Consensus clustering methodology to improve molecular stratification of non-small cell lung cancer.

Recent advances in machine learning research, combined with the reduced sequencing costs enabled by modern next-generation sequencing, paved the way to the implementation of precision medicine through routine multi-omics molecular profiling of tumours. Thus, there is an emerging need of reliable models exploiting such data to retrieve clinically useful information. Here, we introduce an original consensus clustering approach, overcoming the intrinsic instability of common clustering methods based on molecular data. This approach is applied to the case of non-small cell lung cancer (NSCLC), integrating data of an ongoing clinical study (PROMOLE) with those made available by The Cancer Genome Atlas, to define a molecular-based stratification of the patients beyond, but still preserving, histological subtyping. The resulting subgroups are biologically characterized by well-defined mutational and gene-expression profiles and are significantly related to disease-free survival (DFS). Interestingly, it was observed that (1) cluster B, characterized by a short DFS, is enriched in KEAP1 and SKP2 mutations, that makes it an ideal candidate for further studies with inhibitors, and (2) over- and under-representation of inflammation and immune systems pathways in squamous-cell carcinomas subgroups could be potentially exploited to stratify patients treated with immunotherapy.

MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review.

OBJECTIVES: This systematic review aimed to compare the cytotoxicity of root canal filling materials (RCFMs) assessed using tetrazolium salt-based tests (TSBT), including the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, with those obtained using other cell viability assays. METHODS: A search was performed on PubMed, Scopus, Web of Science and OpenGrey up to March 2019, followed by a manual search. According to the Participants, Exposure, Comparator and Outcomes (PECO) criteria, in vitro studies that evaluated the cytotoxic effect of RCFMss on animal and/or human cells through TSBT and at least one other viability assay were compared. The methodological quality of selected papers was assessed using ToxRTool® and SciRAP® . Data were analysed using Wilcoxon's signed-rank test for paired samples and linear weighting kappa. RESULTS: A total of 230 non-duplicated records were identified. After applying the eligibility criteria, 55 studies were selected for methodological evaluation, seven were selected by manual searching, 22 were excluded for methodological reasons, and 40 were included. A total of 410 comparisons were performed between TSBT and distinct cell viability tests (DCVT). MTT had moderate concordance with DCVT using human cells (n = 138 samples) (P = 0.507; k = 0.4225) and animal cells (n = 122 samples) (P = 0.124; k = 0.5775). XTT had good concordance using human (n = 110 samples) (P = 0.507; k = 0.6336) and animal cells (n = 12 samples) (P = 0.564; k = 0.6604). MTT, XTT, WST and MTS assays showed moderate concordance with DCVT (n = 410 samples) (P = 0.375; k = 0.5138) and complete agreement in 226 samples. DISCUSSION: The included studies had methodological heterogeneity that was minimized by the systematic review methodology. CONCLUSIONS: MTT and XTT do not cause over- or underestimation of cell viability during cytotoxicity screening of root canal filling materials, implying that these assays can be considered reliable for this purpose. Nonetheless, the development of protocols for the cytotoxic screening of these materials on 3D tissue-like cultures aiming to improve their predictability in the clinical scenario is suggested.

Aberrant regulation of epigenetic modifiers contributes to the pathogenesis in patients with selenoprotein N-related myopathies.

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.

Gelatin-based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering.

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Analysis of root canal treated primary incisor after trauma: two year outcomes.

BACKGROUND: The low number of clinical studies of traumatized teeth submitted to root canal treatment is completely out of proportion to the seriousness that dental trauma imposes on children in early years. AIM: This study evaluates the outcomes of root canal treatment (RCT) in traumatized primary incisors and identifies the predisposing factors associated with therapy success. METHODS: This is a retrospective study conducted with all dental records of 704 patients who had one or more teeth with traumatic injuries. Patients with irreversible pulp changes in primary teeth leading to RCT with a 24 month follow-up met the inclusion criteria. RESULTS: Twenty-five maxillary incisors of 17 children were evaluated. The children's age at the time of therapy ranged from 24 to 72 months (mean 47.3). Pulp necrosis was the most common disorder (84.0%) and pre-operative periapical lesions were observed in 52.0%. Coronal discoloration was found in 48.0%. The roots were filled with ZOE paste (68.0%) or Guedes-Pinto paste (32.0%). Overall RCT success rate was 68.0%. The absence of pre-operative periapical lesions (p = 0.02) and pathological root resorption (p = 0.02) presented positive association with therapy success. Success was not associated to filling paste (p = 0.49), filling extent (p = 0.44), of discoloration (p = 0.39) nor the patients' age (p = 0.59). CONCLUSIONS: RCT was considered successful in 68.0% of the cases at the 24 month follow-up. Failure of RCT in traumatized primary incisors was associated with pre-operatory periapical lesions and pathological root resorption. The filling paste, the filling extent and the patient's age were unrelated with therapy success.

