Production of a Ric c3 hypo-allergen with no IgE binding or anaphylactogenic activity.

Several studies have been carried out to expand the use of Ricinus communis L. castor bean (Ricinus communis L castor bean.). This oilseed finds appropriate conditions for its development in Brazil, with more than 700 applications. The main allergens of this plant are Ric c1 and Ric c3, that cross-react with various aeroallergens and food allergens such as peanuts, soybeans, corn, and wheat. This study aimed to determine the effect of mutations in Ric c3 amino acid residues known to affect IgE binding and allergy challenges. Based on the Ric c3 structure, B-cell epitopes, and amino acid involved in IgE binding, we produce recombinant mutant protein, mrRic c3, secreted from E. coli. Strategic glutamic acid residues in IgE-biding regions were changed by Leucine. The allergenicity of mrRic c3 was evaluated by determination of IgE, IgG1, and total IgG in immunized Balb/c mice and by degranulation assays of mast cells isolated from Wistar rats. The mrRic c3 presented a percentage of mast cell degranulation close to that seen in the negative control, and the immunization of mice with mrRic c3 presented lower levels of IgE and IgG1 than the group treated with the protein without mutations. The mutant mrRic c3 had an altered structure and reduced ability to stimulate pro-inflammatory responses and bind IgE but retained its ability to induce blocking antibodies. Thus, producing a hypoallergenic mutant allergen (mrRic c3) may be essential in developing new AIT strategies.

Purification, biochemical characterization, and antimicrobial activity of a new lipid transfer protein from Coffea canephora seeds.

Coffee, an agronomical crop of great economic importance, is also among the most commonly traded commodities in worldwide markets. Antimicrobial peptides, which play a role in plant defense, have been identified and isolated particularly from seeds. We isolated and immunolocalized Cc-LTP2, a new lipid transfer protein (LTP) from Coffea canephora seeds. We report its antimicrobial activity against various phytopathogenic fungi of economic importance, and against the bacterium Xanthomonas euvesicatoria. Peptides from C. canephora seeds were initially extracted using acid buffer and subjected to ion-exchange and reverse-phase chromatographies. A purified peptide of approximately 9 kDa, which we named Cc-LTP2, was then subjected to amino acid sequencing. The analyses showed that it was similar to LTPs isolated from various plants. The tissue and subcellular localization of C. canephora LTPs indicated that they were located in cell walls and intracellular palisade parenchyma, mainly in large vacuoles. The results of immunohistochemistry and histochemistry superposed from C. canephora seed tissues showed that LTPs and lipid bodies are present in organelles, supporting the hypothesis that LTPs from seeds are involved in lipid mobilization during germination. Cc-LTP2 did inhibit the development of the phytopathogenic fungi Colletotrichum lindemuthianum, Colletotrichum gloeosporioides, Fusarium solani, Fusarium lateritium, and Colletotrichum sp, but did inhibit X. euvesicatoria. Cc-LTP2 also increased membrane permeability and induced endogenous production of reactive oxygen species in all the fungi tested.

Influence of Adhesive Core Buildup Designs on the Resistance of Endodontically Treated Molars Restored With Lithium Disilicate CAD/CAM Crowns.

OBJECTIVE: To assess the influence of adhesive core buildup designs (4-mm buildup, 2-mm buildup, and no buildup/endocrown) on the fatigue resistance and failure mode of endodontically treated molar teeth restored with lithium disilicate computer-aided design/computer-aided manufacturing (CAD/CAM) complete crowns placed with self-adhesive cement. METHODS AND MATERIALS: Forty-five extracted molars were decoronated at the level of the cementoenamel junction and endodontically treated. Specimens received different Filtek Z100 adhesive core buildups (4-mm buildup; 2-mm buildup; and no buildup endocrown preparation) and were restored with Cerec 3 CAD/CAM lithium disilicate crowns (IPS e.max CAD). The intaglio surfaces of restorations (n=15) were conditioned by hydrofluoric acid etching and silane, and prepared teeth were treated with airborne-particle abrasion, followed by cementation with RelyX Unicem 2 Automix. Specimens were then subjected to cyclic isometric loading at 10 Hz, beginning with a load of 200 N (×5000 cycles), followed by stages of 400, 600, 800, 1000, 1200, and 1400 N at a maximum of 30,000 cycles each. Specimens were loaded until failure or to a maximum of 185,000 cycles. The chewing cycle was simulated by an isometric contraction (load control) applied through a 10-mm in diameter composite resin sphere (Filtek Z100). Surviving specimens were axially loaded until failure or to a maximum load of 4500 N (crosshead speed 0.5 mm/min). The failure mode was assessed, and fractures were designated as catastrophic (tooth/root fracture that would require tooth extraction) or reparable (cohesive or cohesive/adhesive fracture of restoration only). Groups were compared using the life table survival analysis (log-rank test at p=0.05). Surviving specimens were loaded to failure and compared with one-way analysis of variance. RESULTS: The survival rates after the fatigue test were 100%, 93%, and 100% for 4-mm, 2-mm, and no buildup (endocrown), respectively and were not statistically different (only one specimen failed with a 2-mm buildup under a crown that cohesively fractured at 1,400 N). Postfatigue load to failure averaged 3181 N for 4-mm buildups (15 specimens), 3759 N for 2-mm buildups (12 specimens), and 3265 N for endocrowns (14 specimens). The 2-mm buildups were associated with higher loads to failure than endocrowns and 4-mm buildups, but no differences were found between 4-mm buildups and endocrowns (p<0.05.) One endocrown and 2 restorations with a 2-mm buildup survived the load-to-failure test (at 4500 N). Only catastrophic fractures occurred after the load-to-failure test. CONCLUSIONS: The buildup design influenced the performance of endodontically treated molars restored with lithium disilicate CAD/CAM complete crowns placed with self-adhesive resin cement. The 2-mm buildups were associated with higher loads to failure than the endocrown and the 4-mm buildup, but all restoration designs survived far beyond the normal range of masticatory forces.

