Ventilator hyperinflation associated with flow bias optimization in the bronchial hygiene of mechanically ventilated patients.

BACKGROUND: Ventilation configurations are of great clinical importance for adequate outcomes in mechanically ventilated patients, and they may even be used as specific physical therapy techniques. OBJECTIVES: To compare the effectiveness of lung hyperinflation through mechanical ventilation (HMV) with HMV plus flow bias optimization regarding respiratory mechanics, hemodynamics, and volume of secretion. METHODS: Patients mechanically ventilated > 24 h were included in this randomized crossover clinical trial. The following techniques were applied: HMV alone (control group) and HMV plus flow bias optimization (intervention group). RESULTS: The 20 included patients underwent both techniques, totaling 40 collections. A total of 52 % were women, the mean age was 60.8 (SD, 15.7) years, and the mean mechanical ventilation time was 4.3 (SD, 3.0) days. The main cause of mechanical ventilation was sepsis (44 %). Expiratory flow bias in optimized HMV was higher. than conventional HMV (p < 0.001). The volume of tracheal secretions collected was higher during optimized than conventional HMV. (p = 0.012). Significant differences in peak flow occurred at the beginning of the technique and a there was a significant decrease in respiratory system resistance immediately and 30 min after applying the technique in the intervention group. CONCLUSIONS: The volume of tracheal secretions collected was higher during optimized HMV, and, HMV with flow bias optimization resulted in lower respiratory system resistance and flow peaks and produced expiratory flow bias.

Childhood Hypophosphatasia Associated with a Novel Biallelic ALPL Variant at the TNSALP Dimer Interface.

The goal of this study was to perform a clinical and molecular investigation in an eight-year-old female child diagnosed with hypophosphatasia (HPP). The proband and her family were evaluated by medical and dental histories, biochemical analyses, radiographic imaging, and genetic analysis of the tissue-nonspecific alkaline phosphatase (ALPL) gene. A bioinformatic analysis was performed to predict the structural and functional impact of the point mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) molecule and to define their potential contribution to the phenotype. We identified a novel combination of heterozygous ALPL missense variants in the proband, p.Ala33Val and p.Asn47His, compatible with an autosomal recessive mode of inheritance and resulting in skeletal and dental phenotypes. Computational modeling showed that the affected Asn47 residue is located in the coil structure close to the N-terminal α-helix, whereas the affected Ala33 residue is localized in the N-terminal α-helix. Both affected residues are located close to the homodimer interface, suggesting they may impair TNSALP dimer formation and stability. Clinical and biochemical follow-up revealed improvements after six years of ERT. Reporting this novel combination of ALPL variants in childhood HPP provides new insights into genotype-phenotype associations for HPP and specific sites within the TNSALP molecule potentially related to a childhood-onset HPP and skeletal and dental manifestations. Beneficial effects of ERT are implicated in skeletal and dental tissues.

Medical Research Council Scale Predicts Spontaneous Breathing Trial Failure and Difficult or Prolonged Weaning of Critically Ill Individuals.

BACKGROUND: Handgrip strength is an alternative measure to assess peripheral muscle strength and is correlated with the Medical Research Council (MRC) scale, with promising values for diagnosing ICU-acquired weakness (ICUAW). Because ICUAW has been associated with delayed weaning from mechanical ventilation, we hypothesized that ICUAW evaluated with both the MRC scale score and handgrip strength are associated with failure of a spontaneous breathing trial (SBT) and duration of mechanical ventilation weaning. METHODS: We conducted a prospective observational study in 3 general ICUs with a total of 54 beds at 2 academic hospitals. Adult subjects with > 48 h of mechanical ventilation who were eligible for weaning were included in the study. RESULTS: In the evaluation before the first SBT, the MRC score (P < .001) and handgrip strength (P < .001) were significantly different between subjects extubated after a successful first SBT (simple weaning) and those extubated any time after a failed first SBT (difficult weaning). Only the MRC score discriminated between first SBT success or failure (P < .001); in multivariate analysis, the MRC score was significantly associated with first SBT failure (odds ratio 0.91, 95% CI 0.88-0.97, P < .001) and difficult weaning (odds ratio 0.91, 95% CI 0.87-0.96, P < .001). Handgrip strength exhibited good accuracy in identifying ICUAW. CONCLUSIONS: MRC score was independently associated with SBT failure and difficult or prolonged weaning.

Comparative proteomic analysis of dental cementum from deciduous and permanent teeth.

