The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts.

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Thyroid hormone upregulates MDM2 in rat type I fibre: Implications for skeletal muscle mass regulation.

AIM: Based upon a microarray assay, we have identified that triiodothyronine (T3) upregulates MDM2 gene expression in the rat skeletal muscle. As MDM2 protein is an E3 ligase, we hypothesized that this enzyme could play a role in T3 effects on skeletal muscle mass control. METHODS: To test our hypothesis, male rats (2 months old) were randomly assigned into the following groups: intact controls, treated with 20 physiological doses of T3 for 0.5, 1 and 7 days, or with 5, 20 and 50 physiological doses of T3 for 7 days. For in vitro experiments, myotubes and C2C12 cells were treated with T3 for 3 days. RESULTS: After validation of the microarray finding throughout RT-PCR and confirmation that T3 induces increases in MDM2 protein expression in a dose-dependent manner, we observed that MDM2 was upregulated by T3 exclusively in fibre type I. Moreover, detailed histological evaluation showed that MDM2 overexpression distributes punctiformily along the cross section of the fibre and also inside nuclei. MDM2 colocalizes with PAX7 in control muscle and T3 downregulates this myogenic factor. Pharmacological inhibition of MDM2 in cultured myotubes caused a severe decrease in their diameter (~35%, P < .001 vs Control), enhancing the effect of T3 (from ~12% to ~35%, P < .001) alone upon myotube diameter and mRNA levels of atrogenes. Finally, we observed that FOXO3 (MDM2 target) is kept outside the nucleus under T3 stimulation. CONCLUSION: Our results indicate that MDM2 might be involved in the pro-trophic effects of T3 in skeletal muscle.

Ramos do arco aórtico e da aorta torácica em bicho-preguiça (Bradypus variegatus) / Branches of the aortic arch and thoracic aorta of the sloth (Bradypus variegatus)

As preguiças são mamíferos arborícolas, bem específicos quanto aos hábitos e à morfofisiologia. Essa condição faz delas ainda mais vulneráveis aos impactos ambientais. Com o objetivo de fornecer informações que auxiliem na clínica médico-cirúrgica desses Xenarthras, realizou-se um estudo do arco aórtico e da artéria aorta torácica, a fim de se identificarem seus principais ramos. Foram utilizados 10 animais da espécie Bradypus variegatus, sendo uma fêmea e três machos submetidos a dissecação, por meio da qual foram obtidas medidas de diâmetro e comprimento dos vasos, enquanto três fêmeas e três machos destinaram-se à técnica de radiografia digital com contraste. Constatou-se uma sintopia uniforme do arco, bem como da porção descendente da artéria aorta no tórax. No entanto, a topografia difere consideravelmente. Com base nos ramos, percebeu-se, independentemente do sexo, uma maior variação naqueles originados do arco e dos que emergem da artéria aorta torácica e irrigam estruturas medianas e do antímero direito do tórax. Foram obtidos valores maiores para o diâmetro das artérias na fêmea do que nos machos e, para o comprimento da artéria aorta torácica, foram encontrados valores correspondentes em ambos. Toda essa variação dos bradipodídeos apresenta diferenças em relação a outros mamíferos.(AU)

Sloths are arboreal mammals, very specific, regarding their habits and morphology. This condition makes them even more vulnerable to environmental impacts. In order to provide information to assist in the medical and surgical assistance to these Xenarthras, a study of the aortic arch and thoracic portion of the descending aorta was carried out with the purpose of identify its main branches. Ten animals of the Bradypus variegatus species were used. One female and three males were submitted to dissection, where measurements of vessel diameter and length were obtained, while three females and three males were submitted to the digital contrast x-ray technique. There was a uniform syntopy of the arch, as well as of the descending portion of the aorta in the thorax. However, the topography differs considerably. Based on the branches, regardless of gender, a greater variation was observed in those originating from the arch, and those that emerge from the thoracic aorta and irrigate medial structures and the right antimere of the thorax. Greater values were obtained for the diameter of the arteries in the female than in the males and corresponding values for the length of the thoracic aorta in both. All this variation is possibly related to the adaptive processes of Bradipodidae over time.(AU)

Fracture Load and Phase Transformation of Monolithic Zirconia Crowns Submitted to Different Aging Protocols.

