Together, our results define a novel biological function for 4.1R in muscle differentiation and provide a molecular mechanism by which 4.1R promotes myogenic differentiation.

Serum retinol-binding protein 4 is independently associated with pediatric NAFLD and fasting triglyceride level.

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) is identified as a major liver disease in children. The present study aimed to identify the prevalence and predictors of pediatric NAFLD and the correlation between serum retinol-binding protein 4 (RBP4) levels and metabolic characteristics in children. METHODS: A total of 748 schoolchildren, ages 6 to 12 years, were enrolled in 2009. The body weight and height were measured in the morning before intake. Laboratory tests included overnight fasting serum lipids, insulin, liver enzymes, and RBP4 levels. Hepatic steatosis was determined by ultrasound in 219 volunteers. RESULTS: The rates of NAFLD were 3% in the normal-weight, 25% in the overweight, and 76% in the obese children. Twenty (22%) of obese children had abnormal alanine aminotransferase (ALT) levels. In children with NAFLD, younger age and higher body mass index (BMI), insulin/homeostasis model of assessment, and male sex rate were associated with abnormal liver function. Stepwise increments in BMI, insulin, homeostasis model of assessment, and ALT were found in children with normal livers to simple steatosis, and to steatosis with abnormal ALT. Multiple logistic regression analysis confirmed that serum RBP4 levels (P = 0.048), ALT (P = 0.048), and BMI (P < 0.001) were independently predictors of pediatric NAFLD. Moreover, multiple linear regression analysis revealed that only serum triglycerides levels were positively related to RBP4 levels (P < 0.001). CONCLUSIONS: Higher RBP4 and ALT levels as well as BMI are independently associated with pediatric NAFLD in Taiwan. In addition, an increment in RBP4 levels was positively correlated to hypertriglyceridemia in children.

Multidrug-resistant Salmonella enterica serovar Panama carrying class 1 integrons is invasive in Taiwanese children.

BACKGROUND/PURPOSE: An increase in group D Salmonella isolates with high antimicrobial resistant rates is being seen in Taiwan. This study aimed to determine the multidrug-resistant (MDR, more than three antibiotics) phenotype, genotype, and the correlation between the presence of class 1 integrons and its invasiveness of Salmonella panama and Salmonella enteritidis isolated from children. METHODS: Twenty S. panama and 59 S. enteritidis isolates were examined for minimal inhibitory concentrations of ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline by agar dilution method. The presence of blaPSE-1, floR, aadA2, sul1, and tet(G) resistance genes, class 1 integrons, and Salmonella genomic island 1 (SGI1) was identified by polymerase chain reaction. The adhesion and invasion assays of S. panama to Caco-2 cells were determined using the pour plate method. RESULTS: All S. panama and 15 (25.4%) of the S. enteritidis isolates displayed MDR phenotype. Furthermore, MDR genotype was present in 70.0% of S. panama and 6.8% of S. enteritidis. Class 1 integrons were present in 40.0% of S. panama and 11.9% of S. enteritidis. None contained SGI1 or SGI1 variants. Strains carrying class 1 integrons were more frequently isolated from bacteria with MDR (73.3% vs. 37.5%; odds ratio, 4.6; 95% confidence interval, 1.3-16.0; p=0.01) and isolated from blood and cerebrospinal fluid (46.7% vs. 21.9%; odds ratio, 3.1; 95% confidence interval, 1.0-10.1; p=0.05) than noncarriers. S. panama carrying class 1 integrons were more invasive to Caco-2 cells than those without (p=0.01). CONCLUSION: S. panama and S. enteritidis with class 1 integrons are significantly related to the presence of MDR phenotype. Moreover, S. panama with class 1 integrons may present more invasiveness than those without.

Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure.