BACKGROUND AND OBJECTIVES: Dental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition. METHODS: Protein extracts from deciduous (n = 25) and permanent (n = 12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins. RESULTS: A total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth. CONCLUSIONS: Clear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro.

Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies.

Novel rare frameshift variation in aggressive periodontitis: Exomic and familial-screening analysis.

BACKGROUND: Aggressive periodontitis (AgP), currently periodontitis grade C, presents early onset, rapid progression, and a poorly established genetic association. Thus, this study aimed to identify genetic variants associated with AgP via whole exome sequencing (WES) through a familial screening approach. METHODS: WES was performed in two nuclear families, including a proband and a parent affected by AgP and an unaffected parent and sibling. Common variants among affected individuals, excluding those common to healthy people, from each family, composed the data set associated with AgP. In silico analysis evaluated the impact of each variant on protein structure and protein-protein interactions. Moreover, identified deleterious variants were validated in a populational analysis (n = 96). RESULTS: The missense single nucleotide variations (SNVs) rs142548867 in EEFSEC (c.668C>T), rs574301770 in ZNF136 (c.466C>G), and rs72821893 in KRT25 (c.800G>A) and the frameshift indels rs37146475 in GPRC6A (c.2323-2324insT) and c.1366_1372insGGAGCAG in ELN were identified in AgP and have a predicted functional impact on proteins. In silico analysis indicated that the indel in GPRC6A generates a loss of the C-terminal tail of the Gprca protein. Furthermore, this SNV was significantly associated with AgP in a population-based investigation. CONCLUSION: Novel frameshift variation in GPRC6A (c.2323-2324insT) was identified as a potential genetic alteration associated with AgP occurrence.

Effects of Combined Tracheal Suctioning and Expiratory Pause: A Crossover Randomized Clinical Trial.

AIMS: Our aim is to compare volume of suctioned secretion, respiratory mechanics, and hemodynamic parameters in intubated patients undergoing closed-system endotracheal suctioning alone (control group) versus closed-system tracheal suctioning with an expiratory pause (intervention group). SETTINGS AND DESIGN: Randomized crossover clinical trial. MATERIALS AND METHODS: Patients who had been on mechanical ventilation for more than 24 hours were randomly assigned to receive closed-system suctioning alone or closed-system suctioning with an expiratory pause on the ventilator. The following variables were evaluated: heart rate, respiratory rate, mean arterial pressure, peripheral arterial oxygen saturation, peak inspiratory pressure, mechanical ventilator circuit pressure during aspiration, exhaled tidal volume, dynamic compliance, resistance, and weight of suctioned secretion. STATISTICAL ANALYSIS: Compared using the paired t-test and general linear model analysis of variance for normally distributed variables (as confirmed by the Kolmogorov-Smirnov test). The Wilcoxon test was used for variables with a nonparametric distribution, while the Chi-square test and Fisher's exact test were used for categorical variables. RESULTS: The sample comprised 31 patients (mean age, 61.1 ± 18.2 years). The amount of secretion suctioned was significantly higher in the intervention group than in the control group (1.6 g vs 0.45 g; p = 0.0001). There were no significant changes in hemodynamic parameters or respiratory mechanics when comparing pre- and postprocedure time points. CONCLUSION: The combination of closed-system endotracheal suctioning and an expiratory pause significantly increased the amount of secretion suctioned compared to conventional suctioning without expiratory pause. KEY MESSAGES: Combination of closed-system endotracheal suctioning and an expiratory pause significantly increased the amount of secretion suctioned. HOW TO CITE THIS ARTICLE: Martins LFG, Naue WS, Skueresky AS, Bianchi T, Dias AS, Forgiarini Junior LA. Effects of Combined Tracheal Suctioning and Expiratory Pause: A Crossover Randomized Clinical Trial. Indian J Crit Care Med 2019;23(10):454-457.

A novel combination of biallelic ALPL mutations associated with adult hypophosphatasia: A phenotype-genotype association and computational analysis study.

