In vivo and in silico anti-inflammatory mechanism of action of the semisynthetic (-)-cubebin derivatives (-)-hinokinin and (-)-O-benzylcubebin.

(-)-Cubebin (CUB), isolated from seeds of Piper cubeba, was used as starting material to obtain the derivatives (-)-hinokinin (HK) and (-)-O-benzyl cubebin (OBZ). Using paw edema as the experimental model and different chemical mediators (prostaglandin and dextran), it was observed that both derivatives were active in comparison with both negative (5% Tween® 80 in saline) and positive (indomethacin) controls. The highest reduction in the prostaglandin-induced edema was achieved by OBZ (66.0%), while HK caused a 59.2% reduction. Nonetheless, the dextran-induced paw edema was not significantly reduced by either of the derivatives (HK or OBZ), which inhibited edema formation by 18.3% and 3.5%, respectively, in contrast with the positive control, cyproheptadine, which reduced the edema by 56.0%. The docking analysis showed that OBZ presented the most stable ligand-receptor (COX-2 - cyclooxygenase-2) interaction in comparison with CUB and HK.

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation.

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, ß-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and ß-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and ß-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and ß-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.

Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway.

The epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10 min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells.

Efficacy of sealing occlusal caries with a flowable composite in primary molars: A 2-year randomized controlled clinical trial.

OBJECTIVES: This randomized controlled clinical trial evaluated the efficacy of sealing carious dentin in controlling the progression of lesions in primary molars for 2-year follow-up. MATERIALS AND METHODS: Children (6.79 ±â€¯1.81 years, n = 28) presenting primary molars with occlusal caries in the outer half of dentine were randomized and allocated into 2 groups: test (sealing caries with a flowable resin - SC) and control (partial removal of caries followed by restoration - PRC). The primary outcomes were: the clinical success of restorations evaluated by USPHS criteria and the radiographic analysis of caries progression. The children anxiety was evaluated by a Facial Image Scale; and the time required to perform the treatments was registered. RESULTS: In 21 patients evaluated after 2 years, 48 primary molars were analyzed. Clinically, there was no difference between the groups. There was no difference between treatments (p = 0.848) considering lesion progression. The anxiety level did not change after the two interventions (p = 0.650). The treatment time of SC (9.03 ±â€¯1.91 min) was lower (p = 0.002) than the PRC time (17.13 ±â€¯5.26 min). CONCLUSION: Sealing carious dentin may be used in dentistry since it did not alter the children anxiety, reduced the chair time and demonstrated clinical success rate and no radiographic difference in relation to the partial caries removal followed by restoration.

Application of electrospray ionization mass spectrometry fingerprinting associated with macroscopic and histological analysis for Plantago major herbal infusions quality control.

The present study demonstrates the need to conduct an integrative quality control of teas marketed as Plantago major. In the present study, quality control checks were performed through macroscopic, microscopic and chemical analyses of commercial brands. Leaves with parts not allowed for consumption were found in all brands. Six brands showed histological similarity <50% with the authentic P. major. Eight brands demonstrated low chemical similarity (40 to 60%), indicating that they do not have the same compounds from the authentic P. major. All commercial samples are contaminated with aflatoxins, indicative of fungicide contamination and consequent degradation of phenolic compounds and reduction of antioxidant activity from the commercial samples. The difference in the present study is the integrative application of methods for success in the quality control of P. major, which can be applied to other species. Thus, more rigorous inspection actions in the production of teas to guarantee the therapeutic benefit of these products.

Avaliação da profundidade da cárie proximal nos exames radiográfico convencional e digitalizado / Evaluation of the size of proximal caries in radiography and digitized

Introdução: A cárie dentária é uma doença multifatorial que compreende vários fatores biológicos e sociais. A superfície proximal dos dentes é uma região de difícil visualização que pode esconder pequenas lesões cariosas no esmalte dentário, impossibilitando o diagnóstico através de inspeções visuais e táteis. Objetivo: O objetivo deste trabalho foi avaliar a profundidade da cárie proximal nos exames radiográficos convencionais e digitais, comparando as profundidades das lesões consideradas nestes exames às do exame histológico. Método: Foram utilizados exames radiográficos interproximais de 40 dentes humanos, 20 pré-molares e 20 molares, com alterações clínicas em uma das superfícies proximais, como lesões de mancha branca ou acastanhada e pequenas cavitações. Três profissionais especializados em radiologia odontológica com mais de cinco anos de experiência clínica mediram a profundidade das lesões pelos exames radiográfico e digital das amostras. Para obter os resultados, utilizou-se a técnica de análise de variância (ANOVA). Resultados: Constatou-se um nível de significância de 5% nas mensurações dos exames radiográficos convencionais e digitalizados, mostrando a fidelidade das imagens radiográficas em relação a real profundidade da lesão. Conclusão: Conclui-se que os exames de imagem avaliados foram eficientes na determinação da profundidade das lesões de cárie proximal.

