Chemical composition and biological activities of the essential oil from Eugenia stipitata McVaugh leaves.

In the present study, the volatile components and cytotoxic, antibacterial, antioxidant, and antiprotozoal activities of the essential oil obtained from the leaves of Eugenia stipitata McVaugh (Myrtaceae) grown in the Brazilian Northeast region (Araripe) were investigated. The essential oil was obtained by hydrodistillation. The leaves of E. stipitata provided an oil yield of 0.13 ± 0.01% (w/w). The volatile compounds in the essential oil of E. stipitata were analysed using gas chromatography, and the volatile chemical composition was mainly composed of ß-eudesmol (15.28%), γ-eudesmol (10.85%), elemol (10.21%) and caryophyllene oxide (6.65%). The essential oil of E. stipitata was highly selective against Leishmania braziliensis and L. infantum promastigotes. The essential oil exhibited good antibacterial activity. E. stipitata essential oil showed low free-radical scavenging activity. Our results suggest that the E. stipitata essential oil is a relevant source of the primary compounds required for the development of antibacterial and antiprotozoal drugs.

Investigation of factors related to biofilm formation in Providencia stuartii.

Providencia stuartii is one of the Enterobacteriaceae species of medical importance commonly associated with urinary infections, which can also cause other ones, including uncommon ones, such as liver abscess and septic vasculitis. This bacterium stands out in the expression of intrinsic and acquired resistance to antimicrobials. Besides, it uses mechanisms such as biofilm for its persistence in biotic and abiotic environments. This study investigated the cellular hydrophobicity profile of clinical isolates of P. stuartii. It also analyzed genes related to the fimbrial adhesin in this species comparing with other reports described for other bacteria from Enterobacteriaceae family. The investigated isolates to form biofilm and had a practically hydrophilic cell surface profile. However, fimH and mrkD genes were not found in P. stuartii, unlike observed in other species of Enterobacteriaceae. These results show that P. stuartii has specificities regarding its potential for biofilm formation, which makes it difficult to destabilize the infectious process and increases the permanence of this pathogen in hospital units.

Nutritional and technological potential of Umbu (Spondias tuberosa Arr. Cam.) processing by-product flour.

Antioxidants present in many fruit residues can play an essential role in the prevention of diseases. The aim of this study was to determine nutritional and mineral composition, fatty acids profile, anti-nutrients, bioactive compounds, antioxidant activity and technological properties of flour from residues generated by umbu fruit processing. Nutritional composition showed high levels of dietary fiber, especially insoluble fiber (56.67%). The flour can be classified as a good source of calcium, phosphorus and magnesium, and an excellent source of iron, zinc and copper. Palmitic and stearic saturated fatty acids and oleic and linoleic unsaturated fatty acids were identified. No potentially toxic substances were detected. Significant values of ascorbic acid (44.78 mg/100g), carotenoids (463.73 µg/100g) and flavonoids (37.85 mg QE/100g) were found, as well as very high levels of phenolic compounds (20357.26 mg GAE/100g). Strong antioxidant activity was detected in the flour by three methods (ABTS, DPPH and FRAP) demonstrating a linear positive correlation between phenolic content and antioxidant activity. The flour showed high absorption of water, oil, emulsifying capacity and emulsion stability presenting a great potential for use in foods, especially meat, bakery and dairy products. These results indicate promising prospects to full use of umbu as a functional ingredient.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Evaluating glucose and mannose profiles in Candida species using quantum dots conjugated with Cramoll lectin as fluorescent nanoprobes.

Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 µm) was smaller when compared to C. albicans (4.0 µm) and C. parapsilosis sensu stricto (3.8 µm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.

Evaluating the glycophenotype on breast cancer tissues with quantum dots-Cramoll lectin conjugates.