Hypophosphatasia (HPP) is an inherited metabolic disorder that causes defective skeletal and dental mineralization. HPP exhibits a markedly heterogeneous range of clinical manifestations caused by dysfunction of the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP), resulting from loss-of-function mutations in the ALPL gene. HPP has been associated with predominantly missense mutations in ALPL, and a number of compound heterozygous genotypes have been identified. Here, we describe a case of a subject with adult-onset HPP caused by a novel combination of missense mutations p.Gly473Ser and p.Ala487Val, resulting in chronic musculoskeletal pain, myopathy, persistent fatigue, vomiting, and an uncommon dental phenotype of short-rooted permanent teeth. Pedigree and biochemical analysis indicated that severity of symptoms was correlated with levels of residual ALP activity, and co-segregated with the p.Gly473Ser missense mutation. Bioinformatic analysis to predict the structural and functional impact of each of the point mutations in the TNSALP molecule, and its potential contribution to the clinical symptoms, revealed that the affected Gly473 residue is localized in the homodimer interface and predicted to have a dominant negative effect. The affected Ala487 residue was predicted to bind to Tyr479, which is closely located the N-terminal α-helix of TNSALP monomer 2, suggesting that both changes may impair dimer stability and catalytic functions. In conclusion, these findings assist in defining genotype-phenotype associations for HPP, and further define specific sites within the TNSALP molecule potentially related to neuromuscular manifestations in adult HPP, allowing for a better understanding of HPP pathophysiology.

Comparison of bronchial hygiene techniques in mechanically ventilated patients: a randomized clinical trial. / Comparação entre técnicas de higiene brônquica em pacientes mecanicamente ventilados: ensaio clínico randomizado.

OBJECTIVE: To compare the effects of vibrocompression and hyperinflation with mechanical ventilator techniques alone and in combination (hyperinflation with mechanical ventilator + vibrocompression) on the amount of aspirated secretion and the change in hemodynamic and pulmonary parameters. METHODS: A randomized clinical trial with critically ill patients on mechanical ventilation conducted in the intensive care unit of a university hospital. The patients were randomly allocated to receive one of the bronchial hygiene techniques for 10 minutes (vibrocompression or hyperinflation with mechanical ventilator or hyperinflation with mechanical ventilator + vibrocompression). Afterwards, the patients were again randomly allocated to receive either the previous randomly allocated technique or only tracheal aspiration. The weight of aspirated secretions (in grams), ventilatory mechanics and cardiopulmonary data before and after the application of the techniques were analyzed. The tracheal reintubation frequency and time and mortality on mechanical ventilation were also evaluated. RESULTS: A total of 93 patients (29 vibrocompression, 32 hyperinflation with mechanical ventilator and 32 hyperinflation with mechanical ventilator + vibrocompression) on mechanical ventilation for more than 24 hours were included. The hyperinflation with mechanical ventilator + vibrocompression group was the only one that presented a significant increase in aspirated secretions compared to tracheal aspiration alone [0.7g (0.1 - 2.5g) versus 0.2g (0.0 - 0.6g), p value = 0.006]. CONCLUSION: Compared to tracheal aspiration alone, the combination of hyperinflation with mechanical ventilator + vibrocompression techniques was most efficient for increasing the amount of aspirated secretions.

OBJETIVO: Comparar a eficiência das técnicas de vibrocompressão e hiperinsuflação com ventilador mecânico de forma isolada e a associação das duas técnicas (hiperinsuflação com ventilador mecânico + vibrocompressão), na quantidade de secreção aspirada e na alteração de parâmetros hemodinâmicos e pulmonares. MÉTODOS: Ensaio clínico randomizado com pacientes críticos em ventilação mecânica, realizado na unidade de terapia intensiva de um hospital universitário. Os pacientes foram randomizados para receber uma das técnicas de higiene brônquica por 10 minutos (vibrocompressão, ou hiperinsuflação com ventilador mecânico, ou hiperinsuflação com ventilador mecânico + vibrocompressão). Após, foram novamente randomizados para receber inicialmente a técnica (previamente randomizada) ou apenas a aspiração isolada. Foram analisados o peso de secreção aspirada (em gramas), dados de mecânica ventilatória e cardiopulmonares, antes e após a aplicação das técnicas. A frequência de reintubação traqueal, o tempo de ventilação mecânica e a mortalidade, também foram avaliados. RESULTADOS: Foram incluídos 93 pacientes (29 vibrocompressão, 32 hiperinsuflação com ventilador mecânico e 32 hiperinsuflação com ventilador mecânico + vibrocompressão) em ventilação mecânica por mais de 24 horas. O grupo hiperinsuflação com ventilador mecânico + vibrocompressão foi o único que apresentou aumento significativo da secreção aspirada, quando comparado a aspiração isolada 0,7g (0,1 - 2,5g) versus 0,2g (0,0 - 0,6g), com valor de p = 0,006. CONCLUSÃO: Quando comparada à aspiração isolada, a associação das técnicas hiperinsuflação com ventilador mecânico + vibrocompressão foi mais eficiente na quantidade de secreção aspirada.