Introduction: Dental caries is a multifactorial disease that comprises several biological and social factors. The proximal surface of the teeth is a region of difficult visualization that can hide small carious lesions in the dental enamel, making diagnosis through visual and tactile inspection infeasible. Objective: The objective of this study was to evaluate the depth of proximal caries in the conventional and digitized radiographic examinations, comparing the depths of the lesions considered in these examinations to those of the histological examination. Method: Interproximal radiographic examinations of 40 human teeth, 20 premolars and 20 molars, with clinical alterations on one of the proximal surfaces, such as white or brown spot lesions and small cavitations, were used. Three professionals specialized in dental radiology with more than five years of clinical experience measured the depth of the lesions by radiographic examination of the samples. To obtain the results, we used the technique of analysis of variance (ANOVA). Results: A level of significance of 5% was found in conventional and digitized radiographic measurements, showing the fidelity of the radiographic images in relation to the actual depth of the lesion. Conclusion: It was concluded that the imaging tests evaluated were efficient in determining the depth of proximal caries lesions.

Evaluation of the position of unerupted mandibular third molars with and without root dilacerations: a study on panoramic radiographs.

BACKGROUND: Factors that can directly influence the extraction of third molars include the position of the tooth and the presence of root dilacerations. Knowledge of these features favors an accurate therapeutic evaluation of third molars; therefore, the aim of this study was to evaluate, using panoramic radiographs, the positioning of unerupted third molars with and without root dilacerations and to verify a possible association between these two variables. METHODS: In this study, 16,136 panoramic radiographs were analyzed, including 1756 lower third molars, in which the positioning was assessed according to the Winter classification and the presence of root dilacerations was determined. The data obtained from the assessments of the frequencies of the positions and the presence of root dilacerations of impacted mandibular third molar was described. A χ2 test was applied to verify a possible association between the variables. RESULTS: The results indicated that the most frequent position was mesioangular (44.5%), followed by horizontal (24.9%), vertical (17.4%), distoangular (12.5%), inverted (0.4%), and linguoangular (0.3%). Of the mandibular third molars evaluated, 35% had root dilacerations. The chi-square test revealed a significant association between the position of the tooth and the presence of root dilacerations (P<0.0001, χ2=34.28). The frequency of root dilaceration was statistically higher for the vertical (45.5%) and distoangular positions (40.9%). CONCLUSIONS: The mesioangular position was the most prevalent location for lower third molars, and the highest frequencies of root dilacerations were observed in the vertical and distoangular positions. Knowledge about the prevalence of root dilacerations and the significant association between the position of the third molars and root dilacerations will allow safer surgical planning for dental extractions of third molars.

Local analgesic effect of tramadol is mediated by opioid receptors in late postoperative pain after plantar incision in rats.

Tramadol is a drug used to treat moderate to severe pain. It is known to present a peripheral effect, but the local mechanisms underlying its actions remain unclear. The role of peripheral opioid receptors in postoperative pain is not well understood. In the present study, we examined the peripheral opioid receptors to determine the local effect of tramadol in a plantar incision pain model. Rats were subjected to plantar incision and divided into four groups on postoperative day (POD) 1: SF_SF, 0.9% NaCl injected into the right hindpaw; SF_TraI, 0.9% NaCl and tramadol injected into the right hindpaw; SF_TraC, 0.9% NaCl and tramadol injected into the contralateral hindpaw; and Nal_Tra, naloxone and tramadol injected into the ipsilateral hindpaw. To determine the animals' nociceptive threshold, mechanical hyperalgesia was measured before incision, on POD1 before treatment and at 15, 30, 45, and 60 minutes after the incision. The same procedure was repeated on the POD2. The expression levels of µ-opioid receptor (MOR) and δ-opioid receptor (DOR) were obtained through immunoblotting assays in the lumbar dorsal root ganglia (L3-L6) in naïve rats and 1, 2, 3, and 7 days after the incision. Our results showed that the plantar incision was able to cause an increase in mechanical hyperalgesia and that tramadol reversed this hyperalgesia on POD1 and POD2. Tramadol injections in the contralateral paw did not affect the animals' nociceptive threshold. Naloxone was able to antagonize the tramadol effect partially on POD1 and completely on POD2. The DOR expression increased on POD2, POD3, and POD7, whereas the MOR expression did not change. Together, our results show that tramadol promoted a local analgesic effect in the postoperative pain model that was antagonized by naloxone in POD2, alongside the increase of DOR expression.