BACKGROUND: Human heart failure is associated with decreased cardiac voltage-gated Na+ channel current (encoded by SCN5A), and the changes have been implicated in the increased risk of sudden death in heart failure. Nevertheless, the mechanism of SCN5A downregulation is unclear. A number of human diseases are associated with alternative mRNA splicing, which has received comparatively little attention in the study of cardiac disease. Splicing factor expression profiles during human heart failure and a specific splicing pathway for SCN5A regulation were explored in this study. METHODS AND RESULTS: Gene array comparisons between normal human and heart failure tissues demonstrated that 17 splicing factors, associated with all major spliceosome components, were upregulated. Two of these splicing factors, RBM25 and LUC7L3, were elevated in human heart failure tissue and mediated truncation of SCN5A mRNA in both Jurkat cells and human embryonic stem cell-derived cardiomyocytes. RBM25/LUC7L3-mediated abnormal SCN5A mRNA splicing reduced Na+ channel current 91.1±9.3% to a range known to cause sudden death. Overexpression of either splicing factor resulted in an increase in truncated mRNA and a concomitant decrease in the full-length SCN5A transcript. CONCLUSIONS: Of the 17 mRNA splicing factors upregulated in heart failure, RBM25 and LUC7L3 were sufficient to explain the increase in truncated forms and the reduction in full-length Na+ channel transcript. Because the reduction in channels was in the range known to be associated with sudden death, interruption of this abnormal mRNA processing may reduce arrhythmic risk in heart failure.

Hypoalbuminaemia is an independent predictor for hemophagocytic lymphohistiocytosis in childhood Epstein-Barr virus-associated infectious mononucleosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal condition in children with Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). This study aimed to identify commonly available clinical and laboratory predictors that might help clinicians decide to perform the bone marrow and immunological tests for HLH in paediatric EBV-associated IM. METHODS: A retrospective case-control study of patients aged <18 yr diagnosed with EBV-associated IM and HLH from 1991 to 2010 in a tertiary medical centre was conducted. A diagnosis of HLH was defined as fulfilling the criteria of the guidelines of the HLH-2004 protocol of the Histiocyte Society and consisted of at least evidence of hemophagocytosis in a bone marrow biopsy. RESULTS: A total of 177 IM and 27 HLH patients were enrolled. The mean age was 5.3 yr with a female-to-male ratio of 1.06. The most common characteristics (>70% of patients) were fever, lymphadenopathy and hepatomegaly. In addition to the diagnostic criteria of HLH including fever, splenomegaly, cytopenia, hyperferritinaemia, hypertriglyceridemia and/or hypofibrinogenaemia, children with HLH had a significantly higher rate of prolonged fever >10 d, hepatomegaly, jaundice, general malaise, elevated aspartate aminotransferase, lactate dehydrogenase, C-reactive protein and hypoalbuminaemia compared to those with IM (all P < 0.01). Multiple logistic regression confirmed that hypoalbuminaemia (OR = 23.1, P = 0.01) was an independent predictor of paediatric HLH, with a high sensitivity (96%) and a good negative likelihood ratio (0.06) in patients with EBV-associated IM. CONCLUSIONS: Hypoalbuminaemia is a unique characteristic and potentially a valuable predictor for HLH in paediatric EBV-associated IM.

Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5' exons of protein 4.1R gene.

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3' splice sites (ss) of exon 2'/2. Here, we demonstrate that exon 1 and 2'/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3' ss and exon 1B to the proximal 3' ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter-driven splicing patterns but differed from 1A-promoter-driven splicing patterns, suggesting that promoter identity affects exon 2'/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2'/2 junction CAGAGAA, a sequence that overlaps the distal U2AF(35)-binding 3' ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3' ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3' ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.

Septic shock and hypofibrinogenemia predict a fatal outcome in childhood acute acalculous cholecystitis.