Monolithic zirconia crowns have many favorable properties and may potentially be used to solve dental problems such as chipping. However, monolithic zirconia crown resistance can be affected by its phase transformation when subjected to low temperatures, humidity, and stress. This study evaluated the fracture load and phase transformation of monolithic zirconia crowns submitted to different thermal and mechanical aging tests. Seventy monolithic zirconia crowns were randomly divided into the following five groups: control, no treatment; hydrothermal aging at 122°C, two bar for one hour; thermal fatigue, 104 cycles between 5°C and 55°C, dwell time, 30 seconds; and mechanical fatigue, 106 cycles with a load of 70 N, sliding of 1.5 mm at 1.4 Hz; and combination of mechanical plus thermal fatigue. Fracture load was measured with a universal testing machine. Surface changes and fracture mode and origin were examined with a scanning electron microscope. Monoclinic phase content was evaluated by x-ray diffraction. The fracture load was analyzed using one-way analysis of variance at a level of 5%, and Weibull distribution was performed. No statistically significant differences were observed in the mean fracture load and characteristic fracture load among the groups (p>0.05). The Weibull modulus ranged from 6.2 to 16.6. The failure mode was similar for all groups with the crack origin located at the contact point of the indenter. Phase transformation was shown at different surfaces of the crown in all groups (1.9% to 8.9%). In conclusion, monolithic zirconia crowns possess high fracture load, structural reliability, and low phase transformation.

Effect of antimicrobial agents incorporated into resilient denture relines on the Candida albicans biofilm.

OBJECTIVE: The antimicrobial action of five drugs incorporated in temporary denture relines on the fungal biofilm was evaluated. MATERIALS AND METHODS: A Candida albicans biofilm (SC5314) was formed on specimens (10 × 1 mm) of materials (Trusoft and Softone) modified or not (control) by the drugs (nystatin, miconazole, ketoconazole, itraconazole, and chlorhexidine diacetate). Cell viability was determined spectrophotometrically by the tetrazolium salt reduction assay (XTT) after 24 h, 48 h, and 7 and 14 days of incubation. The minimum inhibitory concentrations (MICs) were those which inhibited 90% or more of fungal growth. Fungal susceptibility was confirmed by confocal laser scanning microscopy analysis. RESULTS: The MICs of drugs incorporated in the materials were 0.032, 0.256, 0.128, 0.256, and 0.064 g ml(-1) for nystatin, miconazole, ketoconazole, itraconazole, and chlorhexidine, respectively. Images from nystatin, chlorhexidine, and ketoconazole demonstrated no viable cells. CONCLUSIONS: The antimicrobials incorporated in the resilient materials inhibited fungal growth during 14 days, with lower MICs for nystatin and chlorhexidine.

Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma.

OBJECTIVE: The aim of this study was to evaluate the composition of the salivary pellicle (SP) and multispecies biofilm developed on titanium nitrided by cold plasma. METHODS: Titanium discs were allocated into a control group (Ti) and an experimental group (TiN - titanium-nitrided by cold plasma). The disc surface topography was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemical composition of the disc surface was determined by X-ray photoelectron spectroscopy (XPS). Stimulated, clarified, and filtered saliva was used to form pellicles on the discs. Proteome analysis of the adsorbed SP proteins was performed by liquid chromatography-mass spectrometry (LC-MS). The surface free energy (SFE) was evaluated before and after SP formation. A multispecies biofilm composed of Actinomyces naeslundii, Streptococcus oralis, Streptococcus mutans, Fusobacterium nucleatum, Veillonella dispar, and Candida albicans was developed on the SP-coated discs. Viable microorganism counts were determined. The biomass and average thickness of biofilms were analyzed by confocal laser-scanning microscopy (CLSM) with COMSTAT software. The biofilm organization was visualized by SEM. RESULTS: The surface topography was similar in both groups. The SFE of the TiN group did not differ from that of the Ti group (p>0.05), although the adsorption of pellicle proteins increased the SFE in both pellicle-coated groups (p<0.001). Different proteins were identified on the Ti and TiN surfaces. The amount of biofilm was similar for both groups (p=0.416), but the counts of F. nucleatum and S. oralis were higher in the TiN group (p<0.001). Similar biofilms were characterized by the COMSTAT data, CLSM images, and SEM images. CONCLUSION: The titanium nitrided by cold plasma exhibited differences in SP composition and multispecies microbial biofilm population compared to the control titanium surface.

Effects of undecylenic acid released from denture liner on Candida biofilms.