Cytoplasmic-targeted parvalbumin blocks the proliferation of multipotent mesenchymal stromal cells in prophase.

INTRODUCTION: Multipotent mesenchymal stromal cells (MSCs) have gained considerable interest because of their potential use in the treatment of a variety of diseases and injuries. Although remarkable advancements have been made in clinical studies, substantial concerns still regard the safety of MSCs. Some evidence suggests that MSCs can spontaneously generate a population of cells with tumorigenic potential. Thus, studying the molecular mechanisms that control the proliferation of MSCs may be a necessary step toward the development of strategies for safe clinical practice. Ca(2+) is a second messenger that mediates a wide range of cellular responses, including the regulation of cell proliferation, but little is known about its function in MSCs. The aim of this study was to investigate the effects of targeted Ca(2+) buffering on MSCs proliferation in vitro. METHODS: Here, we used an adenoviral (Ad) vector encoding the Ca(2+) chelator protein parvalbumin (PV) fused to a nuclear exclusion signal (NES) and the Discosoma red fluorescent protein (DsRed) to investigate the function of cytoplasmic Ca(2+) signals on MSC proliferation. Confocal microscopy was used to demonstrate that PV-NES-DsRed was expressed in the cytoplasm. Ca(2+) signaling was monitored by using Fluo-4-AM. Fluorescence-activated cell sorting (FACS) analysis of cells that were stained with propidium iodide was used as a quantitative measure of cell death. The mitotic index was assessed by immunofluorescence, and the expression of cyclins was examined with Western blot. RESULTS: Our results show that the Ad-PV-NES-DsRed fusion protein decreased serum-induced Ca(2+) signaling and blocked the proliferation of rat adipose-derived MSCs (AT-MSCs) in prophase. FACS analysis revealed that Ad-PV-NES-DsRed did not induce cell death in AT-MSCs. Furthermore, Western blot analysis demonstrated that Ad-PV-NES-DsRed reduced extracellular signal-regulated kinase (Erk1/2) phosphorylation and cyclin B1 expression. Buffering cytosolic Ca(2+) did not alter the expression of cyclins A/D1/D2/D3/E and E2. CONCLUSIONS: Our results show that cytoplasmic Ca(2+) signals are important for AT-MSCs progression beyond prophase because of their effects on Erk phosphorylation and cyclin B1 expression.

SET domain-containing Protein 4 (SETD4) is a Newly Identified Cytosolic and Nuclear Lysine Methyltransferase involved in Breast Cancer Cell Proliferation.

Cancer is comprised of a multitude of epigenetic abnormalities, including the global loss and regional gain of DNA methylation as well as alterations in histone methylation. Here, we characterize a new methyltransferase, SET domain-containing protein 4 (SETD4), which is involved in breast carcinogenesis. Quantitative real-time PCR (qPCR) showed elevated expression levels of SETD4 in several breast cancer cell lines. SETD4 overexpression was confirmed by western blot analysis suggesting a correlation between high expression of SETD4 and a lack of the estrogen receptor (ER) in breast cancer. In addition, cell fractionation studies and confocal immunofluorescence revealed the nuclear and non-nuclear localization of this new protein. SETD4 knockdown in breast cancer cell lines significantly suppressed their proliferation and delayed the G1/S cell cycle transition without affecting apoptosis. Furthermore, western blot analysis showed that knockdown of SETD4 decreased cyclin D1 expression, revealing the involvement of SETD4 in cell cycle regulation. These data imply that SETD4 plays a crucial role in breast carcinogenesis and could be a novel molecular target for the development of new strategies for the diagnosis and treatment of breast cancer.