Short communication: polymicrobial community in teeth associated with severe early-childhood caries.

AIM: This was to characterise the microbial diversity in the complex dental plaque of children with severe early-childhood caries (S-ECC), using the denaturing gel gradient electrophoresis (DGGE) technique. METHODS: The DGGE technique was used as a diagnostic tool to analyse samples from the oral cavity of a patient with S-ECC. Dental plaque samples from a 3-year-old child with S-ECC were taken from the primary central maxillary incisor (biofilm on vestibular surface), primary maxillary molar (biofilm on vestibular surface), primary central maxillary incisor (dentine), primary maxillary molar (dentine) and saliva and then analysed by PCR-DGGE. RESULTS: Three bands occurred in all samples, moreover, 86% of similarity was observed in the pattern of bands between incisor and molar teeth biofilm samples, including four similar bands. CONCLUSION: DGGE is a valuable tool for differentiating the microbial composition of the oral plaque in S-ECC children.

ZOE paste pulpectomies outcome in primary teeth: a systematic review.

OBJECTIVE: To perform a systematic review in which the clinical research question for primary teeth with irreversible pulpal pathosis was "how pulpectomies with zinc oxide eugenol (ZOE) paste performed compared to other materials in their clinical and radiographic outcomes after twelve months or more follow-up period. STUDY DESIGN: A literature survey of the electronic database (1950-2010) used the Medical Subject Headings and free text terms. Forty three references were retrieved and inclusion criteria were applied; 15 articles remained for full-text evaluation. From these, two were selected for data extraction regarding quality characteristics and results. RESULTS: Selected studies showed moderate or high risk of bias. The overall success of pulpectomy was 80.0% (Calcicur), 60.0% (Sealapex) and varied from 85.0% to 100.0% (ZOE) and 89.0% to 100.0% (Vitapex). Solely Calcicur presented success rate significantly lower when compared to ZOE and Vitapex. These pastes lead to overfilled canals and particles of extruded ZOE were still evident even after the evaluation period. Resorption of Vitapex, Calcicur and Sealapex within the root canal was also reported. CONCLUSIONS: In primary teeth with irreversible pulpal changes ZOE pulpectomies yielded similar outcome than Vitapex and Sealapex, although there was no agreement with regard to filling materials' resorption.

Zinc oxide-eugenol paste retained in gingival mucosa after primary teeth pulpectomy.

AIM: Long-term follow-up evaluations of pulpectomy in primary teeth have revealed retention of ZOE filling particles in the periapical area even after root resorption. CASE REPORT: This paper reports a case of a child submitted to pulpectomy with ZOE paste in primary teeth. After 28 months, the filling particles remained, having migrated to the alveolar bone from the gingival vestibular mucosa during permanent dentition eruption. Aesthetics required periodontal surgical removal of the paste particles. Primary teeth submitted to pulpectomy should be evaluated carefully both clinically and radiographically to verify radicular and ZOE filling paste resorption. The consequences of retained particles during permanent dentition eruption are unknown.

Microhardness of dentine underlying ART restorations in primary molars: an in vivo pilot study.

OBJECTIVE: To evaluate the microhardness of dentine underlying glass ionomer restorations made with Atraumatic Restorative Treatment. DESIGN: An experimental single-centre study. SETTING: University Department, Brazil, 2001. MATERIALS AND METHODS: Sixteen children were selected showing primary molars containing carious cavities (n = 29), which were restored using Fugi IX. Subsequently, some teeth were extracted respecting Nolla stage 7 or 8 of the permanent successor, and categorised according to the post-restoration time in the mouth: G1 (baseline, immediate extraction), G2 (30 days), G3 (90 days) and G4 (180 days). Four teeth were excluded because they were not in time of normal exfoliation. The 25 extracted teeth were resin embedded, sectioned mesiodistally and prepared metallographically. Knoop microhardness analysis (Micromet 2003; 10g for 1.5 sec.) produced identations in three areas of dentine: Zone 1 (just below the restoration), Zone 3 (as close as possible to the pulp) and Zone 2 (intermediate region between 1 and 3). Non-parametric statistical tests were done--at a significance level of 5%. RESULTS: Four teeth were excluded. No significant difference was noted among the zones, inside the groups. Zones 1 and 2 showed a considerable increase of microhardness according to the time, except for Group 3 (p > 0.05). CONCLUSION: Although microhardness had increased, it was not sufficiently equal to the microhardness of the healthy dentine, regardless of the depth evaluated.

Case report: ultrasonic cavity preparation -- an alternative approach for caries removal in paediatric dentistry.