Comparação entre técnicas de higiene brônquica em pacientes mecanicamente ventilados: ensaio clínico randomizado / Comparison of bronchial hygiene techniques in mechanically ventilated patients: a randomized clinical trial

RESUMO Objetivo: Comparar a eficiência das técnicas de vibrocompressão e hiperinsuflação com ventilador mecânico de forma isolada e a associação das duas técnicas (hiperinsuflação com ventilador mecânico + vibrocompressão), na quantidade de secreção aspirada e na alteração de parâmetros hemodinâmicos e pulmonares. Métodos: Ensaio clínico randomizado com pacientes críticos em ventilação mecânica, realizado na unidade de terapia intensiva de um hospital universitário. Os pacientes foram randomizados para receber uma das técnicas de higiene brônquica por 10 minutos (vibrocompressão, ou hiperinsuflação com ventilador mecânico, ou hiperinsuflação com ventilador mecânico + vibrocompressão). Após, foram novamente randomizados para receber inicialmente a técnica (previamente randomizada) ou apenas a aspiração isolada. Foram analisados o peso de secreção aspirada (em gramas), dados de mecânica ventilatória e cardiopulmonares, antes e após a aplicação das técnicas. A frequência de reintubação traqueal, o tempo de ventilação mecânica e a mortalidade, também foram avaliados. Resultados: Foram incluídos 93 pacientes (29 vibrocompressão, 32 hiperinsuflação com ventilador mecânico e 32 hiperinsuflação com ventilador mecânico + vibrocompressão) em ventilação mecânica por mais de 24 horas. O grupo hiperinsuflação com ventilador mecânico + vibrocompressão foi o único que apresentou aumento significativo da secreção aspirada, quando comparado a aspiração isolada 0,7g (0,1 - 2,5g) versus 0,2g (0,0 - 0,6g), com valor de p = 0,006. Conclusão: Quando comparada à aspiração isolada, a associação das técnicas hiperinsuflação com ventilador mecânico + vibrocompressão foi mais eficiente na quantidade de secreção aspirada.

ABSTRACT Objective: To compare the effects of vibrocompression and hyperinflation with mechanical ventilator techniques alone and in combination (hyperinflation with mechanical ventilator + vibrocompression) on the amount of aspirated secretion and the change in hemodynamic and pulmonary parameters. Methods: A randomized clinical trial with critically ill patients on mechanical ventilation conducted in the intensive care unit of a university hospital. The patients were randomly allocated to receive one of the bronchial hygiene techniques for 10 minutes (vibrocompression or hyperinflation with mechanical ventilator or hyperinflation with mechanical ventilator + vibrocompression). Afterwards, the patients were again randomly allocated to receive either the previous randomly allocated technique or only tracheal aspiration. The weight of aspirated secretions (in grams), ventilatory mechanics and cardiopulmonary data before and after the application of the techniques were analyzed. The tracheal reintubation frequency and time and mortality on mechanical ventilation were also evaluated. Results: A total of 93 patients (29 vibrocompression, 32 hyperinflation with mechanical ventilator and 32 hyperinflation with mechanical ventilator + vibrocompression) on mechanical ventilation for more than 24 hours were included. The hyperinflation with mechanical ventilator + vibrocompression group was the only one that presented a significant increase in aspirated secretions compared to tracheal aspiration alone [0.7g (0.1 - 2.5g) versus 0.2g (0.0 - 0.6g), p value = 0.006]. Conclusion: Compared to tracheal aspiration alone, the combination of hyperinflation with mechanical ventilator + vibrocompression techniques was most efficient for increasing the amount of aspirated secretions.

Membrane proteome characterization of periodontal ligament cell sets from deciduous and permanent teeth.