OBJECTIVES: The aim of the study was to investigate the etiology, clinical presentation, and risk factors for poor prognosis of acute acalculous cholecystitis (AAC) in children. PATIENTS AND METHODS: Children younger than 18 years diagnosed as having AAC were analyzed retrospectively from 2000 to 2009. The demographic and clinical characteristics, etiology, and outcomes were recorded. AAC was defined as a gallbladder wall thickness of >3.5 mm in sonogram with a duration of symptoms <1 month. The severity of sonographic findings was scored, with 1 point each given for wall thickness >3.5 mm, gallbladder distention, sludge, and pericholecystic fluid. RESULTS: A total of 109 children (boys:girls 1:2, median age 4.9 years) were diagnosed. The most common clinical presentation was fever (88%), followed by hepatomegaly (72%). The rates of elevated alanine aminotransferase and thrombocytopenia were 72% and 65%, respectively. The most common causative etiology was infectious diseases (74%). All of the patients were treated nonoperatively. Sixteen (15%) patients died. Children with mortality had a significantly higher rate of septic shock (P < 0.001), anemia (P = 0.01), thrombocytopenia (P = 0.04), hypofibrinogenemia (P = 0.002), the presence of pericholecystic fluid (P = 0.04), and higher sonographic scores (P = 0.04) than those with survival. Multiple logistic regression analysis confirmed that the presence of septic shock (P = 0.004) and hypofibrinogenemia (P = 0.014) were independent risk factors that predict mortality. CONCLUSIONS: Childhood AAC is usually secondary to a variety of etiologies, especially during the course of infectious diseases. The presence of septic shock and a low value of fibrinogen determine a fatal outcome in childhood AAC.

The etiology and treatment outcome of iron deficiency and iron deficiency anemia in children.

SUMMARY: This study aimed to evaluate the frequency of the diverse causes of iron deficiency (ID) and iron deficiency anemia (IDA) and to investigate the treatment outcomes in children. ID was defined as a serum ferritin level<12 microg/L and a transferrin saturation<10%. IDA was established as ID combined with a low hemoglobin level judged by age and gender-specific reference intervals. A total of 116 ID patients were categorized into 4 groups: group I:<2 years old (n=45), group II: 2 to 10 years old (n=13), group III:>10 years old, male (n=18), and group IV: >10 years old, female (n=40). One hundred of them (86.2%) were diagnosed with IDA. The most common causes of ID were inadequate intake in group I (55.6%) and blood loss in groups II (46.1%) and IV (37.5%). Helicobacter pylori-associated ID mainly occurred in children more than 10 years old. Forty-five of 57 (78.9%) IDA patients who had underlying diseases treatment and/or iron supplementation for 3 months recovered their hemoglobin levels (follow-up range: 6-27 mo). In conclusion, the peak incidences of childhood ID were ages under 2 years old and 10-18 years old. Different age groups and sexes showed characteristic etiologies. The outcomes of childhood ID were good.

Etiology and treatment of childhood peptic ulcer disease in Taiwan: a single center 9-year experience.

BACKGROUND/PURPOSE: Peptic ulcer disease (PUD) in children is relatively rare as compared with adults. This study aimed to assess the etiology, clinical and histological characteristics, and treatment of PUD in children. METHODS: All children aged < 18 years with an endoscopic diagnosis of PUD were enrolled in a tertiary referral center. The demographic data, clinical, endoscopic, and histological findings were compared between patients with different causes of PUD. RESULTS: From 1234 endoscopic examinations, 67 (5.4%) children (median age, 11.4 years) with gastric ulcer (GU; n=27) or duodenal ulcer (DU; n=40) were included. Thirty-two (47.7%) of them had Helicobacter pylori infection and 11 (16.5%) had previous use of non-steroidal anti-inflammatory drugs (NSAIDs). Non-H. pylori, non-NSAID PUD was found in 24 (35.8%) patients. Children with H. pylori-related PUD had a significantly higher mean age, antral chronic inflammatory score, rate of familial PUD, and presence of DU and nodular gastritis than those with NSAID-related and non-H. pylori, non-NSAID PUD (p < 0.01). In contrast, children with NSAID-related PUD had a higher rate of upper gastrointestinal bleeding, associated with acute febrile disease, than those with H. pylori-related and non-H. pylori, non-NSAID PUD (p < 0.05). All but two patients with non-H. pylori, non-NSAID PUD were disease free after H. pylori eradication and proton pump inhibitor treatment for 1-2 months. CONCLUSION: In children, H. pylori-related PUD is associated with familial peptic ulcer and the presence of DU. However, short-term NSAID use is correlated highly with GU. The outcome of childhood PUD is good.

Decreased leukocytes and other characteristics of laboratory findings of influenza virus infections in children.

