CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants are more resistant to Botrytis cinerea.

MAIN CONCLUSION: CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.

Venom comparisons of endemic and micro-endemic speckled rattlesnakes Crotalus mitchellii, C. polisi and C. thalassoporus from Baja California Peninsula.

A high diversity of rattlesnake species can be found in the Baja California peninsula and the island of the Gulf of California, nevertheless, their venom has been poorly evaluated. The aim of this work was to present the first characterization of endemic Crotalus mitchellii, micro endemic C. polisi and C. thalassoporus venoms. All samples provoke human plasma coagulation showing doses in the rank of 2.3-41.0 µg and also produce rapid hydrolysis of the alpha chain of bovine fibrinogen while the beta chain is attacked at larger incubation periods by C. polisi and especially by C. thalassoporus. Phospholipase activity ranging from 23.2 to 173.8 U/mg. The venoms of C. thalassoporus and C. polisi show very high hemorrhagic activity (from 0.03 to 0.31 µg). A total of 130 toxin-related proteins were identified and classified into ten families. Crotalus mitchellii venom was characterized by high abundance of crotoxin-like and other phospholipase proteins (34.5%) and serine proteinases (29.8%). Crotalus polisi showed a similar proportion of metalloproteinases (34%) and serine proteinases (22.8%) components with important contribution of C-type lectins (14.3%) and CRiSP (14.0%) proteins. Venom of C. thalassoporus is dominated by metalloproteases that amount to more than 66% of total toxin proteins. These results provide a foundation for comprehending the biological, ecological and evolutionary significance of venom composition of speckled rattlesnake from the Baja California peninsula.

High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

Impact of serial systemic infection on Candida albicans virulence factors.

Aim: To evaluate changes in virulence and pathogenicity approaches from Candida albicans after successive passages in a murine model of systemic candidiasis. Materials & methods: Phenotypic assays were performed using colonies recovered from animals infected serially, totalizing five passages. Results: A progressive infection was observed along the passages, with increased fungal burden and the presence of greater inflammatory areas in the histopathological findings. Recovered strains exhibited increased filamentation and biofilm abilities, along with modulation of phospholipase and proteinase activities. Conclusion: Repeated contact between yeast and host increased the expression of virulence factors. Furthermore, a correspondence between phenotypic profile and proteomic data obtained previously was observed.

Screening and Immobilization of Interfacial Esterases from Marine Invertebrates as Promising Biocatalyst Derivatives.

Interfacial esterases are useful enzymes in bioconversion and racemic mixture resolution processes. Marine invertebrates are few explored potential sources of these proteins. In this work, aqueous extracts of 41 species of marine invertebrates were screened for esterase, lipase, and phospholipase A activities, being all positive. Five extracts (Stichodactyla helianthus, Condylactis gigantea, Stylocheilus longicauda, Zoanthus pulchellus, and Plexaura homomalla) were selected for their activity values and immobilized on Octyl-Sepharose CL 4B support by interfacial adsorption. The selectivity of this immobilization method for interfacial esterases was evidenced by immobilization percentages ≥ 94% in almost all cases for lipase and phospholipase A activities. Six pharmaceutical-relevant esters (phenylethyl butyrate, ethyl-2-hydroxy-4-phenyl-butanoate, 2-oxyranylmethyl acetate (glycidol acetate), 7-aminocephalosporanic acid, methyl-prostaglandin F2α, and methyl-6-metoxy-α-methyl-2-naphtalen-acetate -naproxen methyl ester-) were bioconverted by at least three of these biocatalysts, with the lowest conversion percentage of 24%. In addition, three biocatalysts were used in the racemic mixture resolution of three previous compounds. The S. helianthus-derived biocatalyst showed the highest enantiomeric ratios for glycidol acetate (2.67, (S)-selective) and naproxen methyl ester (8.32, (R)-selective), and the immobilized extract of S. longicauda was the most resolutive toward the ethyl-2-hydroxy-4-phenyl-butanoate (8.13, (S)-selective). These results indicate the relevance of such marine interfacial esterases as immobilized biocatalysts for the pharmaceutical industry.

