Crucial role of the NSE1 RING domain in Smc5/6 stability and FANCM-independent fork progression.

The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.

Adaptation, reliability and validity of health-related quality of life questionnaires: Disabkids chronic and specific diabetes disease in children and adolescents with diabetes mellitus type 1.

INTRODUCTION: It is recommended to periodically evaluate the health-related quality of life (HRQoL) in children and adolescents with type 1 diabetes mellitus (DM1). Despite this, no specific paediatric HRQoL instrument for DM1 has been validated in Spanish. OBJECTIVES: Multicentre, prospective descriptive study in children and adolescents with DM1 with the aim of carrying out cross-cultural adaptation to Spanish and evaluating the reliability and validity of the DISABKIDS chronic disease and diabetes-specific HRQoL questionnaires, using reverse translation. MATERIAL AND METHODS: Sociodemographic variables were compiled together with the most recent capillary glycated haemoglobin (HbA1c) value and HRQoL questionnaires were handed out to 200 Spanish children and adolescents with DM1 aged between 8 and 18 years of age under evaluation in 12 different hospitals. RESULTS: The mean score on the HRQoL questionnaire (patient version) for chronic disease was 80.32 (13.66), being significantly lower (P = .04) in patients with a shorter duration of the disease (≤5 years): 78.34 (13.70) vs. 82.60 (13.36). The mean score of the DM1-specific modules was: 60.81 (16.23) for disease impact and 65.59 (26.19) for treatment impact. The mean HbA1c value was 7.08 (0.79), with no differences (P > .05) noted in the mean score of the HRQoL instruments in patients with HbA1c ≤7% vs. HbA1c >7%. The Cronbach α value varied between 0.72 and 0.90. CONCLUSIONS: The Spanish versions of the DISABKIDS HRQoL instruments meet the proposed objectives of semantic equivalence and internal consistency, making it possible to periodically assess HRQoL in these patients. The good average glycaemic control presented by the patients may explain why no difference was found in the HRQoL instruments based on the HbA1c value.

Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas.

Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets.

Antitumor Effects of Ral-GTPases Downregulation in Glioblastoma.

Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.

Subclinical hypothyroidism in childhood, treatment or only follow-up?

BACKGROUND: Subclinical hypothyroidism (SH) is defined as serum levels of thyroid-stimulating hormone (TSH) above the upper limit with normal concentrations of free T4 (fT4). Its management remains challenging. The aim of the study was to evaluate clinical and laboratory findings as well as the clinical course of children with SH followed in a third level hospital. Sixty-five patients aged between 2 and 18 years old were retrospectively studied. METHODS: The patients were followed for a median period of 9 months (range 6 months to 24 months). Those who normalized TSH levels were discharged (Group 1). If TSH persisted mildly elevated (5-10µUI/mL) with normal fT4 and negative TPOAb/TgAb, they were classified as Group 2 and followed semi-annually without treatment. Those patients whose TSH raised ≥10µUI/mL or who maintained TSH 5-10µUI/mL and positive TPOAb/TgAb were considered suitable for thyroxin therapy (Group 3, G3). RESULTS: In 89% of our patients, TSH concentrations spontaneously reverted to normality or remained stable without treatment (Groups 1 and 2), whereas less than 11% progressed to clinical hypothyroidism (Group 3). Baseline TSH was significantly lower in group 1 than in group 3. In group 3 the prevalence of female sex (71%) was higher and TPO antibodies were present in 85% of patients. The risk of developing overt hypothyroidism in patients with positive anti-thyroid antibodies respect to those who normalized TSH was 45 (95%CI 6.5-312.5). CONCLUSION: Baseline TSH, female sex and the presence of thyroid autoimmunity were the best predictors of the evolution to SH over time.

Evaluation of Tumor Interstitial Fluid-Extraction Methods for Proteome Analysis: Comparison of Biopsy Elution versus Centrifugation.