BACKGROUND AND PURPOSE: An outbreak of influenza A and influenza B appeared at the beginning and end of 2006 in southern Taiwan. We conducted this study to test whether laboratory findings could differentiate influenza A from influenza B infection. METHODS: All children aged 16 years or less, who had nasal and/or throat swabs sent from inpatient or outpatient settings at National Cheng Kung University Hospital for the diagnosis of influenza infection from January 2005 to February 2007, were considered eligible subjects. Retrospective chart review of clinical and laboratory data was performed. RESULTS: 274 patients were enrolled, 151 with influenza A and 123 with influenza B, of whom 127 (46.4%) received laboratory examinations. The peak month of influenza A and influenza B infections was January 2006 and January 2007, respectively. Children with influenza B infections were older than those with influenza A infections (p<0.001). Influenza B-infected patients were more likely to have myalgia (p=0.004) than those with influenza A infections. Furthermore, children with influenza B infections tended to have lower leukocyte counts (6383 +/- 3970/mm3 vs 7639 +/- 3476/mm3, p=0.004), and higher serum creatine kinase level (p=0.002) than those with influenza A infections. The clinical outcomes were usually favorable. CONCLUSIONS: The clinical features of influenza B and influenza A infections are similar. However, decreased leukocytes and increased serum creatine kinase can be used as adjunctive criteria for diagnosis of influenza B infection before viral culture results are available.

Mitotic regulation of protein 4.1R involves phosphorylation by cdc2 kinase.

The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2 kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135 isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135 is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.

Fiberoptic bronchoscopic view change of laryngopharyngeal tissues by different airway supporting techniques: comparison of patients with and without open mouth limitation.

STUDY OBJECTIVE: To investigate the changes in view of the laryngopharyngeal tissues on the fiberoptic bronchoscope (FOB) with different techniques of supporting the airway. DESIGN: Prospective, observational, stratified study. SETTING: University hospital. PATIENTS: 40 ASA physical status I, II, and III men undergoing elective oromaxillofacial surgery during general anesthesia with nasotracheal intubation. INTERVENTIONS: Patients were allocated into the normal mouth-opening group (Group N) or the limited mouth-opening group (Group L) to determine the grade of view of the laryngopharyngeal tissues exposed on the FOB, with 5 different airway supporting techniques: original airway position (OA), triple airway (TA), jaw thrust with opened mouth (JTO), jaw thrust with teeth protrusion (JTP), and head tilt with chin lift (HT). MEASUREMENT AND MAIN RESULTS: An adequate airway support was defined as having nearly full visibility of the entire glottic inlet. The investigator graded the vision of both anterior and posterior laryngopharyngeal tissues of each patient. All subjects experienced adequate airway support with the TA and HT airway supporting techniques. The TA airway supporting technique significantly moved the posterior laryngeal tissues more upward in Group N than Group L (P = 0.027). The JTP airway supporting technique provided adequate airway support for 14 of the 20 patients in Group N but only for two of the 20 Group L patients (P < .001). CONCLUSION: Both the TA and HT techniques provided adequate airway support for patients with and without limited mouth opening.

Protein 4.1R Exon 16 3' Splice Site Activation Requires Coordination among TIA1, Pcbp1, and RBM39 during Terminal Erythropoiesis.

Exon 16 of protein 4.1R encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability. Its expression during erythroid differentiation is regulated by alternative pre-mRNA splicing. A UUUUCCCCCC motif situated between the branch point and the 3' splice site is crucial for inclusion. We show that the UUUU region and the last three C residues in this motif are necessary for the binding of splicing factors TIA1 and Pcbp1 and that these proteins appear to act in a collaborative manner to enhance exon 16 inclusion. This element also activates an internal exon when placed in a corresponding intronic position in a heterologous reporter. The impact of these two factors is further enhanced by high levels of RBM39, whose expression rises during erythroid differentiation as exon 16 inclusion increases. TIA1 and Pcbp1 associate in a complex containing RBM39, which interacts with U2AF65 and SF3b155 and promotes U2 snRNP recruitment to the branch point. Our results provide a mechanism for exon 16 3' splice site activation in which a coordinated effort among TIA1, Pcbp1, and RBM39 stabilizes or increases U2 snRNP recruitment, enhances spliceosome A complex formation, and facilitates exon definition through RBM39-mediated splicing regulation.