Denture liners (DL) are easily colonized by Candida spp. In an attempt to prevent biofilm colonization, manufacturers have incorporated undecylenic acid (UDA) into DL. In this in vitro study, the effects of UDA released from DL on Candida biofilms were investigated. The concentrations of UDA released from commercial DL were determined by gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) and minimum fungistatic concentration (MFC) tests were performed for C. albicans or C. glabrata, with UDA for comparison with the concentrations released from DL. Specimens of DL with (experimental group) and without UDA (control group) were fabricated, and Candida biofilms were developed on DL surfaces. Biofilms were evaluated by cell counts, metabolic activity, structure, and secretion of proteinase or phospholipase. The concentrations of UDA released were within the MIC and MFC ranges. In the presence of UDA, C. albicans biofilms were thinner and had lower numbers of viable and active cells, although no significant enzymatic changes were observed relative to the control group (p > 0.05). In contrast, C. glabrata biofilms exhibited higher cell counts and greater metabolic activity and also increased proteinase activity in the presence of UDA relative to the control group (p < 0.05). Overall, UDA did not prevent Candida biofilm formation.

Effect of acidulated phosphate fluoride gel application time on enamel demineralization of deciduous and permanent teeth.

Although the effect of acidulated phosphate fluoride gel (APF gel) on caries reduction in permanent teeth is based on evidence, the relevance of the clinical application time is still under debate. Also, the effect of 4- versus 1-min application has not been evaluated in deciduous enamel. In a blind, crossover, in situ study of 14 days, 16 adult volunteers wore palatal appliances containing slabs of human permanent and deciduous enamel. At the beginning of each phase, the slabs were submitted to one of the following treatments: no APF application (negative control); APF gel (1.23% F) application for 1 or 4 min. Biofilm accumulation on the slab surface was allowed and the slabs were subjected eight times a day to 20% sucrose, simulating a high cariogenic challenge condition. On the 15th day of each phase, fluoride retained as CaF(2) and fluorapatite (FAp) was determined on the enamel of the slabs and demineralization was assessed by cross-sectional microhardness. Fluoride as CaF(2) and FAp, formed by APF gel application on the enamel slabs not subjected to the cariogenic challenge, was also determined. APF gel reduced demineralization in both enamel types (p < 0.05), but the difference between 1 and 4 min was not statistically significant (p > 0.05). CaF(2) and FAp formed and retained on deciduous and permanent enamel was significantly higher in APF gel groups (p < 0.05), but no significant difference was found between 1 and 4 min (p > 0.05). The findings suggest that 1 min of APF gel application provides a similar effect on inhibition of demineralization as 4 min, for both permanent and deciduous enamel.

Patients' satisfaction and functional assessment of existing complete dentures: correlation with objective masticatory function.

The aim of this study was to evaluate the correlation between patients' and dentist's assessment of dentures and to correlate these variables with objective measures of masticatory function. A sample of 28 edentulous individuals was selected, all wearing both complete dentures for at least 6 months and with no signs or symptoms of temporomandibular joint disorders. They rated their level of satisfaction with their dentures from 0 to 100 by means of a visual analogue scale, and dentures were scored by a dentist from 0 to 9 considering functional aspects. Tooth wear was evaluated on the posterior teeth of dentures considering the lack of occlusal anatomy. Masticatory performance and swallowing threshold tests were performed with an artificial test food (Optocal), and the median particle size was determined by the sieving method. The results showed that the mean satisfaction value of volunteers with their dentures was 49·1 and the median score of dentist's evaluation was 6. The Spearman correlation coefficient revealed no significant correlation between patients' and dentist's assessment of dentures (P>0·05). The median particle size for masticatory performance and swallowing thresholds was 5·5 ± 1·0 and 4·9 ± 1·2 mm, respectively. Data of both masticatory tests showed no significant correlation with patients' satisfaction or with dentist's evaluation of dentures. There was no difference of food comminution between subjects with and without excessive posterior tooth wear. It can be concluded that dentist's and patients' assessment of dentures were not correlated, and no correlation was observed between these variables and masticatory function.

Kinetics of monofluorophosphate hydrolysis in a bacterial test plaque in situ.

Models to evaluate the anticaries potential of fluoride (F) formulations containing monofluorophosphate (MFP) should consider the release of F ion to the oral environment by its enzymatic hydrolysis. This was tested in situ, using a test plaque of a strain of Streptococcus mutans which presents high MFPase activity at pH 5.0. The test plaque was exposed to non-F or MFP (1,450 microg F/g) dentifrices and the fluid phase of the plaque was analyzed after 15, 30, 45 and 75 min. MFP concentration in the plaque fluid decreased over time after exposure to MFP dentifrice, but F ion reached 134.9 +/- 32.0 microM at 15 min and decreased significantly only at 75 min, suggesting continuous MFP hydrolysis by the test plaque.

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms.

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.

Effects of essential oils on Aedes aegypti larvae: alternatives to environmentally safe insecticides.

