Clonagem e expressão do gene que codifica o inibidor de quimotripsina de Erythrina velutina WILLD. - caracterização e avaliação de seu potencial farmacológico

Proteinases are enzymes distributed widely founded in several organisms and perform many different functions, from maintaining homeostasis to the worsening of some diseases such as cancer, autoimmune diseases and infections. The proteins responsible of controlling the action of these enzymes are the inhibitors, that are classified based on their target proteases and are founded since simple organisms, such as bacteria, to higher organisms, such as larger plants and mammals. Plant proteinase inhibitors act by reducing or inactivating the activity of target proteases, thus, these proteins have been studied as potential tools in the treatment of diseases related to protease activities. In this context, an inhibitor of chymotrypsin from Erythrina velutina, called EvCI was previously purified and it was observed that this protein plays in vitro anticoagulant activity and anti-inflammatory activity in in vivo model. Aiming to reduce the environmental impact caused by the purification EvCI in high amounts and to facilitate the process of obtaining this protein, the recombinant chymotrypsin inhibitor from Eryhrina velutina was produced after cloning and expression in Escherichia coli. The bacteria were grown in LB medium and after induction of the expression this material was subjected to procedures for cell lysis and the product was applied on Nickel-affinity column. The proteins adsorbed were digested by thrombin and applied on Chymotrypsin-Sepharose affinity column, obtaining the purified inhibitor, named recEvCI. After electrophoresis, the recombinant inhibitor showed an approximately molecular mass of 17 kDa, and reduced the chymotrypsin and elastase activities in vitro. The recombinant inhibitor was sequenced and was found similar amino acids residues when compared to other inhibitors deposited in the database, with some modifications. recEvCI showed high stability under pH variations and reducing conditions, maintaining its activity around 80%. This protein increased the blood coagulation time in vitro by acting on the intrinsic pathway and did not show cytotoxicity against strains of mouse 3T3 fibroblasts and RAW 264.7 macrophages. recEvCI showed microbicide activity related to release of nitric oxide and consequently the activation of macrophages, futhermore having proinflammatory effects assessed by increased release of TNF-α. These results indicate that recEvCI can be biotechnologically used as a new tool in the control of coagulation-related diseases as well as can be an activating agent of the immune system in immunosuppressed individuals

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

A novel mutation of AFG3L2 might cause dominant optic atrophy in patients with mild intellectual disability

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Atividade de enzimas ligadas ao ciclo do nitrogênio em solo submetido a frequentes aplicações de lodo de esgoto.

RESUMO: Grande proporção do nitrogênio no solo está na forma de material proteináceo, cuja quebra é regulada pela atividade de proteases extracelulares. Outra enzima envolvida no ciclo do nitrogênio, a urease, é responsável pela hidrólise da uréia a amônia e CO2. Neste trabalho, avaliou-se o efeito da suplementação do solo com diferentes doses e tipos de lodo (ETE de Barueri e ETE de Franca) na atividade das enzimas protease e urease. As aplicações de lodo ao solo iniciaram-se em 1999 a taxas que variaram na dose recomendada, tomando-se como base os requerimentos da planta em N, até uma taxa 8 vezes maior. Nos anos de 2004 e 2005 os lodos não foram aplicados. Em 2006 e 2007 somente foi aplicado o lodo de Franca. Os resultados deste trabalho referem-se às coletas de solo feitas no ano agrícola 2007/2008. A atividade da protease aumentou com o aumento da dose de lodo de Franca. Por outro lado, houve decréscimo da atividade da protease com o aumento da dose do lodo de Barueri. Na maior dose deste lodo a atividade foi semelhante à obtida no tratamento com fertilização mineral. A menor atividade daquela enzima foi obtida no tratamento testemunha. A atividade da urease foi maior nos tratamentos com o lodo de Franca independentemente da dose aplicada. A atividade das duas enzimas envolvidas no ciclo do nitrogênio foram sensíveis indicadores da qualidade de solo tratado com lodo de esgoto.