Influence of no-ferrule and no-post buildup design on the fatigue resistance of endodontically treated molars restored with resin nanoceramic CAD/CAM crowns.

OBJECTIVES: To evaluate the influence of adhesive core buildup designs-4-mm buildup, 2-mm buildup, and no buildup (endocrown)-on the fatigue resistance and failure mode of endodontically treated molar teeth restored with resin nanoceramic (RNC) CAD/CAM complete crowns placed with self-adhesive resin cement. METHODS AND MATERIALS: Forty-five extracted molars were decoronated at the level of the cementoenamel junction, and the roots were endodontically treated. Specimens received different Filtek Z100 adhesive core buildups (4-mm buildup, 2-mm buildup, and no buildup, endocrown preparation) and were restored with Cerec 3 CAD/CAM RNC crowns (Lava Ultimate). Restorations (n=15) and prepared teeth were treated with airborne-particle abrasion, followed by cementation with RelyX Unicem 2 Automix. Specimens were then subjected to cyclic isometric loading at 10 Hz, beginning with a load of 200 N (for 5000 cycles), followed by stages of 400, 600, 800, 1000, 1200, and 1400 N at a maximum of 30,000 cycles each. Specimens were loaded until failure or to a maximum of 185,000 cycles (10-mm-diameter composite resin sphere antagonist). The failure mode was assessed: "catastrophic" (tooth/root fracture that would require tooth extraction), "possibly reparable" (cohesive/adhesive failure with fragment and minor damage, chip or crack, of underlying tooth structure), or "reparable" fracture (cohesive or cohesive/adhesive fracture of restoration only). Groups were compared using the life table survival analysis. Intact specimens were loaded to failure and compared with one-way analysis of variance. RESULTS: All specimens survived the fatigue test until the 800 N-step. The survival rates for 4-mm, 2-mm, and no buildup (endocrown) were 53%, 87%, and 87%, respectively, and were not statistically different even though crowns with 2-mm buildups only started to fail at 1200 N. Minor cohesive chips were detected in many samples despite having survived all 185,000 cycles. Postfatigue load-to-failure ranged from 2969 N with 4-mm buildup (eight specimens), 2794 N for 2-mm buildup (13 specimens), and 2606 N for endocrowns (13 specimens) and were also not statistically different. There were only two catastrophic failures during the fatigue test and small subgingival delamination fractures and cracks (only with 4-mm buildup). All specimens in the load-to-failure test exhibited nonrestorable catastrophic fractures. CONCLUSIONS: There was no influence of the buildup design on the performance of endodontically treated molars restored with RNC CAD/CAM complete crowns placed with self-adhesive cement. All restoration designs survived the normal range of masticatory forces. Failure mode tended to be more favorable with the 2-mm buildup or no buildup (endocrown).

Peroxidase is involved in Pepper yellow mosaic virus resistance in Capsicum baccatum var. pendulum.

Pathogenesis-related proteins (PRs) are among the defense mechanisms of plants that work as an important barrier to the development of pathogens. These proteins are classified into 17 families according to their amino acid sequences, serology, and/or biological or enzyme activity. The present study aimed to identify PRs associated with the pathosystem of Capsicum baccatum var. pendulum: Pepper yellow mosaic virus (PepYMV). Forty-five-day-old plants from accession UENF 1624, previously identified as resistant to PepYMV, were inoculated with the virus. Control and infected leaves were collected for analysis after 24, 48, 72, and 96 h. The inoculated and control plants were grown in cages covered with anti-aphid screens. Proteins were extracted from leaf tissue and the presence of ß-1,3-glucanase, chitinase, peroxidase, and lipid transport protein was verified. No difference was observed between the protein pattern of control and infected plants when ß-1,3-glucanase, chitinase, and lipid transport protein were compared. However, increased peroxidase expression was observed in infected plants at 48 and 72 h after inoculation, indicating that this PR is involved in the response of resistance to PepYMV in C. baccatum var. pendulum.