BACKGROUND: Physiological roles for the periodontal ligament (PDL) include tooth eruption and anchorage, force absorption, and provision of proprioceptive information. Despite the advances in understanding the biology of PDL cells, there is a lack of information regarding the molecular signature of deciduous (DecPDL) and permanent (PermPDL) PDL tissues. Thus, the present study was designed to characterize the membrane proteome of DecPDL and PermPDL cells. METHODS: Primary PDL cells were obtained (n = 6) and a label-free quantitative proteome of cell membrane-enriched components was performed. Proteome findings were validated by quantitative polymerase chain reaction and Western blot assays in fresh human tissues (n = 8) and primary cell cultures (n = 6). In addition, confocal microscopy was used to verify the expression of target factors in the PDL cell cultures. RESULTS: Comparative gene ontology enrichment analysis evidenced that most stickling differences involved "endomembrane system" (PICALM, STX4, and LRP10), "hydrolase activity" (NCSTN and XRCC6), "protein binding" (PICALM, STX4, GPNMB, VASP, extended-synaptotagmin 2 [ESYT2], and leucine-rich repeat containing 15 [LRRC15]), and "isomerase activity" (FKBP8). Data are available via ProteomeXchange with identifier PXD010226. At the transcript level, high PICALM in DecPDL and ESYT2 and LRRC15 in PermPDL were confirmed in fresh PDL tissues. Furthermore, Western blot analysis confirmed increased levels of PICALM, LRRC15, and ESYT2 in cells and/or fresh tissues, and confocal microscopy confirmed the trends for PICALM and LRRC15 expression in PDL cells. CONCLUSION: We report the first comprehensive characterization of the membrane protein machinery of DecPDL and PermPDL cells, and together, we identified a distinct molecular signature for these cell populations, including unique proteins for DecPDL and PermPDL.

Variable expressivity and novel PTEN mutations in Cowden syndrome.

Cowden syndrome (CS) is a phosphatase and tensin homolog gene (PTEN)-associated condition characterized by multiple mucocutaneous hamartomas and an increased risk of malignancies. We reported an isolated case and another of several individuals in one family affected by CS. The isolated case showed typical features, including fibrocystic breast disease, benign thyroid nodules, and multiple papillomatous lesions in the face and oral cavity, and the cause was a novel nonsense mutation-guanine (G) to thymine (T) transition at position 940 (c.940 G>T)-in PTEN. In the family, the proband showed erythema nodosum, duodenal ulcer, intestinal polyps, cervical lipoma, renal cysts, and glaucoma, whereas multiple members of her family were found to have intestinal polyps, and a sister had been diagnosed with breast cancer at early age. An intronic mutation-T>G transition at the +32 position of intron 8 (c.1026+32 T>G)-was found in this family, with in silico analysis revealing the creation of a new donor splice site. This study confirmed the involvement of PTEN in CS and the variable clinical expressivity of disease.

Computational analysis for GNAQ mutations: New insights on the molecular etiology of Sturge-Weber syndrome.

Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G>A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gß subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.

Leucine-Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires Flotillin-1 Mediated Endocytosis.

Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide-LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by flotillin-1. J. Cell. Physiol. 232: 556-565, 2017. © 2016 Wiley Periodicals, Inc.

Apoio em rede: a Rede HumanizaSUS conectando possibilidades no ciberespaço / Apoyo en red: HumanizaSUS, la red que conecta posibilidades en el ciberespacio / Network Support: HumanizaSUS Network connecting possibilities in cyberspace

Este trabalho é um texto analítico sobre a experiência da Rede HumanizaSUS como espaço virtual de conexão das práticas e saberes do Sistema Brasileiro de Saúde (SUS) e de experimentação da função apoio em rede. Para isso, partindo do referencial teórico da Inteligência Coletiva, apresentaremos algumas dimensões do apoio experimentado na Rede HumanizaSUS como uma tecnologia de intervenção em rede para o fortalecimento dos espaços virtuais do SUS ou do CiberespaSUS.

Este es un texto analítico sobre la experiencia de la red HumanizaSUS como un espacio de conexión virtual de las prácticas y los saberes del SUS (Sistema Brasileño de Salud), así como de la experimentación de funciones de apoyo en red. Para esto, a partir del marco teórico de la Inteligencia Colectiva, presentamos algunas de las dimensiones de apoyo experimentadas en HumanizaSUS como una tecnología de intervención en red para el fortalecimiento de los espacios virtuales del SUS o de los CiberespaSUS.

The paper of the HumanizaSUS Network (Rede HumanizaSUS) as a virtual environment connecting practices and knowledge about SUS (Brazilian National Health System), conveying the support function in a network. Based on the theoretical framework of Collective Intelligence, we present some dimensions of support experienced on Rede HumanizaSUS as a network intervention technology for strengthening the virtual environment of SUS or CiberespaSUS.

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern.

UNLABELLED: Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. OBJECTIVE: This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105(+) cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). MATERIAL AND METHODS: Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105(+) cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. RESULTS: PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. CONCLUSIONS: These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities.

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities. .