In vitro degradation and angiogenesis of the porous calcium silicate-gelatin composite scaffold.

Calcium silicate-based materials have attracted a great deal of interest due to their osteogenesis and have been used as implant materials for bone repair and regeneration. The purpose of this study was to use gelatin with and without genipin cross-linking for controlling degradation, improving mechanical properties, and enhancing angiogenesis of calcium silicate bioceramics. The in vitro degradation of gelatin-containing scaffolds was analysed in a simulated body fluid (SBF) solution. Human mesenchymal stem cells (hMSCs) were used to examine angiogenesis. The results indicated that the gelatin-containing scaffolds showed a diametral tensile strength of about 2 MPa and a porosity of about 60% falling within the range of values reported for the cancellous bone. Apatite precipitation occurred on all scaffold surfaces after soaking in SBF for 1 week. The gelatin-containing scaffold without cross-linking exhibited a greater weight loss and porosity than the control without gelatin. The cross-linking agent, genipin, significantly improved the mechanical stability of the composite scaffold. The gelatin enhanced the viable cell populations. More importantly, gelatin actively promoted the secretion of angiogenic factors such as von Willebrand factor and angiopoietin-1 in hMSCs. It is concluded that combination of calcium silicate and gelatin may synergistically enhance clinically desirable functions in terms of controlled degradation, improved mechanical properties, and enhanced angiogenesis.

Stem cell differentiation-induced calcium silicate cement with bacteriostatic activity.

Calcium-based bone cements are widely used in dental and orthopaedic surgery. Those based on calcium phosphate (CPCs) or calcium silicate (CSCs) have a number of favourable properties that encourage their clinical use in bone defect repair. The purpose of the present study was to compare the in vitro osteogenesis and bacteriostatic activity of BoneSource CPCs with home-made CSCs, particularly in regard to their facility for cell differentiation. Cement in vitro osteogenic activity was evaluated by incubating the cement specimens with human mesenchymal stem cells (hMSCs). The bacteriostatic activity of the two cements against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacterial strains was assessed using a bacteriostasis ratio assay and by inhibition zone examination. Compared with CPC, CSC was shown to promote greater proliferation and osteogenic differentiation (alkaline phosphatase and osteocalcin), and the formation of mineralization nodules of hMSCs. It is worth noting that CSC could effectively induce hMSC differentiation, even when the culture medium did not contain osteogenetic differentiation agents. Compared with CPC, CSC also showed significantly greater bacteriostatic activity, as revealed by inhibition zones and the bacteriostasis ratio. Our findings suggest that CSC is a useful bioactive material for bone repair in terms of inducing cell differentiation, and may be considered an alternative to CPCs.

Role of the P38 pathway in calcium silicate cement-induced cell viability and angiogenesis-related proteins of human dental pulp cell in vitro.

INTRODUCTION: This study investigated that calcium silicate (CS) cement may influence the behavior of human dental pulp cells (hDPCs) via mitogen-activated protein kinase pathway, in particular p38. We have addressed that Si ion released from CS cement can influence osmolarity in the medium, which may stimulate hDPC viability and induce angiogenesis-related proteins through stimulation of the nitric oxide synthase and nitric oxide secretion. METHODS: The hDPCs was cultured with CS cement to angiogenesis. Then, cell viability, ion concentration, osmolality, nitric oxide secretion, the von Willebrand factor, and angiopoietin-1 protein expression were examined. RESULTS: CS cement elicited a significant (P < .05) increase of 15%, 20%, and 19% in viability compared with control on days 1, 3, and 5 of cell seeding, respectively. The CS cement consumed calcium and phosphate ions and released more Si ions in medium. The CS significantly (P < .05) increased the osmolality to 303.52 ± 3.07, 315.03 ± 5.80, and 319.95 ± 4.68 mOsm/kg for 1, 3, and 5 days, respectively. P38 was activated through phosphorylation; the phosphorylation kinase was investigated in our cell system after culture with CS cement. Moreover, expression levels for angiopoietin-1 and von Willebrand factor in hDPCs on CS cement were higher than those of the CS + p38 inhibitor (SB203580) group (P < .05) at all of the analyzed time points. CONCLUSIONS: This study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signaling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on CS cement.