The analysis of tumor interstitial fluid (TIF) composition is a valuable procedure to identify antimetastatic targets, and different laboratories have set up techniques for TIF isolation and proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force (10 000g) was required to obtain TIF from cSCC by centrifugation. Second, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77-80%) than among samples (50%). These results suggest that there exists an elevated consistence of TIF composition independently of the method used. However, we observed a 3-fold increase of extracellular proteins in nonoverlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies.

Impact of design and length on the accuracy of closed tray transfer copings.

BACKGROUND: The aim of this study was to evaluate the accuracy of two closed-tray transfer copings for implant impressions (a new design vs. an old design) in two different lengths (short and long). MATERIAL AND METHODS: Four groups of transfer copings (NS - new short, NL - new long, OS - old short and OL - old long) were tested. An epoxy resin model was prepared of missing teeth 1.4, 1.5 and 1.6. Two Alpha-Bio analogues were placed in position of teeth 1.4 and 1.6, at a 10o angulation. Two calibrated operators took 10 closed-tray impressions for each group with polyether in a Rim-Lock impression tray. RESULTS: After measuring and comparing impressions, a significant difference was found between the two new transfer copings and the old short transfer coping. CONCLUSIONS: The new transfer coping design significantly improved impression accuracy. An adequate transfer coping design for the closed-tray impression technique can help to achieve clinically acceptable impressions for two-unit implant supported bridges. Key words:Closed tray, impression coping, transfer coping, implant impression.

Cytoplasmic cyclin D1 regulates glioblastoma dissemination.

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4-retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development of Periapical Lesions in Endodontically Treated Teeth with and without Periodontal Involvement: A Retrospective Cohort Study.

INTRODUCTION: The purpose of this study was to investigate the incidence of apical periodontitis (AP) in endodontically treated teeth with and without periodontal involvement. METHODS: The records of 602 patients with 775 root canal-treated teeth were initially examined. Only teeth with adequate root canal filling, adequate coronal restoration, and no AP (periapical index = 1) were selected for further investigation. A total of 194 teeth were included in this cohort study. Age, sex, history of diabetes mellitus, smoking, hypertension, and immunodeficiency disorders were recorded. Two groups were made according to the periodontal status of the patients. The control group included periodontally healthy patients and the periodontal group patients with periodontal disease receiving nonsurgical periodontal treatment. After an observation period of at least 2 years, the incidence of AP was scored using the periapical index. The relationship between patients' variables and AP was conducted using the Cohen kappa test, the chi-square test, odds ratio (OR), and logistic regression analysis. RESULTS: Newly emerged AP was found in 14% of periodontally involved teeth and in 3% of nonperiodontal involved teeth (P < .05, OR = 5.19, 95% confidence interval). The periodontal condition and hypertension were the only significant factors associated with the presence of AP in the follow-up after univariate logistic regression. Adjusting for hypertension, multivariate logistic regressions showed that periodontal status remained significant (OR = 5.25, 95% CI, P < .05). CONCLUSIONS: The risk of developing AP in endodontically treated teeth is 5.19 times higher for patients with periodontal disease compared with patients without periodontal disease.

Six-month color change and water sorption of 9 new-generation flowable composites in 6 staining solutions.