Molecular interactions between p-coumaric acid and snake venom toxins.

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.

Virulence factors of Candida spp. obtained from blood cultures of patients with candidemia attended at tertiary hospitals in Northeast Brazil.

BACKGROUND: Yeasts of the Candida genus are one of the most common causes of bloodstream infections associated with high rates of morbidity and mortality, mainly affecting immunocompromised patients. We aimed to identify yeasts obtained from blood cultures of patients interned at tertiary hospitals in Brazil. METHODS: We evaluated some of the major virulence factors of Candida spp., including the ability to adhere to human buccal epithelial cells, biofilm formation, hemolytic and phospholipase activity. RESULTS: We analyzed 70 isolates of Candida spp. obtained from March 2011 and March 2015. Candida spp. showed different peculiarities in terms of expression of virulence factors evaluated in vitro. C. albicans strains were more adherent to HBEC than all the other Candida species. C. tropicalis strains were considered strong biofilm producers. Strains belonging to the C. parapsilosis species complex were able to produce hemolysins, while C. glabrata was also able to lyse erythrocytes and to produce phospholipase. CONCLUSION: These results suggest that Non-Candida albicans Candida species are also able to express virulence factors which play an important role in bloodstream infectious caused by these yeasts.

Lipase and Phospholipase Activity Methods for Marine Organisms.

Lipases are very important enzymes having a role in fat digestion and lipid metabolism in marine animals, plants, and microorganisms. The methods for measuring lipase and phospholipase activity have been applied in several studies; however, considering that lipases are water-soluble molecules and their substrates are generally water-insoluble molecules, several steps are required for measuring their digestion products. After a general review of the main type of methods used in marine lipase studies, and experimental procedures, a proposal of new or improved methods is described in order to facilitate the lipase activity measurements in marine organisms.

Rational Design Strategy as a Novel Immobilization Methodology Applied to Lipases and Phospholipases.

Immobilization of lipases and phospholipases, mainly on water-insoluble carriers, helps in their economic reusing and in the development of continuous bioprocesses. Design of efficient lipase and phospholipase-immobilized systems is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop efficient immobilized systems. We conceived a new strategy for the rational design of immobilized derivatives (RDID) in favor of the successful synthesis of optimal lipase and phospholipase-immobilized derivatives, aiming the prediction of the immobilized derivative's functionality and the optimization of load studies. The RDID strategy begins with the knowledge of structural and functional features of synthesis components (protein and carrier) and the practical goal of the immobilized product. The RDID strategy was implemented in a software named RDID1.0. The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.

Overexpression of a phospholipase (OsPLDα1) for drought tolerance in upland rice (Oryza sativa L.).

This work aimed to evaluate the drought tolerance of transformed plants of the cultivar BRSMG Curinga that overexpress the rice phospholipase D α1 (OsPLDα1) gene. The productivity of independent transformation event plants of the OsPLDα1 gene was evaluated in an experiment where 19 days of water deficit were applied at the reproductive stage, a very strict growing condition for upland rice. The non-genetically modified cultivar (NGM) under drought treatment reduced productivity by 89% compared with that under irrigated treatment, whereas transformed plants (PLDα1_E2) reduced productivity by only 41%. After the drought treatment, the PLDα1_E2 plants productivity was five times greater than that of the NGM plant. Moreover, no adverse effects on growth and development of the transgenic plants were observed. Seven days after the resumption of irrigation, PLDα1_E2 plants had higher stomatal conductance, greater photosynthetic rate, and transpiration rate than did NGM plants, as well as a higher expression level of the OsPLDα1 gene. A delay in the senescence process was observed in these PLDα1_E2 plants, and this was determined for the recovery of photosynthesis, with greater expression of the Rubisco and lower expression of the SOD. This finding was suggestive of decreased oxidative stress, probably due to gas exchange by the partial closure of the stomata of these transformed plants, which prevented the formation of reactive oxygen species. OsPLDα1 gene overexpression resulted in a reduction in production loss under severe water deficit and revealed a possibility for the development of upland rice cultivars that are more tolerant to extreme drought conditions.

Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential.

