Effect of experimental dentin etchants on dentin bond strength, metalloproteinase inhibition, and antibiofilm activity.

OBJECTIVE: this study evaluated dentin microtensile bond strength (µTBS) and failure modes (at 24 h and one year), bonding interface regarding hybridization, surface morphology regarding demineralization, in situ metalloproteinase (MMP) activity, and antibacterial effect of three dentin etchants compared to 35% phosphoric acid (PA). MATERIALS AND METHODS: The Adper Single Bond 2 adhesive (3 M Oral Care) was applied on moist dentin etched with PA (control) or on air-dried dentin etched with 3% aluminum nitrate + 2% oxalic acid (AN), 6.8% ferric oxalate + 10% citric acid (FO), or 10% citric acid (CA). The µTBS test used 40 human teeth (n = 10). Failure modes and surface morphology were analyzed by scanning electron microscopy (n = 3), while bonding interface morphology and MMP activity were evaluated by laser scanning confocal microscopy (n = 3). Antibacterial activity was evaluated against S. Mutans biofilm by means of viable cells count (CFU/mL). RESULTS: PA presented the highest bond strengths regardless of aging time. PA, AN, and CA showed stable bond strengths after one year of storage. Adhesive and mixed failures were predominant in all groups. Thin hybrid layers with short resin tags were observed for the experimental etchants. The AN-based etchant was able to inhibit MMP activity. All tested etchants presented antibacterial activity against S. Mutans biofilm. SIGNIFICANCE: This study suggests different dentin etchants capable of inhibiting MMP activity while also acting as cavity disinfectants.

Cathepsin B and Plasma Kallikrein Are Reliable Biomarkers to Discriminate Clinically Significant Hepatic Fibrosis in Patients with Chronic Hepatitis-C Infection.

We aimed to determine the biomarker performance of the proteolytic enzymes cathepsin B (Cat B) and plasma kallikrein (PKa) and transforming growth factor (TGF)-ß to detect hepatic fibrosis (HF) in chronic hepatitis C (CHC) patients. We studied 53 CHC patients and 71 healthy controls (HCs). Hepatic-disease stage was determined by liver biopsies, aminotransferase:platelet ratio index (APRI) and Fibrosis (FIB)4. Hepatic inflammation and HF in CHC patients were stratified using the METAVIR scoring system. Cat-B and PKa activities were monitored fluorometrically. Serum levels of TGF-ß (total and its active form) were determined using ELISA-like fluorometric methods. Increased serum levels of Cat B and PKa were found (p < 0.0001) in CHC patients with clinically significant HF and hepatic inflammation compared with HCs. Levels of total TGF-ß (p < 0.0001) and active TGF-ß (p < 0.001) were increased in CHC patients compared with HCs. Cat-B levels correlated strongly with PKa levels (r = 0.903, p < 0.0001) in CHC patients but did not correlate in HCs. Levels of Cat B, PKa and active TGF-ß increased with the METAVIR stage of HF. Based on analyses of receiver operating characteristic (ROC) curves, Cat B and PKa showed high diagnostic accuracy (area under ROC = 0.99 ± 0.02 and 0.991 ± 0.007, respectively) for distinguishing HF in CHC patients from HCs. Taken together, Cat B and PKa could be used as circulating biomarkers to detect HF in HCV-infected patients.

Semysinthetic biflavonoid Morelloflavone-7,4',7″,3‴,4‴-penta-O-butanoyl is a more potent inhibitor of Proprotein Convertases Subtilisin/Kexin PC1/3 than Kex2 and Furin.

BACKGROUND: Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. METHODS: Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. RESULTS: Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 µM and Ki = 0,59 µM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1ß secretion in Caco-2 cells. CONCLUSIONS: BF4 is a potent and selective inhibitor of PC1/3. GENERAL SIGNIFICANCE: BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

BRAF and NRAS mutated melanoma: Different Ca2+ responses, Na+/Ca2+ exchanger expression, and sensitivity to inhibitors.

Calcium is a ubiquitous intracellular second messenger, playing central roles in the regulation of several biological processes. Alterations in Ca2+ homeostasis and signaling are an important feature of tumor cells to acquire proliferative and survival advantages, which include structural and functional changes in storage capacity, channels, and pumps. Here, we investigated the differences in Ca2+ homeostasis in vemurafenib-responsive and non-responsive melanoma cells. Also, the expression of the Na+/Ca2+ exchanger (NCX) and the impact of its inhibition were studied. For this, it was used B-RAFV600E and NRASQ61R-mutated human melanoma cells. The intracellular Ca2+ chelator BAPTA-AM decreased the viability of SK-MEL-147 but not of SK-MEL-19 and EGTA sensitized NRASQ61R-mutated cells to vemurafenib. These cells also presented a smaller response to thapsargin and ionomycin regarding the cytosolic Ca2+ levels in relation to SK-MEL-19, which was associated to an increased expression of NCX1, NO basal levels, and sensitivity to NCX inhibitors. These data highlight the differences between B-RAFV600E and NRASQ61R-mutated melanoma cells in response to Ca2+ stimuli and point to the potential combination of clinically used chemotherapeutic drugs, including vemurafenib, with NCX inhibitors as a new therapeutic strategy to the treatment of melanoma.