Color match and water sorption are two factors that affect restorative materials. Discoloration is essential in the lifespan of restorations. The aim of this study was to evaluate color change and water sorption of nine flowable composites at multiple time points over 6 months. 60 samples of each composite were divided into two groups (Color Change and Water Sorption/Solubility). Each Color Change group was divided into six subgroups, which were immersed in distilled water (DW), coffee (CF), Coca-Cola (CC), red wine (RW), tea (TE) and orange juice (OJ). The color was measured at the baseline, 1, 2, 3 and 4 weeks, and 3 and 6 months and color change values (ΔE) were calculated. Each Water Sorption [WS]/Solubility [WL] group was tested according to ISO 4049:2009. The data were evaluated using two-way ANOVA, Fisher's post-hoc test and Pearson's correlation test. The composite with the lowest ΔE differed for each solution: Filtek™ Bulk Fill in DW (∆E = 0.73 (0.17-1.759)); Vertise Flow in CF (∆E = 14.75 (7.91-27.41)), in TE (∆E = 7.27 (2.81-24.81)) and OJ (∆E = 3.17 (0.87-9.92)); Tetric EvoFlow® in CC (∆E = 1.27 (0.45-4.02)); and Filtek™ Supreme XTE in RW (∆E = 8.88 (5.23-19.59)). RW caused the most discoloration (∆E = 23.62 (4.93-51.36)). Vertise Flow showed the highest water sorption (WS = 69.10 ± 7.19). The Pearson test showed statistically significant positive correlations between water sorption and solubility and between water sorption and ∆E; the positive solubility-∆E correlation was not statistically significant. The findings suggest that water sorption is one factor associated with the ability of composites to discolor; however, discoloration is a multifactorial problem.

Six-month color change and water sorption of 9 new-generation flowable composites in 6 staining solutions

Abstract Color match and water sorption are two factors that affect restorative materials. Discoloration is essential in the lifespan of restorations. The aim of this study was to evaluate color change and water sorption of nine flowable composites at multiple time points over 6 months. 60 samples of each composite were divided into two groups (Color Change and Water Sorption/Solubility). Each Color Change group was divided into six subgroups, which were immersed in distilled water (DW), coffee (CF), Coca-Cola (CC), red wine (RW), tea (TE) and orange juice (OJ). The color was measured at the baseline, 1, 2, 3 and 4 weeks, and 3 and 6 months and color change values (ΔE) were calculated. Each Water Sorption [WS]/Solubility [WL] group was tested according to ISO 4049:2009. The data were evaluated using two-way ANOVA, Fisher's post-hoc test and Pearson's correlation test. The composite with the lowest ΔE differed for each solution: Filtek™ Bulk Fill in DW (∆E = 0.73 (0.17-1.759)); Vertise Flow in CF (∆E = 14.75 (7.91-27.41)), in TE (∆E = 7.27 (2.81-24.81)) and OJ (∆E = 3.17 (0.87-9.92)); Tetric EvoFlow® in CC (∆E = 1.27 (0.45-4.02)); and Filtek™ Supreme XTE in RW (∆E = 8.88 (5.23-19.59)). RW caused the most discoloration (∆E = 23.62 (4.93-51.36)). Vertise Flow showed the highest water sorption (WS = 69.10 ± 7.19). The Pearson test showed statistically significant positive correlations between water sorption and solubility and between water sorption and ∆E; the positive solubility-∆E correlation was not statistically significant. The findings suggest that water sorption is one factor associated with the ability of composites to discolor; however, discoloration is a multifactorial problem.

Color Stability of Teeth Restored with Biodentine: A 6-month In Vitro Study.

INTRODUCTION: White mineral trioxide aggregate (WMTA) has been reported to cause dental discoloration. A previous study on the color stability of 5 calcium silicate-based materials investigated the color stability of Biodentine (Septodont, Saint-Maur-des-Fossés, France) in different experimental environments; however, no data are available on the color stability of teeth restored with Biodentine. In this study, we assessed the color stability under artificial light of ex vivo human teeth restored coronally with WMTA or Biodentine. METHODS: Cavities were prepared on coronal tooth specimens and restored with WMTA + composite (n = 16), Biodentine + composite (n = 16), or composite alone (control, n = 3). Color was assessed spectrophotometrically at 6 time points (initial, 1 week, 2 weeks, 1 month, 3 months, and 6 months), and color difference values were calculated. Statistical analysis was performed using analysis of variance and the Fisher least significant difference test for which P < .05 was considered statistically significant. RESULTS: The WMTA group showed discoloration at 1 week, which increased over time. The Biodentine and control groups showed color stability and were not significantly different from one another. CONCLUSIONS: Teeth treated with WMTA exhibited discoloration, whereas those treated with Biodentine maintained color stability throughout the study. However, further in vivo studies are necessary to corroborate these results.

