Hemoglobin Protects Enamel against Intrinsic Enamel Erosive Demineralization.

INTRODUCTION: This study investigated the changes in the acquired enamel pellicle (AEP) proteome when this integument is formed in vivo after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), and a statherin-derived peptide (StN15), or their combination and then exposed to an intrinsic acid challenge. The effectiveness of these treatments in preventing intrinsic erosion was also evaluated. METHODS: Ten volunteers, after prophylaxis, in 5 crossover phases, rinsed with the following solutions (10 mL, 1 min): control (deionized water-H2O) - group 1, 0.1 mg/mL CaneCPI-5 - group 2, 1.0 mg/mL HB - group 3, 1.88 × 10-5M StN15 - group 4, or a blend of these - group 5. Following this, AEP formation occurred (2 h) and an enamel biopsy (10 µL, 0.01 m HCl, pH 2.0, 10 s) was conducted on one incisor. The biopsy acid was then analyzed for calcium (Arsenazo method). The vestibular surfaces of the other teeth were treated with the same acid. Acid-resistant proteins in the residual AEP were then collected and analyzed quantitatively via proteomics. RESULTS: Compared to control, treatment with the proteins/peptide, mixed or isolated, markedly enhanced acid-resistant proteins in the AEP. Notable increases occurred in pyruvate kinase PKM (11-fold, CaneCPI-5), immunoglobulins and submaxillary gland androgen-regulated protein 3B (4-fold, StN15), Hb, and lysozyme C (2-fold, StN15). Additionally, a range of proteins not commonly identified in the AEP but known to bind calcium or other proteins were identified in groups treated with the tested proteins/peptide either in isolation or as a mixture. The mean (SD, mM) calcium concentrations released from enamel were 3.67 ± 1.48a, 3.11 ± 0.72a, 1.94 ± 0.57b, 2.37 ± 0.90a, and 2.38 ± 0.45a for groups 1-5, respectively (RM-ANOVA/Tukey, p < 0.05). CONCLUSIONS: Our findings demonstrate that all treatments, whether using a combination of proteins/peptides or in isolation, enhanced acid-resistant proteins in the AEP. However, only HB showed effectiveness in protecting against intrinsic erosive demineralization. These results pave the way for innovative preventive methods against intrinsic erosion, using "acquired pellicle engineering" techniques.

Abl depletion via autophagy mediates the beneficial effects of quercetin against Alzheimer pathology across species.

Alzheimer's disease is the most common age-associated neurodegenerative disorder and the most frequent form of dementia in our society. Aging is a complex biological process concurrently shaped by genetic, dietary and environmental factors and natural compounds are emerging for their beneficial effects against age-related disorders. Besides their antioxidant activity often described in simple model organisms, the molecular mechanisms underlying the beneficial effects of different dietary compounds remain however largely unknown. In the present study, we exploit the nematode Caenorhabditis elegans as a widely established model for aging studies, to test the effects of different natural compounds in vivo and focused on mechanistic aspects of one of them, quercetin, using complementary systems and assays. We show that quercetin has evolutionarily conserved beneficial effects against Alzheimer's disease (AD) pathology: it prevents Amyloid beta (Aß)-induced detrimental effects in different C. elegans AD models and it reduces Aß-secretion in mammalian cells. Mechanistically, we found that the beneficial effects of quercetin are mediated by autophagy-dependent reduced expression of Abl tyrosine kinase. In turn, autophagy is required upon Abl suppression to mediate quercetin's protective effects against Aß toxicity. Our data support the power of C. elegans as an in vivo model to investigate therapeutic options for AD.

Protein-based engineering of the initial acquired enamel pellicle in vivo: Proteomic evaluation.

OBJECTIVE: To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. MATERIALS AND METHODS: In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10-5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. RESULTS: Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2-2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7-25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. CONCLUSION: Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. CLINICAL RELEVANCE: Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP.

Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis.

The Sphenophorus levis (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC (Vmax = 1017.60 ± 135.55, Km = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (Ki = 38.52 ± 1.20 µM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.

Solutions and Gels Containing a Sugarcane-Derived Cystatin (CaneCPI-5) Reduce Enamel and Dentin Erosion in vitro.

The effect of solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) on the protection against enamel and dentin erosion in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 135 and 153/group for enamel and dentin, respectively) that were treated with solutions or chitosan gels containing 0.1 or 0.25 mg/mL CaneCPI-5. The positive controls for solutions and gels were Elmex Erosion Protection™ solution and NaF gel (12,300 ppm F), respectively. Deionized water and chitosan gel served as controls, respectively. The solutions were first applied on the specimens for 1 min and the gels for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.1% citric acid pH 2.5/90 s, artificial saliva/2 h, and artificial saliva overnight). The solutions and gels were applied again during pH cycling, 2 times/day for 1 min and 4 min, respectively, after the first and last erosive challenges. Enamel and dentin losses (µm) were assessed by contact profilometry. Data were analyzed by 2-way ANOVA and Tukey's test (p < 0.05). All the treatments significantly reduced enamel and dentin loss in comparison with controls. Both CaneCPI-5 concentrations had a similar protective effect against enamel erosion, but only the higher concentration was as effective against dentin erosion as the positive control. Regarding the vehicles, only the 0.1 mg/mL gel performed worse than the positive control for dentin. CaneCPI-5 reduced enamel and dentin erosion to a similar extent as the fluoride-containing vehicles. However, dentin requires higher CaneCPI-5 concentrations, in the case of gels. Solutions or gels containing CaneCPI-5 might be a new approach to protect against dental erosion.

Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization.

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60 s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500 ppm F, as NaF), (4) 0.025 mg/ml CaneCPI-5, and (5) 0.05 mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8 h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60 s) with the solutions for 5 days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.

A sugarcane cystatin (CaneCPI-5) alters microcosm biofilm formation and reduces dental caries.

The antimicrobial and anticaries effects of CaneCPI-5 were evaluated. Ninety bovine enamel samples were treated for 60 s with either phosphate-buffered-saline (PBS), 0.12% chlorhexidine (CHX), 0.05 mg ml-1 CaneCPI-5, 0.1 mg ml-1 CaneCPI-5 or 0.5 mg ml-1 CaneCPI-5. They were incubated with inoculum (human saliva + McBain's saliva) for the first 8 h. From then until the end of the experiment, the enamel was exposed to McBain saliva with sucrose and, once a day, for 5 days, they were treated with the solutions. At the end of the experimental period, resazurin and viable plate count assays were performed. Enamel demineralization was also measured. All concentrations of CaneCPI-5 and CHX significantly reduced the activity of biofilms compared with PBS. For viable plate counts, all treatments similarly reduced the lactobacilli and total streptococci; for the mutans streptococci, 0.05 mg ml-1 CaneCPI-5 performed better than CHX. All CaneCPI-5 concentrations significantly reduced the integrated mineral loss. This study represents the first step regarding the use of CaneCPI-5 within the concept of acquired enamel pellicle and biofilm engineering to prevent dental caries.

hRNAi-mediated knock-down of Sphenophorus levis V-ATPase E in transgenic sugarcane (Saccharum spp interspecific hybrid) affects the insect growth and survival.

KEY MESSAGE: Transgenic sugarcane expressing V-ATPase subunit E dsRNA affects growth and survival of Sphenophorus levis. Plants being sessile organisms are constantly confronted with several biotic and abiotic stresses. Sugarcane (Saccharum spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to Sphenophorus levis (sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with V-ATPase E dsRNA in S. levis larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the V-ATPase E gene of S. levis in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.

Sugarcane cystatins: From discovery to biotechnological applications.

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.

Acquired pellicle protein-based engineering protects against erosive demineralization.

OBJECTIVES: To evaluate, in vivo: 1) proteomic alterations in the acquired enamel pellicle (AEP) after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), statherin-derived peptide (StN15) or their combination before the formation of the AEP and subsequent erosive challenge; 2) the protection of these treatments against erosive demnineralization. MATERIALS AND METHODS: In 5 crossover phases, after prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with: deionized water-1, 0.1 mg/mL CaneCPI-5-2, 1.0 mg/mL HB-3, 1.88 × 10-5 M StN15-4 or their combination-5. AEP was formed (2 h) and enamel biopsy (10 µL, 1%citric acid, pH 2.5, 10 s) was performed on one incisor for calcium analysis. The same acid was applied on the vestibular surfaces of the remaining teeth. The acid-resistant proteins within the remaining AEP were collected. Samples were quantitatively analyzed by label-free proteomics. RESULTS: Treatment with the proteins/peptide, isolated or combined, increased several acid-resistant proteins in the AEP, compared with control. The highest increases were seen for PRPs (32-fold, StN15), profilin (15-fold, combination), alpha-amylase (9-fold; StN15), keratins (8-fold, CaneCPI-5 and HB), Histatin-1 (7-fold, StN15), immunoglobulins (6.5-fold, StN15), lactotransferrin (4-fold, CaneCPI-5), cystatins, lysozyme, protein S-100-A9 and actins (3.5-fold, StN15), serum albumin (3.5-fold, CaneCPI-5 and HB) and hemoglobin (3-fold, StN15). Annexin, calmodulin, keratin, tubulin and cystatins were identified exclusively upon treatment with the proteins/peptide, alone or combined. Groups 2, 3 and 4 had significantly lower Ca released from enamel compared to group 1 (Kruskal-Wallis/Dunn's, p < 0.05). CONCLUSIONS: Treatment with CaneCPI-5, HB or StN15 remarkably increases acid-resistant proteins in the AEP, protecting against erosion. CLINICAL SIGNIFICANCE: Our results show, for the first time, that treatment with proteins/peptide remarkably increases acid-resistant proteins in the AEP, protecting against erosive demineralization. These findings open an avenue for a new preventive approach for erosive demineralization, employing acquired pellicle engineering procedures that may in the future be incorporated into dental products.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion.

BACKGROUND: Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. METHODS: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. RESULTS: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. CONCLUSIONS: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)

Minor snake venom proteins: Structure, function and potential applications.

Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most abundant toxins have been extensively studied in the last decades and some of them have already been used for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored, even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of more sensitive techniques in the last few years allowed the discovery of new venom components and the in-depth study of some already known minor proteins. This review summarizes information regarding some structural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules or applications for the already known venom components.