CD44 gene polymorphisms and environmental factors on oral cancer susceptibility in Taiwan.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the fourth leading cause of male cancer death in Taiwan. Exposure to environmental carcinogens is the primary risk factor for developing OSCC. CD44, a well-known tumor marker, plays a crucial role in tumor cell differentiation, invasion, and metastasis. This study investigated CD44 single-nucleotide polymorphisms (SNPs) with environmental risk factors to determine OSCC susceptibility and clinicopathological characteristics. METHODOLOGY/PRINCIPAL FINDINGS: Real-time polymerase chain reaction (PCR) was used to analyze 6 SNPs of CD44 in 599 patients with oral cancer and 561 cancer-free controls. We determined that the CD44 rs187115 polymorphism carriers with the genotype AG, GG, or AG+GG were associated with oral cancer susceptibility. Among 731 smokers, CD44 polymorphisms carriers with the betel-nut chewing habit had a 10.30-37.63-fold greater risk of having oral cancer compared to CD44 wild-type (WT) carriers without the betel-nut chewing habit. Among 552 betel-nut chewers, CD44 polymorphisms carriers who smoked had a 4.23-16.11-fold greater risk of having oral cancer compared to those who carried the WT but did not smoke. Finally, we also observed that the stage III and IV oral cancer patients had higher frequencies of CD44 rs187115 polymorphisms with the variant genotype (AG+GG) compared with the wild-type (WT) carriers. CONCLUSION: Our results suggest that gene-environment interactions between the CD44 polymorphisms and betel quid chewing and tobacco smoking increase the susceptibility to oral cancer development. Patients with CD44 rs187115 variant genotypes (AG+GG) were correlated with a higher risk of oral cancer development, and these patients may possess greater chemoresistance to advanced- to late-stage oral cancer than WT carriers do. The CD44 rs187115 polymorphism has potential predictive significance in oral carcinogenesis and also may be applied as factors to predict the clinical stage in OSCC patients.

Aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of pentose phosphate pathway.

Obesity has become a major public health problem of global significance. Today, aspirin remains the most commonly used medication for the treatment of pyrexia, pain, inflammation and antiplatelet. The present study aims at evaluating the possible existence of a putative p53-dependent pathway underlying the aspirin-induced inhibition of adipogenesis. Cell migration assay was identified by the ability to migrate through Transwell insert. Oil Red O staining was employed to quantify adipose accumulation. The concentration of glucose and triglyceride were measured by using assay kits. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Aspirin significantly inhibited preadipocyte migration and adipose accumulation. The p53-p21 signaling and the expression of differentiation marker glycerol-3-phosphate dehydrogenase were increased in a dose-dependent manner. It indicated that aspirin induced adipocyte differentiation through p53-p21 pathway. The oncogenic ERK 1/2 MAPK signaling was induced, whereas, the expression of adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (A-FABP) and inflammatory factors cyclooxygenase-2 (Cox-2), tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were inhibited. Aspirin negatively regulated the pentose phosphate pathway (PPP) by inhibiting the expression of rate-limiting enzyme glucose-6-phosphate dehydrogenase. Knockdown the expression of oncogenic ERK 1/2 MAPK by using 10 µM PD98059 significantly increased triglyceride synthesis, adipose accumulation and activated PPP, however, decreased glucose uptake. Diverted the glucose flux to PPP, rather than increased glucose uptake, was associated with adipogenesis. Down-regulated the expression of tumor suppressor p53 by 10 µM pifithrin-α (PFTα) alone had no effect on adipose accumulation. However, administration of aspirin accompanied with PFTα abolished aspirin-induced inhibition of adipogenesis. We demonstrated that aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of PPP. Blockade PPP may be a novel strategy for obesity prevention and therapy. Moreover, when use aspirin in therapeutic strategy, the p53 status should be considered.