Análisis espectrofotométrico de la decoloración dental inducida por MTA blanco y Biodentine / Spectrophotometric analysis of tooth discoloration induced by white MTA and biodentine

Objetivo: Evaluar el potencial de decoloración del ProRoot BMTA y del Biodentine en dientes humanos bajo condiciones de iluminación artificial. Material y métodos: 19 dientes humanos fueron divididos en dos grupos experimentales (n=8) y un grupo control (n=3). Los dientes fueron seccionados1 mm por debajo de la unión amelocementaria (UAC), se les extirpó la pulpa cameral y se les preparó una cavidad. Las cavidades de los grupos experimentales fueron obturadas con ProRoot BMTA(R) y con Biodentine(R), en el grupo control no se colocó ningún material. Tras 48 horas, todas las cavidades se obturaron con composite y se colocaron 10 cm debajo de una lámpara, al 100% de humedad. El color fue registrado utilizando un espectrofotómetro en cuatro momentos: tras la colocación de material, tras la restauración coronal, una y dos semanas después de la restauración. Resultados: Se observaron diferencias significativas entre los dos grupos experimentales. El grupo BMTA mostró un cambio de color mayor que el de Biodentine, a las 48 horas, a la semana y a las 2 semanas. A las 48 horas, el BMTA presentó oscurecimiento, que fue aumentado con el tiempo, revelando diferencias significativas entre los tres intervalos. No se observaron diferencias significativas entre el Biodentine y el grupo control durante todo el tiempo. Conclusiones: Debido al hecho de que el Biodentine mostró una mayor estabilidad de color que el BMTA, este se podría recomendar en tratamientos que requieran de materiales de silicato cálico (CSM) en zonas de compromiso estético. Sin embargo, se necesitan estudios a más largo plazo

Objective: To evaluate the discoloration potential of ProRoot WMTA and Biodentine in human teeth under artificial lighting conditions. Methods: 19 human teeth were divided into two experimental groups (n=8) and one control group (n=3). The teeth were sectioned 1 mm below the CEJ, the pulps were extirpated and a cavity was prepared. The experimental groups’ cavities were filled with ProRoot WMTA(R) and with Biodentine(R), no material was placed in the control group. After 48 hours, all the cavities were sealed with composite and the specimens were placed 10 cm below a lamp in a 100% humidity environment. Color values were recorded using a spectrophotometer at four time points: after the material placement, after the coronal restoration, at one and two weeks after restoration. Results: Significant differences were observed between the experimental groups. WMTA group showed a more perceptible discoloration than did Biodentine at 48 hours, 1 week and 2 weeks. At 48-hour time point WMTA presented a discoloration, which increased over time, showing significant differences between the three time intervals. No significant differences were observed between Biodentine and the control group in the three time intervals. Conclusions: Due to the fact that Biodentine showed greater color stability than did WMTA, Biodentine is recommended in treatments requiring calcium silicate materials (CSMs) for esthetically sensitive areas. However, more long-term studies are needed to verify our findings

Evaluation of the effectiveness of an in-office bleaching system and the effect of potassium nitrate as a desensitizing agent.