PURPOSE: Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex. METHODOLOGY: Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated.Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible-dose-dependent to itraconazole, the other two C. orthopsilosis were susceptible-dose-dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible-dose-dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were ß-haemolytic, and all C. metapsilosis were α-haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure. CONCLUSION: These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.

Proteopeptidomic, Functional and Immunoreactivity Characterization of Bothrops moojeni Snake Venom: Influence of Snake Gender on Venom Composition.

Venom composition varies across snakes from all taxonomic levels and is influenced by the snakes’ age, habitat, diet, and sexual dimorphism. The present study reports the first in-depth investigation of venom composition in male and female Bothrops moojeni (B. moojeni) snakes (BmooM and BmooF, respectively) through three proteomics approaches associated with functional, cytotoxic, and immunoreactivity characterization. Compared with BmooM venom, BmooF venom exhibited weaker hyaluronidase, metalloproteinase, and phospholipase activity; stronger recognition by anti-bothropic serum; 1.4-fold stronger cytotoxicity; and greater number of peptides. The increased L-amino acid oxidase expression probably accounted for the stronger immunoreactivity and cytotoxicity of BmooF venom. BmooF and BmooM venom shared only 19% peptides. Some venom components were gender-specific, such as phospholipases B, phospholipase inhibitor, and hyaluronidases in BmooM, and cysteine-rich secretory proteins in BmooF. In conclusion, we describe herein the first proteomics study of B. moojeni snake venom and an in-depth characterization of gender-specific differences in venom composition. Altogether, our findings not only stress the importance of considering the snake’s gender during antivenom production, but also help to identify new potential drugs and biotechnological tools.

Virulence factors of fluconazole-susceptible and fluconazole-resistant Candida albicans after antimicrobial photodynamic therapy.

This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and LED light on the virulence factors of fluconazole-susceptible (CaS) and fluconazole-resistant (CaR) Candida albicans. Standardized suspensions of strains were prepared (107), and after 48 h of biofilm formation, these strains were incubated with PDZ (100 mg/L) for 20 min and exposed to LED light (660 nm, 37.5 J/cm2). Additional samples were treated with PDZ or light only, and the control consisted of biofilms that received no treatment. After aPDT, the cells were recovered and the virulence factors were evaluated. To analyze the capacity of adhesion, cells were recovered after aPDT and submitted to the adhesion process in the bottom of a 96-well plate. After this, metabolic activity tests (XTT assay) and cell viability (colony forming units per milliliter, CFU/mL) were applied. To evaluate the biofilm-forming ability after aPDT, the cells recovered were submitted to biofilm formation procedures, and the biofilm formed was evaluated by XTT, CFU/mL, and total biomass (crystal violet) tests. Lastly, the capacity for synthesizing protease and phospholipase enzymes after aPDT was evaluated by fluorimetric tests. Data were analyzed by two- or three-way ANOVA tests (p ≤ 0.05). It was verified that aPDT reduced the viability of both strains, fluconazole-susceptible and fluconazole-resistant C. albicans. It was also observed that the CaR strain had lower susceptibility to the aPDT when compared with the CaS strain. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability and enzymatic production.