BACKGROUND: Since the invention and application of rotating instruments, the operative treatment of carious lesions has often resulted in considerable removal of tooth structure. Moreover, conventional caries removal and cavity preparation by a combination of the use of a turbine and a handpiece with a bur presents disadvantages to very young patients, whose perception of drilling is unpleasant. This report concerns an alternative approach for caries removal in a child using ultrasound and discusses the advantages and disadvantages of this technique. CASE REPORT: A 2-year-old male child was brought by his mother to a private paediatric dental clinic in Rio de Janeiro, Brazil. The mother's main complaint was the presence of dental caries in her son's anterior teeth. Intraoral clinical examination revealed that the patient had two carious lesions in the maxillary central incisors on mesial surfaces. Cavity preparation was performed using ultrasound and restoration with a light-curing resin. FOLLOW-UP: One year and a half after the restorative procedure the patient presented with the restored teeth in perfect condition.

Case report: the importance of oral manifestations in diagnosing iron deficiency in childhood.

AIM: The aim of this article is to report a case of iron deficiency diagnosed in a child after routine oral examination. CASE REPORT: A 5-year-old male child of African descent was brought to the paediatric dental clinic of a public university in Rio de Janeiro, Brazil. His mother's main complaint was her child's decayed teeth and sensitivity in the tongue every time he ate spicy or hot food. Anamnesis revealed chronic respiratory problems due to allergy, two previous episodes of anaemia and hospitalization about 15 months before the dental visit because of severe primary herpetic gingivostomatitis. Soft tissue examination revealed his tongue had various patches of atrophic mucosa characterizing absence of papillae in these areas. The child's dietary assessment indicated that he never ate meat or vegetables. Haematological investigation showed that the child probably had an iron deficiency, although the full blood count was not totally compatible with anaemia. A rapid initial recovery was quite noticeable after the beginning of oral therapy with ferrous sulphate, as remission of tongue sensitivity as well as papillae neoformation were observed.

Microscopic investigation of artificially demineralized surface enamel exposed to controlled intra-oral periods.

BACKGROUND: Arrest and regression of enamel caries is generally followed by clinical surface changes in enamel. The aim of this study was to evaluate microscopic surface changes in demineralized enamel submitted to toothbrushing with a fluoridated dentifrice or to a fluoridated gel in situ. METHODS: Enamel blocks demineralized by an acidic gel, were built in plates of five volunteers during three periods of 14 days each: exposure to the oral cavity (C+); brushing with a fluoridated dentifrice three times a days (B); application of a 2 per cent sodium fluoride gel and exposure to the oral cavity (F). Eight blocks were kept in 100 per cent humidity under refrigeration as negative controls (C-). RESULTS: Stereomicroscopic examination revealed that 15, 47.4 and 30 per cent of samples from C+, B and F presented enamel cavitation after the experimental period. The demineralized enamel area in these groups decreased considerably compared to C-, both in cavitated and non-cavitated samples, although it was statistically significant only in cavitated samples from B (p=0.003; t test). Scanning electron microscopy revealed that C+ and F retained irregularities caused by the acid challenge, while B presented a more leveled profile of the enamel surface. CONCLUSIONS: Exposure of demineralized enamel to intra-oral periods reduced the demineralized enamel area. Tooth brushing enhanced this reduction, because of surface abrasion.

Neumomediastino espontáneo. A propósito de un caso / Spontaneous pneumomediastinum. A case report

No disponible

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.

Evaluation of the palate dimensions of patients with perennial allergic rhinitis.

OBJECTIVE: The purpose of this study was to compare the transverse and vertical palate dimensions of two groups of children, one diagnosed as having perennial allergic rhinitis (PAR) and the other without any respiratory pathology. DESIGN: Case-control study. SETTING: Institute of Paediatrics and Child Care of the Federal University of Rio de Janeiro. SAMPLE AND METHODS: The sample consisted of 101 children with the diagnoses of PAR (mean age 6.5) and 91 without any respiratory pathology (mean age 7.3). From 192 patients, 65% was in mixed dentition phase and 35% in primary dentition. Each subject underwent an intra-oral clinical examination using three-dimensional Korkhaus compass to measure the intermolar distance, intercanine distance and palate depth. RESULTS: The allergic group showed greater averages of palate depth (P = 0.00), both in the primary dentition phase (11.53 mm x 10 mm) and in the mixed dentition (11.96 mm x 10.21 mm). There was no statistically significant difference for intermolar and intercanine distances, either in primary or mixed dentition (P > 0.05). CONCLUSION: The transverse dimension of the palate did not vary significantly between the groups, which seems to confirm that the main influence of alteration of the breathing pattern from nasal to mouth occurs on the vertical plane.