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

Flavonoids, coumarins and other polyphenolic compounds are powerful antioxiants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for blood coagulation disorders, more specifically to control some serine protease enzymes, the mechanism of anti-inflammatory activity of coumarins is unknown, unlike that of flavonoids. Although their controlling effect on serine proteases is well acknowledged, their action on secretory phospholipase A2 (sPLA2) remains obscure. The present study describes the interaction between umbelliferone (7-HOC) and the sPLA2 from Crotalus durissus collilineatus venom. In vitro inhibition of sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this enzyme and CD spectra revealed that it strongly decreased sPLA2 alphahelical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated sPLA2s, which were used in pharmacological experiments to compare their biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw edema model, in which 7-HOC presented an effect similar to those of dexamethasone and cyproheptacline against the pro-inflammatory effect induced by native sPLA2 on the mouse paw edema, mast cell degranulation and skin edema. on the other hand, 7-HOC exhibited a more potent inhibitory effect on sPUL2 than that of p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom. (C) 2008 Published by Elsevier Ltd.

Do improved pastures affect enzymatic activity and C and N dynamics in soils of the montado system ?

Vast montado areas are threatened by degradation, as the result of a long history of land use changes. Since improved pastures have been installed aiming soil quality improvement and system sustainability, it is crucial to evaluate the effects of these management changes on soil organic matter status and soil biological activity, as soil quality indicators. Therefore, a 35-yr old improved pasture and a natural pasture were studied, considering areas beneath tree canopy and in the open. Total organic C, total N, hot water soluble (HWS) and particulate (POM) C, microbial biomass C (MBC) and N (MBN), C mineralization rate (CMR) and net N mineralization rate (NMR) were determined. In addition, for a 1-yr period, soil β-glucosidase, urease, proteases and acid phosphomonoesterase were periodically determined. Improved pasture promoted the increase of soil C and N through POM-C increment, particularly beneath the trees canopies. The two study pastures did not show differences regarding soil microbial biomass, but variations in CMR, HWS-C and N availability (proteases and urease activities) suggest divergent soil microbial communities. Tree regulator role on C, N and P transformation processes in soil was confirmed

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

Background The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. Conclusions This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Associação entre o proteassoma e o inibidor de protease BTCI : caracterização fisico-química e efeitos moleculares em células de câncer de mama (MCF-7)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2010.

Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment.

Mangrove ecosystems are environments subject to substantial degradation by anthropogenic activities. Its location, in coastal area, interfacing the continents and the oceans makes it substantially important in the prospection for biotechnological applications. In this study, we assessed the diversity of culturable bacteria present over the seasons at two depths (0-10 and 30-40 cm) in a mangrove sediment and in a transect area from the land to the sea. In total, 238 bacteria were isolated, characterized by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and further identified, by Fatty Acid Methyl Esther (FAME-MIDI), into the orders of Vibrionales, Actinomycetales and Bacillales. Also the ability of the isolates in producing economically important enzymes (amylases, proteases, esterases and lipases) was evaluated and the order Vibrionales was the main enzymatic source.

Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo

The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins: KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period, and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples.

Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio-economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host-pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population.

An investigation of the relationship between the components of tumour microenvironment, signal transduction pathways and outcome in breast cancer