Evaluation of an anatomic dual-laminate composite resin shade guide.

BACKGROUND: Color assessment in aesthetic dentistry is one of the most challenging steps for direct restorative treatment. Shade selection tools should be able to mimic closely the materials and layering technique used in the final restoration, hence the development of prefabricated anatomic dual-laminate shade guides. OBJECTIVE: This study aims to compare different shade selection techniques and determine the suitability of a prefabricated anatomic dual-laminate shade guide and its best mode of use compared to a conventional guide and a layered custom guide. MATERIALS AND METHODS: CIELab coordinates of different shade guides were assessed: Vitapan Classical (tab A2; Vita); Miris2 prefabricated anatomic dual-laminate shade guide, enamel WR tab on top of dentine S3 tab and nothing in-between (M2air) or glycerin gel (M2gly) or water (M2w); custom shade guide using prefabricated silicon moulds, Miris2 enamel WR composite resin moulded directly on dentine S3 pre-polymerised base (M2cus). The average values were obtained to calculate DE and compare the different shade selection techniques. Additional samples and measurements were made to compare Vitapan Classical shade tabs A1, A2 and A3 and all possible combinations of Miris2 and establish the closest matching shade (DE 3.3). RESULTS: High DE values were found (6.51­9.11) when comparing M2air to Vita, M2gly M2w M2cus. Differences appeared acceptable (DE 2.09­2.99) between Vita, M2gly and M2w and M2cus. Seven combinations of M2 were found to match Vita tab A1 and A2 and three Miris2 combinations for Vita A3 (DE 3.3). CONCLUSIONS: The use of Miris2 prefabricated anatomic dual-laminate shade guide with interposition of water or glycerin between the enamel­dentine tabs demonstrated acceptable DE values when compared to Vitapan Classical and custom guides. A chart for matching Vita shades with various combinations of Miris2 enamel/dentine shades was produced to assist the clinician in obtaining acceptable restorations. CLINICAL SIGNIFICANCE: The prefabricated anatomic dual-laminate shade guide is as efficient as a custom shade guide, facilitating clinical steps and saving material when doing compositeresin restorations.

Characterization of Capsicum species using anatomical and molecular data.

Capsicum species are frequently described in terms of genetic divergence, considering morphological, agronomic, and molecular databases. However, descriptions of genetic differences based on anatomical characters are rare. We examined the anatomy and the micromorphology of vegetative and reproductive organs of several Capsicum species. Four Capsicum accessions representing the species C. annuum var. annuum, C. baccatum var. pendulum, C. chinense, and C. frutescens were cultivated in a greenhouse; leaves, fruits and seeds were sampled and their organ structure analyzed by light and scanning electronic microscopy. Molecular accession characterization was made using ISSR markers. Polymorphism was observed among tector trichomes and also in fruit color and shape. High variability among accessions was detected by ISSR markers. Despite the species studied present a wide morphological and molecular variability that was not reflected by anatomical features.

Cloning and characterization of a cowpea seed lipid transfer protein cDNA: expression analysis during seed development and under fungal and cold stresses in seedlings' tissues.

Lipid transfer proteins (LTPs) are antimicrobial peptides (AMPs) involved in the defense of plants against pathogens. Our group has previously characterized and purified a LTP from cowpea (Vigna unguiculata (L.) Walp.) seeds which caused the inhibition of growth of fungal pathogens in vitro. The aim of this work was to obtain the cDNA encoding the cowpea LTP and after cloning, to use the cDNA as a probe for studying its expression profile during the development of cowpea seeds. In this work, the N-terminal sequence of the mature LTP peptide from cowpea was used to produce a degenerated oligonucleotide. This primer allowed the amplification of the LTP cDNA by RT-PCR from mRNA of cowpea seeds. The sequence analysis of the cloned cDNA, named VULTP, showed 494 bp which encoded a polypeptide of 91 amino acids. The deduced peptide presented high homology of similarity to plant LTPs of Vigna radiata var. radiate (94%), Prunus domestica (82%) and Zea mays (72%). The expression profile of the VULTP gene in cowpea was analyzed by Northern blot and revealed that the transcript is not accumulated in adult tissues. Conversely, VULTP mRNA is early and strongly accumulated during seed development. The results obtained to seedling of cowpea demonstrate that the VULTP gene presents differential expression in response to different stress. Further studies will be conducted to try to gain better understanding about the physiological role of this gene in cowpea.

Purification and characterization of a novel peptide with antifungal activity from Bothrops jararaca venom.

Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.