Mutanase from Trichoderma harzianum inductively Produced by Mutan: Short-Term Treatment to Degrade Mature Streptococcus mutans Biofilm.

This study aimed to evaluate the disruptive effect of fungal mutanase against cariogenic biofilm after short-term treatment. For that, mature Streptococcus mutans biofilms (n = 9) were exposed to active or inactivated enzymes produced by Trichoderma harzianum for 1 min, two times per day. Biofilms were analyzed by amount of matrix water-insoluble polysaccharides, bacterial viability, acidogenicity, and morphology by scanning electron microscopy (SEM). The group treated with active enzymes (AE) had a significantly lower amount of insoluble polysaccharides (893.30 ± 293.69) when compared to the negative control group (NaCl, 2192.59 ± 361.96), yet no significant difference was found when comparing to the positive control group (CHX, 436.82 ± 151.07). Also, there was no significant effect on bacteria metabolism and viability (P-value < 0.05). Data generated by the quantitative analysis were confirmed through scanning electron microscopy images. Thus, fungal mutanase degraded the biofilm after a short-term treatment without interfering with bacterial viability and metabolism. Such findings offer insight to the development of routine oral care products containing this input.

Mango peel as a potential enzyme inducer in Trichoderma harzianum: a strategy for cariogenic biofilm degradation and reuse of industrial waste

Water-insoluble exopolysaccharides (I-EPS) are a virulence factor for dental biofilms. It has already been demonstrated that mango pulp induces the secretion of glucan-hydrolytic enzymes in the fungus Trichoderma harzianum, and that they have an effect on I-EPS from young biofilms. Aim: Evaluate the effect of mango peel as an enzyme inducer in T. harzianum, and the effect of enzymes secreted on mature biofilms. Methods: Fractions of the peel (PL) and ethanol-precipitated pulp (PP) of Tommy Atkins mangoes were sterilized and added to a culture medium containing T. harzianum for induction of hydrolytic enzymes. After 192 h, the culture medium was centrifuged and the supernatant (enzyme extract) was used as treatment on S. mutans biofilms (n=9): a) NaCl 0.9 %; b) 0.12 % chlorhexidine digluconate; and c) extract of enzymes induced by PL or PP. Acidogenicity, bacterial viability, quantification of insoluble polysaccharides, and three-dimensional analysis of the biofilm by scanning electron microscopy (SEM) was performed. Data were analyzed by ANOVA followed by the Tukey test (α=5 %). Results: The hydrolytic enzymes did not alter the metabolism or bacterial viability of the biofilm (p<0.05). Although the images obtained by SEM suggest some degree of matrix degradation, the quantification of I-EPS for the PL and PP groups did not differ from the control group (p>0.05), suggesting a slight effect on the disorganization of the mature S. mutans biofilm. Conclusion: The results suggest that mango peel fraction can induce secretion of mutanase by T. harzianum, however in an insufficient amount to generate significant degradation on cariogenic biofilm.

Liquid crystalline phase nanodispersions enable skin delivery of siRNA.

The ability of small interfering RNAs (siRNAs) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for the widespread use of this technology in skin is the lack of an effective delivery system. The aim of the present study was to evaluate nanodispersed systems in liquid crystalline phases that deliver siRNA into the skin. The proposed systems present important properties for the delivery of macromolecules in a biological medium, as they are formed by substances that have absorption-enhancing and fusogenic effects; additionally, they facilitate entrapment by cellular membranes due to their nano-scale structure. The cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations, and after dispersion in aqueous medium, liquid crystalline phase nanodispersions were obtained and characterized by their physicochemical properties. Then, in vitro penetration studies using diffusion cell and pig ear skin were carried out to evaluate the effect of the nanodispersions on the skin penetration of siRNA; based on these results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo for skin penetration, skin irritation, and the ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the treatment of skin disorders; they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment.

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120mmHg for 45min, which was followed by 15min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15min. In the RBM exposed to 3mM phosphate and/or 100µM Ca(2+), C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment.

Comparative evaluation of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans.