The essential oils from leaves of Hyptis fruticosa (Lamiaceae) Salzm., H. pectinata (Lamiaceae) Poit., and Lippia gracilis (Verbenaceae) HBK were investigated for their larvicidal activity against Aedes aegypti and analyzed by GC/MS. Fifty-nine compounds, representing 91.28-98.39% of the essential oils, have been identified. A standard solution was used to make 20 mL solutions ranging from 30 to 2000 ppm. Twenty larvae between third and fourth stages were added to the essential oil solution. A mortality count was conducted 24 h after treatment. Essential oils LC50 and their confidence limits at 95% probability were calculated by the methods of Reed-Muench and Pizzi, respectively. The essential oil of Lippia gracilis showed potent insecticidal effect against Aedes aegypti larvae, the vector of dengue fever. Carvacrol and caryophyllene oxide were the main responsible for the activity of L. gracilis and H. pectinata. Minor compounds are probably acting synergistically to achieve H. fruticosa activity.

In vitro efficacy of extracts of Melia azedarach against the tick Boophilus microplus.

The efficacy of ripe fruit extracts of Melia azedarach L. (Rutales: Meliaceae) was evaluated against the tick, Boophilus microplus (Canestrini) (Acari: Ixodidae). Ripe fruits of M. azedarach dried and powdered were extracted by Soxhlet apparatus successively using hexane, CHCl3 and 96% aqueous ethanol. Larvae and engorged females were immersed in decreasing concentrations from 0.25% to 0.015% of each extract. The mortality of larvae was evaluated 24, 72 and 168 h after treatment. The effectiveness of treatment against engorged females was assessed by measuring egg production. All tested extracts caused mortality of B. microplus larvae, with higher mortality rates observed in CHCl3 (100%) and hexanic extract (98%) than in ethanolic extract (50%) 168 h after treatment. The mortality was dependent on concentration and on time after treatment. Similarly hexanic and CHCl3 extracts showed higher effectiveness (varying from 14% to 100%) against B. microplus engorged females than ethanolic extract (varying from 0% to 46%). Melia azedarach extracts did not kill the adult females, but inhibited partially or totally egg production and embryogenesis. These results show that the less polar the extract of M. azedarach ripe fruits the more its effectiveness against larvae and engorged females of B. microplus. This plant may therefore be useful in the control of resistant B. microplus populations.

Role of positive end-expiratory pressure changes on functional residual capacity in surfactant treated preterm infants.

Both surfactant replacement and positive end-expiratory pressure (PEEP) increase lung volume in infants with respiratory distress syndrome (RDS). We measured pulmonary mechanics and functional residual capacity (FRC) in 21 preterm infants with RDS, > 48 hr post-surfactant therapy (BW, 1,168 +/- 441 g; GA, 28.3 +/- 2.8 weeks; postnatal age, 3-7 days). A non-linear but significant increase in mean FRC was noted as PEEP increased from 2 to 5 cmH2O: 18.4 +/- 4.7 mL/kg at 2 cmH2O; 19.7 +/- 4.3 mL at 3 cmH2O; 22.6 +/- 5.5 ml/kg at 4 cmH2O; and 26.2 +/- 6.2 mL/kg at 5 cmH2O (P < 0.01). Because of the synergistic combined effect on lung volume, surfactant treated neonates should be weaned cautiously from PEEP during ventilatory management. Our study also suggests that the occurrence of inadvertent end-distending pressure during FRC measurement in the ventilated neonate lead to erroneous results.

Tissue culture and plant regeneration in diploid perennial teosinte.

High frequency plant regeneration is described for Zea diploperennis callus from somatic tissues. Small callus fragments were excised after 3-4 passages on primary medium (MS salts containing 2,4-D) and transferred to regeneration medium (without 2,4-D). After 10-15 days, numerous shoots were observed which grew to normal plantlets following separation and transfer to a rooting medium. Presently, ca. 500 plants have been recovered from which approximately 80 plants are growing in small pots in a nursery and ca. 50 plants have already produced normal flowers and seeds under field conditions. The culture of somatic tissues of interspecific hybrids between cultivated corn and teosinte may offer new success for in vitro plant regeneration.

Lysine-ketoglutarate reductase activity in developing maize endosperm.

Lysine-ketoglutarate reductase activity was detected and characterized in the developing endosperm of maize (Zea mays L.). The enzyme showed specificity for its substrates: lysine, alpha-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was close to 7, and the K(m) for lysine and alpha-ketoglutarate were 5.2 and 1.8 millimolar, respectively.