The interaction of sodium trimetaphosphate with collagen I induces conformational change and mineralization that prevents collagenase proteolytic attack.

OBJECTIVES: This study evaluated the cell viability and expression of different major genes involved in mineralization in odontoblast-like cells exposed to sodium trimetaphosphate (STMP). It was also investigated the influence of STMP on the rate of calcium phosphate crystal growth, its anti-proteolytic action against the enzymatic degradation of type I collagen, the binding mechanism of STMP to collagen fibrils, and the potential mechanism to induce collagen stabilization. METHODS: Immortalized rat odontoblast MDPC-23 cells were cultured. Cell viability was assessed by trypan blue staining, and the changes in gene expression balance induced by STMP were assessed by quantitative reverse transcription (qRT) PCR assays. Crystalline particle formation was monitored by light-scattering detectors to estimate pH variation and the radial size of the crystalline particles as a function of reaction time (pH 7.4, 25°C) in the presence of STMP in supersaturated calcium phosphate solution (Ca/P=1.67). Images were obtained under atomic force microscopy (AFM) to measure the particle size in the presence of STMP. A three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams after immersion in STMP solution. The binding mechanism of STMP to collagen fibrils and potential stabilization mechanism was assessed with circular dichroism spectrometry (CD). The data were analyzed statistically (α=0.05). RESULTS: STMP had no significant influence on the cell viability and gene expression of the MDPC-23 cells. STMP greatly increased the rate of crystal growth, significantly increasing the average radial crystal size. AFM corroborated the significant increase of STPM-treated crystal size. Mineralized collagen I fibrils exhibited less collagenase degradation with lower STMP concentration. CD analysis demonstrated changes in the conformational stability after STMP binding to type I collagen. SIGNIFICANCE: The increased resistance of collagen against the proteolytic activity of collagenases appears to be related to the conformational change induced by STMP binding in collagen I and the STMP capacity for promoting biomimetic mineralization in type I collagen fibrils.

Na+/Ca2+ exchangers: Unexploited opportunities for cancer therapy?

Calcium is a well-studied ion that acts as a cofactor in several reactions and as intracellular second messenger. It plays crucial roles in living cells by regulating several processes from cell division to death. The disruption of Ca2+ homeostasis is related to cell and tissue damage and it is involved in several pathological conditions and diseases, including cancer. Tumor cells exhibit several molecular features in relation to normal cells in order to acquire proliferative and survival advantages, and Ca2+ signaling is directly or indirectly involved in these pathways. Thus, changes in the expression of Ca2+ channels and pumps are frequently described in some cancers, including transient receptor potential (TRP) family channels, store- and voltage-gated Ca2+ channels, store release channels, and Ca2+ ATPases. Although the sodium/calcium exchanger (Na+/Ca2+ exchanger; NCX) and the therapeutic potential of its inhibitors have been extensively studied in heart diseases, there are few studies about the molecular and functional aspects of NCX in cancer. Here, the current knowledge about NCX in cancer will be reviewed and possible strategies to target NCX for cancer therapy will be discussed.

Oxidative stress induced by self-adhesive resin cements affects gene expression, cellular proliferation and mineralization potential of the MDPC-23 odontoblast-like cells.