Streptococcus mutans triggers dental caries establishment by two major factors: synthesis of organic acids, which demineralize dental enamel, and synthesis of glucans, which mediate the attachment of bacteria to the tooth surface. Propolis is a natural product that may prevent dental caries. Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, is the most important botanical origin for the production of green propolis (Brazilian propolis) by honeybees. However, whether B. dracunculifolia (Bd) has an anticariogenic effect, like green propolis, remains unknown. Herein, we have made a comparative evaluation of the effects of extracts from green propolis and Bd on the glucan synthesis and acidogenic potential of S. mutans. The inhibitory effects of the extracts on bacterial acid production were evaluated through the potentiometric measurement of pH from bacterial suspensions treated with serial concentrations of both extracts. Besides presenting close inhibitory values at the same concentration range, Bd leaf rinse and green propolis extracts had similar IC(50) values (0.41 and 0.34 mg/ml, respectively). Both extracts produced a bacteriostatic effect on S. mutans cultures at a concentration of 0.40 mg/ml. Estimated inhibitory values of green propolis and Bd leaf rinse extracts on the synthesis of insoluble glucans (IC(50)=12.9 and 25.0 microg/ml, respectively) and soluble glucans (IC(50)=50.4 and 49.1 microg/ml, respectively) were not significantly different from each other at p<0.05. The results demonstrate that Bd leaf rinse and green propolis extracts have similar inhibitory effects on the S. mutans cariogenic factors evaluated herein, and allowed us to suggest that Bd leaves may be a potential source for pharmaceutical products employed for this purpose.

Clinical and biochemical evaluation of the saliva of patients with xerostomia induced by radiotherapy.

Clinical aspects and biochemical properties in the saliva of 21 patients prior to and following radiotherapy for head and neck cancer were evaluated (experimental group) and compared with the same properties in a control group of 21 subjects free of cancer. Salivary flow was evaluated by measuring the time necessary, in seconds, for the output of 2 ml of stimulated saliva; and the buffering capacity changes were determined using a simple colorimetric method. Total salivary protein concentration was determined by the Bradford 4 method. Amylase activity was measured by reducing sugars released from a soluble starch substrate, quantified by the dinitrosalicylic method. The electrophoretic profile was evaluated in polyacrylamide gel (12% SDS-PAGE) using samples of 5 mg of salivary protein. A statistically significant reduction (p < 0.01) of the salivary flow was observed, (162.47 s +/- 28.30 before and 568.71 s +/- 79.75 after irradiation), as well as a reduction in the salivary buffering capacity (pH 5.45 +/- 0.14 before and pH 4.40 +/- 0.15 after irradiation). No statistically significant alteration was observed in total salivary protein concentration. A statistically significant reduction (p < 0.01) of salivary alpha-amylase activity (856.6 ng/mg +/- 88.0 before and 567.0 ng/mg +/- 120.6 after irradiation) was observed. Electrophoretic profile differences in salivary protein bands were also observed after radiotherapy, mainly in the range of molecular weight of 72000 to 55000 Daltons. Clinically, patients presenting xerostomia induced by radiotherapy presented an increase in oral tissue injury.

Clinical and biochemical evaluation of the saliva of patients with xerostomia induced by radiotherapy

Foram avaliados alguns aspectos clínicos e algumas propriedades bioquímicas salivares de 21 pacientes, antes e após o tratamento radioterápico para câncer de cabeça e pescoço (grupo experimental) e de 21 pacientes sem câncer (grupo controle). O fluxo salivar foi avaliado pelo tempo necessário (segundos) para produção estimulada de 2 ml de saliva e a capacidade tamponante determinada frente à utilização de um método colorimétrico simples. A concentração de proteína total salivar foi determinada pelo método de Bradford4. A atividade da amilase foi mensurada através dos açúcares redutores liberados e quantificados pelo método do ácido dinitrossalicílico utilizando a glicose como substrato. O perfil eletroforético foi avaliado em gel de poliacrilamida (SDS-PAGE 12%) para amostras salivares contendo 5 mg de proteína. Foi observada, no grupo experimental, redução estatisticamente significativa (p < 0,01) para o fluxo salivar (162,47 s ± 28,30 antes e 568,71 s ± 79,75 após) e para a capacidade tamponante (pH 5,45 ± 0,14 antes e 4,40 ± 0,15 após). Não foi observada alteração estatisticamente significativa na concentração de proteína. A atividade específica da a-amilase foi significativamente diminuída (p < 0,01) (856,6 ng/mg ± 88,0 antes e 567,0 ng/mg ± 120,6 após). No perfil eletroforético, foram observadas diferenças nas bandas protéicas, principalmente na faixa de peso molecular de 72.000 a 55.000 Da. Clinicamente, os pacientes com xerostomia induzida pela radioterapia apresentaram aumento de lesões na mucosa.

Evaluation of the influence of cetylamine fluoride and sodium fluoride on acid production by S. mutans

The acidogenic character of Streptococcus mutans has been pointed out as one of the most important physiological aspects associated with dental caries establishment. Fluoride is a key component of oral health promotion. Amine fluorides (AmF) have been used in products such as mouth rinses, toothpastes, gels and other topical fluoride agents for over 30 years in several European countries. In Brazil, the use of such substances was limited to toothpaste until August 2000, when a new resolution was published to approve the use of three AmFs in mouth rinses and other formulations for odontological purposes. In this report the influence of cetylamine fluoride (CAmF) and sodium fluoride (NaF) on acid production...