During carcinogenesis, changes in the glycosylation can modulate many biological processes. Thus, the interest in exploring and understanding the roles of carbohydrates as cancer biomarkers has been increasing. Lectins have been applied as useful tools in glycobiology, especially when associated with fluorescent reporters. Therefore, to take advantage of the physicochemical properties of quantum dots (QDs), herein, we conjugated Cramoll, a lectin that recognizes glucose/mannose residues, with those nanoparticles. We applied the conjugates to investigate the glycocode of normal, fibroadenoma (FB), and invasive ductal carcinoma (IDC) human breast tissues. Additionally, we proposed a method to quantitatively evaluate the tissue labeling intensity by a fluorescence microplate assay (FMA). Conjugates showed intense fluorescence and specificity. The lectin activity and secondary structure were also preserved after the conjugation with QDs. Moreover, fluorescence images showed that ductal cells of normal and FB tissues were preferentially labeled by conjugates, whereas both cells and stroma were strongly labeled in IDC. FMA showed in a quantitative, practical, and sensitive way that the level of exposed glucose/mannose residues increased accordingly to the sample malignancy degree. In conclusion, QDs-Cramoll conjugates can be considered effective, specific, and versatile probes to evaluate glycan profiles in normal and transformed tissues, by fluorescence microscopy as well as FMA quantification. Furthermore, FMA showed to be a potential method that can be applied with other fluorescent conjugates.

Titanium dioxide nanotubes functionalized with Cratylia mollis seed lectin, Cramoll, enhanced osteoblast-like cells adhesion and proliferation.

An alternative to accelerate the osseointegration on titanium dioxide nanotubes (TNTs) used in osseointegrated implants is through the functionalization of these nanostructured surfaces with biomolecules. In this work, we immobilized a lectin with recognized mitogenic activity, the Cramoll lectin, extracted from Cratylia mollis seeds, on surfaces modified by TNTs. For the immobilization of Cramoll on TNTs surfaces, we used the Layer-by-Layer technique (LbL) by growing five alternate layers of poly(allylamine) hydrochloride (PAH) and poly(acrylic) acid (PAA); lastly we incubated the lectin, at different concentrations, with the TNTs-LbL. Before and after the immobilization procedures, the substrate surfaces were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and, electrochemical impedance spectroscopy (EIS). We also evaluated the Cramoll activity after immobilization on TNTs by using the lectin interaction with ovalbumin. The lectin did not lose its biological activity, even after immobilization onto nanotubular arrays. In addition, we observed an increase osteoblast-like cell adhesion on the TNTs-LbL-Cramoll system when compared to the bare TNTs surfaces. Moreover, a significative cell proliferation was identified on the substrates when Cramoll was immobilized at concentrations of 80, 160 and 320 µg/mL after 48 h of incubation by using the resazurin assay. Our results suggest that Cramoll was efficiently immobilized on a nanotubular array and this new platform presents a great potential to be tested in implantology.

Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice.

Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.

Anti-Inflammatory Activity of Babassu Oil and Development of a Microemulsion System for Topical Delivery.

Babassu oil extraction is the main income source in nut breakers communities in northeast of Brazil. Among these communities, babassu oil is used for cooking but also medically to treat skin wounds and inflammation, and vulvovaginitis. This study aimed to evaluate the anti-inflammatory activity of babassu oil and develop a microemulsion system with babassu oil for topical delivery. Topical anti-inflammatory activity was evaluated in mice ear edema using PMA, arachidonic acid, ethyl phenylpropiolate, phenol, and capsaicin as phlogistic agents. A microemulsion system was successfully developed using a Span® 80/Kolliphor® EL ratio of 6 : 4 as the surfactant system (S), propylene glycol and water (3 : 1) as the aqueous phase (A), and babassu oil as the oil phase (O), and analyzed through conductivity, SAXS, DSC, TEM, and rheological assays. Babassu oil and lauric acid showed anti-inflammatory activity in mice ear edema, through inhibition of eicosanoid pathway and bioactive amines. The developed formulation (39% A, 12.2% O, and 48.8% S) was classified as a bicontinuous to o/w transition microemulsion that showed a Newtonian profile. The topical anti-inflammatory activity of microemulsified babassu oil was markedly increased. A new delivery system of babassu microemulsion droplet clusters was designed to enhance the therapeutic efficacy of vegetable oil.

Application of Omics Technologies for Evaluation of Antibacterial Mechanisms of Action of Plant-Derived Products.

In the face of increasing bacterial resistance to antibiotics currently in use, the search for new antimicrobial agents has received a boost in recent years, with natural products playing an important role in this field. In fact, several methods have been proposed to investigate the antibacterial activities of natural products. However, given that the ultimate aim is future therapeutic use as novel drugs, it is extremely necessary to elucidate their modes of action, stating the molecular effects in detail, and identifying their targets in the bacterial cell. This review analyzes the application of "omics technologies" to understand the antibacterial mechanisms of bioactive natural products, to stimulate research interest in this area and promote scientific collaborations. Some studies have been specifically highlighted herein by examining their procedures and results (targeted proteins and metabolic pathways). These approaches have the potential to provide new insights into our comprehension of antimicrobial resistance/susceptibility, creating new perspectives for the struggle against bacteria, and leading to the development of novel products in the future.