The inhibitory potential of the condensed-tannin-rich fraction of Plathymenia reticulata Benth. (Fabaceae) against Bothrops atrox envenomation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnobotanical studies have shown that Plathymenia reticulata Benth. (Fabaceae) has been widely used in cases of snake envenomation, particularly in Northern Brazil. In light of this, the aim of this study was to evaluate the inhibitory potential of the condensed-tannin-rich fraction obtained from the bark of P. reticulata against the main biological activities induced by Bothrops atrox venom (BaV). MATERIALS AND METHODS: The chemical composition of the aqueous extract of P. reticulata (AEPr) was first investigated by thin-layer chromatography (TLC) and the extract was then fractionated by column chromatography on Sephadex LH-20. This yielded five main fractions (Pr1, Pr2, Pr3, Pr4 and Pr5), which were analyzed by colorimetry to determine their concentrations of total phenolics, total tannins and condensed tannins and to assess their potential for blocking the phospholipase activity of BaV. The Pr5 fraction was defined as the fraction rich in condensed tannins (CTPr), and its inhibitory potential against the activities of the venom was evaluated. CTPr was evaluated in different in vivo and in vitro experimental protocols. The in vivo protocols consisted of (1) pre-incubation (venom:CTPr, w/w), (2) pre-treatment (orally administered) and (3) post-treatment (orally administered) to evaluate the effect on the hemorrhagic and edematogenic activities of BaV; in the in vitro protocol the effect on phospholipase and coagulant activity using pre-incubation in both tests was evaluated. RESULTS: There was statistically significant inhibition (p<0.05) of hemorrhagic activity by CTPr when the pre-incubation protocol was used [55% (1:5, w/w) and 74% (1:10, w/w)] and when pre-treatment with doses of 50 and 100mg/kg was used (19% and 13%, respectively). However, for the concentrations tested, there was no statistically significant inhibition in the group subjected to post-treatment administered orally. CTPr blocked 100% of phospholipase activity and 63.3% (1:10, w/w) of coagulant activity when it was pre-incubated with BaV. There was a statistically significant reduction (p<0.05) in edema induced by BaV in the oral protocols. Maximum inhibition was 95% (pre-treatment). CONCLUSION: Our findings indicate that CTPr could be a good source of natural inhibitors of the components of snake venom responsible for inducing local inflammation.

Structural Basis of Lipid Targeting and Destruction by the Type V Secretion System of Pseudomonas aeruginosa.

The type V secretion system is a macromolecular machine employed by a number of bacteria to secrete virulence factors into the environment. The human pathogen Pseudomonas aeruginosa employs the newly described type Vd secretion system to secrete a soluble variant of PlpD, a lipase of the patatin-like family synthesized as a single macromolecule that also carries a polypeptide transport-associated domain and a 16-stranded ß-barrel. Here we report the crystal structure of the secreted form of PlpD in its biologically active state. PlpD displays a classical lipase α/ß hydrolase fold with a catalytic site located within a highly hydrophobic channel that entraps a lipidic molecule. The active site is covered by a flexible lid, as in other lipases, indicating that this region in PlpD must modify its conformation in order for catalysis at the water-lipid interface to occur. PlpD displays phospholipase A1 activity and is able to recognize a number of phosphatidylinositols and other phosphatidyl analogs. PlpD is the first example of an active phospholipase secreted through the type V secretion system, for which there are more than 200 homologs, revealing details of the lipid destruction arsenal expressed by P. aeruginosa in order to establish infection.

Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea.

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase-induced expression of defense-related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked-down the expression of SlPLC2 by virus-induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)-defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non-silenced infected plants, while transcripts of the jasmonic acid (JA)-defense gene markers Proteinase Inhibitor I and II (SlPI-I and SlPI-II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea.

(7-Chloroquinolin-4-yl)arylhydrazones: Candida albicans enzymatic repression and cytotoxicity evaluation, Part 2.

OBJECTIVE: This work describes the anti-enzymatic activity of (7-chloroquinolin-4-yl)arylhydrazones against Candida albicans and examines their cytotoxicity. MATERIAL AND METHODS: Ten C. albicans strains [nine isolates and one azole-resistant standard strain (ATCC 62342)] were used to assess the anti-enzymatic activity. Fifteen compounds at sub-antifungal concentrations ranging from 12.5 to 100 µg/ml were assessed after a 30-min exposure. The strains were seeded onto petri dishes with selective agar media for aspartyl proteases (Saps) and phospholipases (PLs). Enzymatic inhibition was measured by the reduction of the precipitation zone (Pz) against untreated strains (positive control). A colorimetric MTT assay was used with 3T3/NIH mouse fibroblasts to evaluate cytotoxicity. Cells were exposed to 15 compounds in concentrations from 6.25 to 100 µg/ml for 24 and 48 h. RESULTS: Four hydrazones showed enzymatic repression values over 40% to Pl and three over 20% to Saps. The cell viability was over 50% at hydrazone concentrations of 25-100 µg/ml. CONCLUSION: These results revealed that select (7-chloroquinolin-4-yl)arylhydrazones may be potential antifungal agents for the control of C. albicans infections.