The aims of the study were to evaluate by spectrophotometer the in vivo colour changes resulting from the application of an in-office tooth bleaching system containing 28% H2O2 by light-emitting diode (LED) activation and to determine whether the application of 5% potassium nitrate 30 min before bleaching decreased tooth sensitivity. Thirty-two individuals were assigned randomly to two groups (n = 16). Group A received 5% potassium nitrate as a desensitizing agent 30 min before bleaching with 28% hydrogen peroxide activated by LED. Group B received glycerin as a placebo and the same bleaching protocol was applied. The colour of the right central incisor of each patient was measured visually and by spectrophotometer before bleaching, immediately thereafter, 15 days and 3 months later. Differences in L* a* b* values were tested with a repeated measures analysis of variance (ANOVA). Differences in ΔΕ values were tested with ANOVA statistical analysis at a 0.05 level of significance. Significant (p < 0.05) differences were detected in L*, as well as in b* values, between initial (I) and post bleaching (PB) and between initial (I) and 3 months post-op. In contrast, there was no significant difference between PB and 3 months post-op. The a* values showed no statistically significant differences among the different time points. Tooth sensitivity decreased significantly when potassium nitrate was applied. In-office bleaching system gave quantitatively stable results over a 3-month period. Tooth sensitivity was reduced significantly, when a desensitizing agent was applied 30 min before treatment, but the efficacy of bleaching decreased.

Influence of light and oxygen on the color stability of five calcium silicate-based materials.

INTRODUCTION: Difficult handling, long setting time, and potential discoloration are important drawbacks of white mineral trioxide aggregate (WMTA). The development of Biodentine, a recently developed calcium silicate-based material (CSM), has overcome some of these shortcomings; however, there are no available data on its color stability. A previous study showed that WMTA discolors under light irradiation in an oxygen-free environment. The present study evaluated the influence of light irradiation and oxygen on the color stability of 5 CSMs. METHODS: Fifteen samples of 5 CSMs (ProRoot WMTA, Angelus WMTA, White Portland Cement [PC], PC with bismuth oxide, and Biodentine) were divided into 5 groups. Each group was exposed to different oxygen and light conditions. A spectrophotometer was used to determine the color of each specimen at 0, 120 seconds, and 5 days. Data were analyzed by using analysis of variance and Tukey honestly significant difference test. RESULTS: The materials PC with bismuth oxide, Angelus WMTA, and ProRoot WMTA showed dark discoloration after light irradiation in an oxygen-free environment, which was statistically significantly different from Biodentine and PC. In groups that were exposed to no light irradiation or to an oxygen atmosphere, all materials showed color stability over time, and no significant differences were observed among them. PC and Biodentine maintained color stability in all conditions over time and showed no significant differences. CONCLUSIONS: The combination of light and anaerobic conditions (similar to those in clinical situations) results in differences in color of the tested CSMs during a period of 5 days, of which Biodentine and PC demonstrated color stability.

Color stability of white mineral trioxide aggregate.

OBJECTIVE: This study aims to evaluate the color stability of white mineral trioxide aggregate (WMTA) after irradiation with three different curing lights and with a fluorescent lamp in an oxygen-free environment. MATERIAL AND METHODS: Thirty samples of WMTA were divided into four experimental groups (three curing light and one fluorescent lamp) and one negative control group. The samples in the curing light groups were immersed in glycerine and were irradiated for 20, 60, and 120 s with a curing light. The samples in the fluorescent lamp group were immersed in glycerine and left on a laboratory shelf below a fluorescent lamp, whereas the negative control group was irradiated with a curing light without immersion in glycerine. A spectrophotometer was used to determine the color of each specimen before and after each light exposure and after 5 days. Data were analyzed using analysis of variance and Fisher's least significant difference test. RESULTS: All the groups showed discoloration except for the negative control group. At 20, 60, and 120 s, there were no significant differences between the Optilux and Bluephase groups (which were the darkest). The Demi group was the curing light experimental group that showed the lowest degree of discoloration (P = 0.0001). No differences were observed between the fluorescent lamp and the negative control groups. After 5 days, the fluorescent lamp group also showed darkening of the sample surface and there were no significant differences between this group and the other three experimental groups (P > 0.05). CONCLUSIONS: WMTA showed dark discoloration after irradiation with a curing light or fluorescent lamp in an oxygen-free environment. CLINICAL RELEVANCE: WMTA may cause tooth discoloration when it is used in a coronal position.