Breast cancer, the most commonly diagnosed type of cancer in women, is a major cause of morbidity and mortality in the western world. Well-established risk factors of breast cancer are mostly related to women’s reproductive history, such as early menarche, late first pregnancy and late menopause. Survival rates have improved due to a combination of factors, including better health education, early detection with large-scale use of screening mammogram, improved surgical techniques, as well as widespread use of adjuvant therapy. At initial presentation, clinicopathological features of breast cancer such as age, nodal status, tumour size, tumour grade, and hormonal receptor status are considered to be the standard prognostic and predictive markers of patient survival, and are used to guide appropriate treatment strategies. Lymphovascular invasion (LBVI), including lymphatic (LVI) and blood (BVI) vessel invasion, has been reported to be prognostic and merit accurate evaluation, particularly in patients with node negative tumours who might benefit from adjuvant chemotherapy. There is a lack of standard assessment and agreement on distinguishing LVI from BVI despite the major challenges in the field. A systematic review of the literatures, examining methods of detection and the prognostic significance of LBVI, LVI and BVI, was carried out. The majority of studies used haematoxylin and eosin (H&E) and classical histochemistry to identify LVI and BVI. Only few recent studies used immunohistochemistry (IHC) staining of the endothelium lining lymphatic and blood vessels, and were able to show clear differences between LVI and BVI. The prognostic significance of LBVI and LVI was well-documented and strongly associated with aggressive features of breast tumours, while the prognostic value and the optimal detection method of BVI were unclear. Assessment and prognostic value of LBVI on H&E sections (LBVIH&E) was examined and compared to that of LVI and BVI detected using IHC with D2-40 for LVI (LVID2–40) and Factor VIII for BVI (BVIFVIII) in patients with breast cancer including node negative and triple negative patients (n=360). LBVIH&E, LVID2–40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node negative patients (206), LBVIH&E, LVID2–40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple negative patients (102), LBVIH&E, LVID2–40 and BVIFVIII were present in 35 (29%), 36 (35%) and 14 (14%) respectively. LBVIH&E, LVID2–40 and BVIFVIII were all significantly associated with tumour recurrence in all cohorts. On multivariate survival analysis, only LVID2–40 and BVIFVIII were independent predictors of cancer specific survival (CSS) in the whole cohort (P=0.022 and P<0.001 respectively), node negative (P=0.008 and P=0.001 respectively) and triple negative patients (P=0.014 and P<0.001 respectively). Assessment of LVI and BVI by IHC, using D2-40 and Factor VIII, improves prediction of outcome in patients with node negative and triple negative breast cancer and was superior to the conventional detection method. Breast cancer is recognised as a complex molecular disease and histologically identical tumours may have highly variable outcomes, including different responses to therapy. Therefore, there is a compelling need for new prognostic and predictive markers helpful of selecting patients at risk and patients with aggressive diseases who might benefit from adjuvant and targeted therapy. It is increasingly recognised that the development and progression of human breast cancer is not only determined by genetically abnormal cells, but also dependent on complex interactions between malignant cells and the surrounding microenvironment. This has led to reconsider the features of tumour microenvironment as potential predictive and prognostic markers. Among these markers, tumour stroma percentage (TSP) and tumour budding, as well as local tumour inflammatory infiltrate have received recent attention. In particular, the local environment of cytokines, proteases, angiogenic and growth factors secreted by inflammatory cells and stromal fibroblasts has identified crucial roles in facilitating tumour growth, and metastasis of cancer cells through lymphatic and/or blood vessel invasion. This might help understand the underlying process promoting tumour invasion into these vessels. An increase in the proportion of tumour stroma and an increase in the dissociation of tumour cells have been associated with poorer survival in a number of solid tumours, including breast cancer. However, the interrelationship between these variables and other features of the tumour microenvironment in different subgroups of breast cancer are not clear. Also, whether their prognostic values are independent of other components of the tumour microenvironment have yet to be identified. Therefore, the relationship between TSP, clinicopathological characteristics and outcome in patients with invasive ductal breast cancer, in particular node negative and triple negative disease was examined in patients with invasive ductal breast cancer (n=361). The TSP was assessed on the haematoxylin and eosin-stained tissue sections. With a cut-off value of 50% TSP, patients with ≤50% stroma were classified as the low-TSP group and those with >50% stroma were classified as the high-TSP group. A total of 109 (30%) patients had high TSP. Patients with high TSP were old age (P=0.035), had involved lymph node (P=0.049), Her-2 positive tumours (P=0.029), low-grade peri-tumoural inflammatory infiltrate (P=0.034), low CD68+ macrophage infiltrate (P<0.001), low CD4+ (P=0.023) and low CD8+ T-lymphocytes infiltrate (P=0.017), tumour recurrence (P=0.015) and shorter