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake.

Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and inflammatory cells were all decreased as compared to venom injection alone. Altogether, data show that fucCS was able to inhibit myotoxicity and inflammation induced by B. jararacussu venom and its phospholipase toxins, BthTX-I and BthTX-II. Thus, fucosylated chondroitin sulfate is a new polyanion with potential to be used as an adjuvant in the treatment of snakebites in the future.

Effect of the crude extract of Eugenia uniflora in morphogenesis and secretion of hydrolytic enzymes in Candida albicans from the oral cavity of kidney transplant recipients.

BACKGROUND: Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. Yeasts virulence factors contribute for both the maintenance of colonizing strains in addition to damage and cause tissue invasion, thus the establishment of infection occurs. The limited arsenal of antifungal drugs for the treatment of candidiasis turn the investigation of natural products mandatory for the discovery of new targets for antifungal drug development. Therefore, tropical countries emerge as important providers of natural products with potential antimicrobial activity. This study aimed to investigate morphogenesis and secretion of hydrolytic enzymes (phospholipase and proteinase) in the presence of the CE of Eugenia uniflora. METHODS: The isolates were tested for their ability to form hyphae in both solid and liquid media under three different conditions: YPD + 20% FBS, Spider medium and GlcNac and the ability to secrete phospholipase and proteinase in the presence of 2000 µg/mL of E. uniflora. RESULTS: The CE of E. uniflora inhibited hypha formation in both liquid and solid media tested. It also impaired hydrolytic enzymes production. CONCLUSIONS: This was the first study to describe the interaction of a natural product with the full expression of three different factors in C. albicans. E. uniflora may be an alternative therapeutic for oral candidiasis in the future.

Práticas de cuidado ao idoso indígena - atuação dos profissionais de saúde / The elderly care practices of indigenous-performance of health / Las prácticas de cuidado de ancianos indígenas de rendimiento de la salud

Este estudo objetivou compreender as práticas de cuidado dos profissionais de saúde que assistem os idosos Kaingang. Estudo qualitativo, apoiado na etnografia, realizado com dez profissionais à que atuam na atenção primária saúde da Terra Indígena Faxinal, Paraná, Brasil. Os dados foram coletados no período de novembro de 2010 a fevereiro de 2012 por meio da observação participante e entrevistas, e, analisados à luz da Teoria Transcultural do Cuidado. Identificaram-se como práticas de cuidado a medicação e imunização, bem como, cuidados da medicina tradicional. Para realização destes cuidados, os profissionais dispunham de estratégias que proporcionavam manutenção dos idosos na assistência. Conclui-se que valores culturais e científicos necessitam integrar a assistência para melhoria da saúde dos idosos indígenas.

This research aims to understand the care practices of health professionals who assist the elderly Kaingang. It is a qualitative study, supported in ethnography, conducted by ten professionals working in primary health care in the indigenous land of Faxinal, Paraná, Brazil. The data was collected from November 2010 to February 2012 by participant observation and interviews, and analyzed based on the Transcultural Care Theory. Was identified the preoccupation of the carers practices with the medication and immunization, as well as traditional medical care. To achieve these, care professionals had strategies that implemented maintenance of older people in care. We conclude that cultural values and integrate scientific need assistance to improve the health of elderly indigenous.

Este estudio tuvo como objetivo entender las prácticas de cuidado de los profesionales de la salud que asisten a los ancianos Kaingang. Estudio cualitativo, apoyado en la etnografía, llevado a cabo con diez profesionales que trabajan en la atención primaria de la salud de la tierra indígena de Faxinal, Paraná, Brasil. Los datos fueron recogidos a partir de noviembre 2010 a febrero 2012 a través de la observación participante y las entrevistas, y analizado con base en la Teoría del Cuidado Transcultural. Se identificaron las prácticas de atención médica y imunizacion,el cuidado de la medicina, así tradicional. Para lograrlo, los profesionales tenían estrategias que proporcionaban el mantenimiento de las personas mayores en su atención. Se concluye que los valores culturales y científicos necesitan ayuda para mejorar la salud de los ancianos indígenas.