HIV-1 Tat protein stimulates in vivo vascular permeability and lymphomononuclear cell recruitment.

HIV-1 Tat protein released by infected cells is a chemotactic molecule for leukocytes and induces a proinflammatory program in endothelial cells (EC) by activating vascular endothelial growth factor (VEGF) receptors expressed on both cell types. Its potential role in causing vascular permeability and leukocyte recruitment was studied in vivo following its s.c. injection in mice. Tat caused a dose-dependent early (15 min) and late (6 h) wave of permeability that were inhibited by a neutralizing Ab anti-VEGF receptor type 2. Tissue infiltration of lymphomononuclear cells, mainly monocytes (76%), was evident at 6 h and persisted up to 24 h. WEB2170, a platelet activating factor (PAF) receptor antagonist, reduced the early leakage by 70-80%, but only slightly inhibited the late wave and cell recruitment. In vitro, Tat induced a dose-dependent flux of albumin through the EC monolayer that was inhibited by Ab anti-vascular VEGF receptor type 2 and WEB2170, and PAF synthesis in EC that was blocked by the Ab anti-VEGF receptor type 2. Lastly, an anti-monocyte chemotactic peptide-1 (MCP-1) Ab significantly reduced the lymphomononuclear infiltration elicited by Tat. In vitro, Tat induced a dose-dependent production of MCP-1 by EC after a 24-h stimulation. These results highlighted the role of PAF and MCP-1 as secondary mediators in the onset of lymphomononuclear cell recruitment in tissues triggered by Tat.

Human endothelial cells expressing polyoma middle T induce tumors.

The middle T oncogene of murine polyomavirus (PymT) rapidly transforms and immortalizes murine embryonic endothelial cells (EC), leading to the formation of vascular tumors in newborn mice, by recruitment of host, non-transformed EC. These tumors are reminiscent of human vascular tumors like cavernous hemangioma, Kaposi's sarcoma or those characterizing Kasabach-Merrit syndrome. Here we investigate the in vitro and in vivo behavior of human primary umbilical cord vein EC expressing PymT. While PymT has been unable to transform human fibroblasts in earlier experiments or controls done here, mT expressing EC (PymT-EC) derived by infection with pLX-PymT retrovirus induce hemangiomas in nu/nu mice. These tumors contain not only human cells but also recruited mouse EC as shown by the presence of human and murine CD31 positive EC. In vitro analysis shows that PymT-EC retain endothelial specific markers like CD31, Von Willebrand factor, and VE-cadherin, and reach the confluence without signs of overgrowth. They are also responsive to vascular endothelial growth factor-A. However, their proliferation rate is increased. The balance between urokinase-type plasminogen activator and plasminogen activator inhibitor-1 is modified; RNA and catalytic activity for the former are elevated while PAI-1 RNA is reduced. In contrast with murine model, where the PymT EC cells become immortal, the effects induced by PymT in human EC are transient. After 12-15 passages, human PymT EC stop proliferating, assume a senescent phenotype, and lose the ability to induce hemangiomas. At the same time both the amount of middle T protein and the level of activation of pp60c-src lower.

Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi's sarcoma cells.

Recent evidence suggesting vascular endothelial growth factor-C (VEGF-C), which is a regulator of lymphatic and vascular endothelial development, raised the question whether this molecule could be involved in Kaposi's sarcoma (KS), a strongly angiogenic and inflammatory tumor often associated with infection by human immunodeficiency virus-1. This disease is characterized by the presence of a core constituted of three main populations of "spindle" cells, having the features of lymphatic/vascular endothelial cells, macrophagic/dendritic cells, and of a mixed macrophage-endothelial phenotype. In this study we evaluated the biological response of KS cells to VEGF-C, using an immortal cell line derived from a KS lesion (KS IMM), which retains most features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice. We show that VEGFR-3, the specific receptor for VEGF-C, is expressed by KS IMM cells grown in vitro and in vivo. In vitro, VEGF-C induces the tyrosine phosphorylation of VEGFR-2, a receptor also for VEGF-A, as well as that of VEGFR-3. The activation of these two receptors in KS IMM cells is followed by a dose-responsive mitogenic and motogenic response. The stimulation of KS IMM cells with a mutant VEGF-C unable to bind and activate VEFGR-2 resulted in no proliferative response and in a weak motogenic stimulation, suggesting that VEGFR-2 is essential in transducing a proliferative signal and cooperates with VEGFR-3 in inducing cell migration. Our data add new insights on the pathogenesis of KS, suggesting that the involvement of endothelial growth factors may not only determine KS-associated angiogenesis, but also play a critical role in controlling KS cell growth and/or migration and invasion.

Tumor necrosis factor-alpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.