Effects of Tityus stigmurus (Thorell 1876) (Scorpiones: Buthidae) venom in isolated perfused rat kidneys.

Scorpions belonging to the Tityus genus are of medical interest in Brazil. Among them, Tityus stigmurus is the main scorpion responsible for stings in the Northeast region. After a sting, the scorpion venom distributes rapidly to the organs, reaching the kidneys quickly. However, there are few studies concerning the renal pathophysiology of scorpion poisoning. In this study, we evaluated the effects of T. stigmurus venom (TsV) on renal parameters in isolated rat kidneys. Wistar rats (n = 6), weighing 250-300 g, were perfused with Krebs-Henseleit solution containing 6 g/100 mL bovine serum albumin. TsV at 0.3 and 1.0 µg/mL was tested, and the effects on perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and electrolyte excretion were analyzed. Effects were observed only at TsV concentration of 1.0 µg/mL, which increased PP (controlPP40' = 92.7 ± 1.95; TsVPP40' = 182.0 ± 4.70* mmHg, *p < 0.05), RVR (controlRVR40' = 3.28 ± 0.23 mmHg; TstRVR40' = 6.76 ± 0.45* mmHg, *p < 0.05), UF (controlUF50' = 0.16 ± 0.04; TstUF50' = 0.60 ± 0.10* mL/g/min,*p < 0.05), GFR and electrolyte excretion, with histological changes that indicate renal tubular injury. In conclusion, T. stigmurus venom induces a transient increase in PP with tubular injury, both of which lead to an augmented electrolyte excretion.

Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach.

BACKGROUND: Finding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels. RESULTS: The bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli, the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels. CONCLUSIONS: These results attest to the adequacy of these proteins extraction methods for proteomic studies.

Natural Green coating inhibits adhesion of clinically important bacteria.

Despite many advances, biomaterial-associated infections continue to be a major clinical problem. In order to minimize bacterial adhesion, material surface modifications are currently being investigated and natural products possess large potential for the design of innovative surface coatings. We report the bioguided phytochemical investigation of Pityrocarpa moniliformis and the characterization of tannins by mass spectrometry. It was demonstrated that B-type linked proanthocyanidins-coated surfaces, here termed Green coatings, reduced Gram-positive bacterial adhesion and supported mammalian cell spreading. The proposed mechanism of bacterial attachment inhibition is based on electrostatic repulsion, high hydrophilicity and the steric hindrance provided by the coating that blocks bacterium-substratum interactions. This work shows the applicability of a prototype Green-coated surface that aims to promote necessary mammalian tissue compatibility, while reducing bacterial colonization.

Tannins possessing bacteriostatic effect impair Pseudomonas aeruginosa adhesion and biofilm formation.

Plants produce many compounds that are biologically active, either as part of their normal program of growth and development or in response to pathogen attack or stress. Traditionally, Anadenanthera colubrina, Commiphora leptophloeos and Myracrodruon urundeuva have been used by communities in the Brazilian Caatinga to treat several infectious diseases. The ability to impair bacterial adhesion represents an ideal strategy to combat bacterial pathogenesis, because of its importance in the early stages of the infectious process; thus, the search for anti-adherent compounds in plants is a very promising alternative. This study investigated the ability of stem-bark extracts from these three species to control the growth and prevent biofilm formation of Pseudomonas aeruginosa, an important opportunistic pathogen that adheres to surfaces and forms protective biofilms. A kinetic study (0-72 h) demonstrated that the growth of extract-treated bacteria was inhibited up to 9 h after incubation, suggesting a bacteriostatic activity. Transmission electron microscopy and fluorescence microscopy showed both viable and nonviable cells, indicating bacterial membrane damage; crystal violet assay and scanning electron microscopy demonstrated that treatment strongly inhibited biofilm formation during 6 and 24 h and that matrix production remained impaired even after growth was restored, at 24 and 48 h of incubation. Herein, we propose that the identified (condensed and hydrolyzable) tannins are able to inhibit biofilm formation via bacteriostatic properties, damaging the bacterial membrane and hindering matrix production. Our findings demonstrate the importance of this abundant class of Natural Products in higher plants against one of the most challenging issues in the hospital setting: biofilm resilience.

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.