CSS (P<0.001). In node negative patients (n=207), high TSP was associated with low CD68+ macrophage infiltrate (P=0.001), low CD4+ (P=0.040) and low CD8+ T-lymphocytes infiltrate (P=0.016) and shorter CSS (P=0.005). In triple negative patients (n=103), high TSP was associated with increased tumour size (P=0.017) high tumour grade (P=0.014), low CD8+ T-lymphocytes infiltrate (P=0.048) and shorter CSS (P=0.041). The 15-year cancer specific survival rate was 79% vs 21% in the low-TSP group vs high-TSP group. On multivariate survival analysis, a high TSP was associated with reduced CSS in the whole cohort (P=0.007), node negative patients (P=0.005) and those who received systemic adjuvant therapy (P=0.016), independent of other pathological characteristics including local host inflammatory responses. Therefore, a high TSP in invasive ductal breast cancer was associated with recurrence and poorer long-term survival. The inverse relation with the tumour inflammatory infiltrate highlights the importance of the amount of tumour stroma on immunological response in patients with invasive ductal breast cancer. Implementing this simple and reproducible parameter in routine pathological examination may help optimise risk stratification in patients with breast cancer. Similarly, the relationship between tumour budding, clinicopathological characteristics and outcome was examined in patients with invasive ductal breast cancer (n=474), using routine pathological sections. Tumour budding was associated with several adverse pathological characteristics, including positive lymph node (P=0.009), presence of LVI (P<0.001), and high TSP (P=0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.002). In node negative patients, a high tumour budding was associated with presence of LVI (P<0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.038). On multivariate survival analysis, tumour budding was associated with reduced CSS (P=0.001), independent of nodal status, tumour necrosis, CD8+ and CD138+ inflammatory cells infiltrate, LVI, BVI and TSP. Furthermore, tumour budding was independently associated with reduced CSS in node negative patients (P=0.004) and in those who have low TSP (P=0.003) and high-grade peri-tumoural inflammatory infiltrative (P=0.012). A high tumour budding was significantly associated with shorter CSS in luminal B and triple negative breast cancer subtypes (all P<0.001). Therefore, tumour budding was a significant predictor of poor survival in patients with invasive ductal breast cancer, independent of adverse pathological characteristics and components of tumour microenvironment. These results suggest that tumour budding may promote disease progression through a direct effect on local and distant invasion into lymph nodes and lymphatic vessels. Therefore, detection of tumour buds at the stroma invasive front might therefore represent a morphologic link between tumour progression, lymphatic invasion, spread of tumour cells to regional lymph nodes, and the establishment of metastatic dissemination. Given the potential importance of the tumour microenvironment, the characterisation of intracellular signalling pathways is important in the tumour microenvironment and is of considerable interest. One plausible signalling molecule that links tumour stroma, inflammatory cell infiltrate and tumour budding is the signal transducer and activator of transcription (STAT). The relationship between total and phosphorylated STAT1 (ph-STAT1), and total and ph-STAT3 tumour cell expression, components of tumour microenvironment and survival in patients with invasive ductal breast cancer was examined. IHC of total and ph-STAT1/STAT3 was performed on tissue microarray of 384 breast cancer specimens. Cellular STAT1 and cellular STAT3 expression at both cytoplasmic and nuclear locations were combined and identified as STAT1/STAT3 tumour cell expression. These results were then related to CSS and phenotypic features of the tumour and host. A high ph-STAT1 and a high ph-STAT3 tumour cell expression was associated with increased ER (P=0.001 and P<0.001 respectively) and PR (all P<0.05), reduced tumour grade (P=0.015 and P<0.001 respectively) and necrosis (all P=0.001). Ph-STAT1 was associated with increased general peri-tumoural inflammatory infiltrate (P=0.007) and ph-STAT3 was associated with lower CD4+ T-lymphocyte infiltrate (P=0.024). On multivariate survival analysis, including both ph-STAT1 and ph-STAT3 tumour cell expression, only high ph-STAT3 tumour cell expression was significantly associated with improved CSS (P=0.010) independent of other tumour and host-based factors. In patients with high necrosis grade, high ph-STAT3 tumour cell expression was independent predictor of improved CSS (P=0.021). Ph-STAT1 and ph-STAT3 were also significantly associated with improved cancer specific survival in luminal A and B subtypes. STAT1 and STAT3 tumour cell expression appeared to be an important determinant of favourable outcome in patients with invasive ductal breast cancer. The present results suggest that STATs may affect disease outcome through direct impact on tumour cells, and the surrounding microenvironment. The above observations of the present thesis point to the importance of the tumour microenvironment in promoting tumour budding, LVI and BVI. The observations from STATs work may suggest that an important driving mechanism for the above associations is the presence of tumour necrosis, probably secondary to hypoxia. Further work is needed to examine the interaction of other molecular pathways involved in the tumour microenvironment, such as HIF and NFkB in patients with invasive ductal breast cancer.