OBJECTIVE: Clinical issues have been raised about problems related to cytotoxic effects caused when applying self-adhesive cement. It was hypothesized that byproducts eluted from self-adhesive cements modulate oxidative stress response, the gene expression of signaling pathways of inflammatory process/transcriptional activators, and the expression and activity of interstitial collagenases, and modify the phenotypic characteristics of cellular proliferation and mineral deposition in odontoblastic-like cells. METHODS: Cements (MaxCem Elite [MAX] and RelyX U200 [U200)]) were mixed, dispensed into moulds, and photoactivated according to the manufacturers' instructions. Immortalized rat odontoblast-like cells (MDPC-23) were cultured and exposed to polymerized specimens of cements for 4 h. Reactive oxidative specimen production and quantification of gene expression were evaluated. Cell proliferation assay and alizarin red staining were also performed to evaluate the disturbance induced by the cements on cellular proliferation and mineralization. RESULTS: Despite their cytotoxic effects, both self-adhesive cements influenced the metabolism in the odontoblast cells on different scales. MAX induced significantly higher oxidative stress in odontoblast cells than U200. Gene expression varied as a function of exposure to self-adhesive cements; MAX induced the expression of pro-inflammatory cytokines such as TNF-α, whereas U200 downregulated, virtually depleted TNF-α expression, also inducing overexpression of the transcriptional factor Runx2. Overexpression of heme oxygenase-1 (HO-1) and thioredoxin reductase 1 (TRXR1) occurred after exposure to both cements, antioxidant genes that are downstream of Keap1-Nrf2-ARE system. MAX significantly induced the overexpression of collagenase MMP-1, and U200 induced the expression of gelatinase MMP-2. MAX significantly inhibited cell proliferation whereas U200 significantly activated cell proliferation. Alizarin red staining revealed significantly decreased mineral deposition especially when exposed to MAX. SIGNIFICANCE: These results support the hypothesis that byproducts of different self-adhesive cements play important roles in the highly orchestrated process which ultimately affect the cellular proliferation and the mineral deposition in odontoblastic-like cells, possibly delaying the reparative dentin formation after cementation of indirect restorations, especially on recently exposed dentin preparations.

Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol.

OBJECTIVES: To evaluate the cytotoxic effects of exposing odontoblast cells to a variety of commercial self-adhesive cements polymerized using different activation modes. METHODS: Five cements: MaxCem Elite (MAX), Bifix SE (BSE), G-Cem LinkAce (GCE), Clearfil SA Luting (CAS), and RelyX U200 (U200) were mixed, dispensed into molds, and distributed in groups, according to polymerization protocols: immediate photoactivation; delayed photoactivation (10min self-curing plus light-activation); and chemical activation (no light exposure). Immortalized rat odontoblast cells (MDPC-23) were cultured. Cell viability was assessed by Trypan Blue staining and total cell death was assessed by annexin V-APC/7-AAD double staining and flow cytometry. Volatilized compounds from polymerized specimens of cements were evaluated by gas chromatography/mass spectrometry (GC-MS). Data was analyzed with 2-way ANOVA/Tukey tests (α=0.05). RESULTS: Exposure to all of the cements tested significantly reduced the cell viability, irrespective of the activation protocol (p<0.05). The least harmful cements were CSA and U200. Total death of cells significantly increased when exposed to BSE, GCE, and MAX, especially when chemically activated (p<0.05). Characteristic apoptotic cells increased after exposure to cements, mainly for MAX, regardless of the activation mode. Chemical activation of MAX also induced necrosis. Moreover, GCE and MAX exhibited higher percentages of late apoptotic/dead cells. Chromatograms revealed 28 compounds released from the cements tested, some of them with known carcinogenic effects. Selection of self-adhesive cements and polymerization protocols affect the cytotoxicity and cell viability of odontoblastic cells. CLINICAL SIGNIFICANCE: Despite the simplified cementation protocol, care is needed when cementing indirect restorations with self-adhesive cements, especially on recently exposed dentin. This category of material may cause differential cytotoxic effects and should be considered when selecting a cement. This is particularly true in clinical cases of light attenuation, where the polymerization depends on chemical activation, inducing higher cytotoxic damages when using some of the cements tested.

Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen Xylella fastidiosa, Involved in the Synthesis of Bacterial Lipopolysaccharide.

Xylella fastidiosa is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce's disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to X. fastidiosa subsp. pauca, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of X. fastidiosa genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as XfUxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the X. fastidiosa uxs gene affects the LPS structure among CLS-related X. fastidiosa strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts.

Co-distribution of cysteine cathepsins and matrix metalloproteases in human dentin.

It has been hypothesized that cysteine cathepsins (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin. Activities of CTs and MMPs in dentin extracts were evaluated spectrofluorometrically. In addition, in situ gelatinolytic activity of dentin was assayed by zymography. The results revealed the distribution of CT-B and CT-K along the dentin organic matrix and also indicated co-occurrence of MMPs and CTs in that tissue. The enzyme kinetics studies showed proteolytic activity in dentin extracts for both classes of proteases. Furthermore, it was observed that, at least for sound human dentin matrices, the activity of MMPs seems to be predominant over the CTs one.

Heparan sulfate mediates trastuzumab effect in breast cancer cells.

BACKGROUND: Trastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components--heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)--in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab. METHODS: The cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate. RESULTS: Breast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in these resistant cells. CONCLUSION: Trastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.

Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans.

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.