Revisión sistemática utilidad de asa y calcio con o sin vitamina d en la prevención de cáncer colorrectal y la recurrencia de pólipos adenomatosos en paciente con riesgo de padecer esta patología

Introducción: El cáncer colorrectal es una patología con alto impacto en la salud pública, debido a su prevalencia, incidencia, severidad, costo e impacto en la salud mental y física del individuo y la familia. Ensayos clínicos realizados en pacientes con antecedente de infarto al miocardio que consumían ácido acetil salicílico (asa), calcio con y sin vitamina D, mostraron asociación entre el consumo de estos medicamentos y disminución en la incidencia en cáncer colorrectal y pólipos adenomatosos. Objetivo: Evaluar la literatura sobre el uso de asa, calcio con y sin vitamina D con relación a su impacto en la prevención del cáncer colorrectal y pólipos adenomatosos. Métodos: Se realizó revisión sistemática buscando ensayos clínicos realizados en pacientes con factores de riesgo para cáncer colorrectal y pólipos adenomatosos que usaron asa, calcio con y sin vitamina D fueron incluidos. Resultados: se escogieron 105 para la revisión sistemática. Conclusiones: Es necesario desarrollar más estudios que lleven a evaluar el efecto protector de la aspirina, calcio y vitamina D. En los artículos revisados la aspirina a dosis de 81 a 325 mg día se correlaciona con reducción de riesgo de aparición de CRC aunque la dosis ideal, el tiempo de inicio y la duración de la ingesta continua no son claros. Hacen falta estudios que comparen poblaciones con ingesta de asa a diferentes dosis.

Characterization and Technological Features of Autochthonous Coagulase-Negative Staphylococci as Potential Starters for Portuguese Dry Fermented Sausages

The manufacture of dry fermented sausages is an important part of the meat industry in Southern Europeancountries. These products are usually produced in small shops from a mixture of pork, fat, salt, and condiments andare stuffed into natural casings. Meat sausages are slowly cured through spontaneous fermentation by autochthonousmicrobiota present in the raw materials or introduced during manufacturing. The aim of this work was to evaluate thetechnological and safety features of coagulase-negative staphylococci (CNS) isolated from Portuguese dry fermented meatsausages in order to select autochthonous starters. Isolates (n = 104) obtained from 2 small manufacturers were identifiedas Staphylococcus xylosus, Staphylococcus equorum, Staphylococcus saprophyticus,andStaphylococcus carnosus. Genomically diverseisolates (n = 82) were selected for further analysis to determine the ability to produce enzymes (for example, nitrate-reductases, proteases, lipases) and antibiotic susceptibility. Autochthonous CNS producing a wide range of enzymes andshowing low antibioresistance were selected as potential starters for future use in the production of dry fermented meatsausages.