Utilização de material de origem coralina em regeneração óssea guiada

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas MonizA perda óssea, fisiológica ou patológica, é um processo inerente à vida do ser humano. O envelhecimento, o trauma, doenças infeciosas, degenerativas ou oncológicas, entre outros fatores, motivam a necessidade de terapêuticas regenerativas do tecido duro mineralizado especializado que designamos por osso, sendo esta necessidade por demais premente nas estruturas maxilares, onde avultam especialmente razões relacionadas com os dentes e os seus tecidos de suporte. Procedimentos regenerativos ósseos podem incluir a utilização de osso autólogo, produtos modificados com origem em osso humano ou animal, e ainda materiais naturais ou de síntese, todos com diferentes capacidades e desempenhos, o que motiva o estudo aturado por forma a otimizar a sua utilização. Pesquisas desenvolvidas permitiram a identificação e a purificação de extratos de coral, produzindo biomateriais com capacidades regenerativas sobre o tecido ósseo.Bone loss, physiological or pathological, is an inherent process in human life. Aging, trauma, infectious, degenerative or oncologic diseases, among other factors, motivate the need for regenerative therapies of the specialized mineralized hard tissue that we call bone, being this need too urgent in the maxillary structures, where there are especially reasons related to the teeth and their supporting tissues. Bone regenerative procedures can include the use of autologous bone, modified products from human or animal bone, and also natural or synthetic materials, all with different capacities and performances, which motivate the thorough study in order to optimize its use. Researches developed have allowed the identification and purification of coral extracts, producing biomaterials with regenerative capacities over bone tissue

Production, Recovery and Characterization of an Enterocin with Anti-Listerial Activity Produced by Enterococcus hirae OS1

 Background and objective: Lactic acid bacteria, used in food processing for a long time, can produce various metabolites during their growth, including bacteriocins. These antimicrobials, used as natural bio-preservatives, enhance the food safety. The objective of this study was to assess lactic acid bacteria bacteriocins with anti-listerial activity and optimize their production and recovery process.Material and methods: Isolate was identified using conventional assays (morphological and biochemical characteristics) and 16S rRNA gene sequencing. Lactic acid bacteria bacteriocins were characterized based on their physicochemical properties (nature, pH stability and thermo-resistance). The production process was based on optimization of media components (growth media and addition of glucose, nitrogen source and tween 20) and culture conditions (temperature, pH, agitation and inoculum size). Furthermore, optimization of the recovery process was studied using ion exchange chromatography on amberlite IRC-50 (effects of resin size and NaCl eluent concentration).Results and conclusion: Enterococcus hirae OS1 was isolated from Moroccan raw cow milk as bacteriocinogenic strain. After optimization of the bacteriocin production process, results showed that the key parameters for increasing of production included temperature of 30°C, pH of 6.5 and inoculum size of 5%. Production with whey-based and economic food-grade substrate allowed high production of enterocin OS1 (1,600 AU ml-1) (P<0.001). Simultaneous addition of glucose (1%) and Tween 20 (1%) increased enterocin titer significantly (5,866 AU ml-1) (P<0.01). Recovery efficiency increased with use of 70% amberlite IRC-50 resin and elution with 2M NaCl. Indeed, recovery of 75.4% of bacteriocin was achieved in comparison to 15.7% of bacteriocin without optimization. This promises achieving high quantities of enterocin at low costs.Conflict of interest: The authors declare no conflict of interest

La movilidad transfonteriza: el caso de Melilla-Nador

This article aims to study and analyze the border region of Melilla (Spain) - Nador (Morocco) highlighting the conflict of cross-border mobility and emphasizing peaceful relations between individuals and/or citizens. However, the definition of border diluted from neighboring areas when we approach the centers of power. This border is a multicultural geological fault defined both a colonial border between Spain and Morocco in economic terms between Europe and Africa, a geopolitical division between the north and south of the Mediterranean and, primarily, a religious conflict between Christianity and Islam. The border region between Melilla-Nador has become a multicultural diversity and variability space, as a rehearsal interdisciplinary space. Concluding that the transboundary speech of people who practice the movement between both sides and examining narratives define a population without fear of exchange where national identity is giving way to the border and/or transborder identity Resumen: Este artículo pretende estudiar y analizar la región fronteriza Melilla (España) - Nador (Marruecos) destacando el conflicto de la movilidad transfronteriza y acentuando las relaciones pacíficas entre los individuos y/o ciudadanos. Sin embargo, la definición de frontera se diluye desde las zonas limítrofes mientras nos aproximamos a los centros de poder. Esta frontera es una falla geológica, multicultural, que se define a la vez una frontera colonial entre España y Marruecos, en términos económicos entre Europa y África, una división geopolítica entre el norte y el sur del Mediterráneo y un conflicto religioso principalmente entre cristianismo e islam. La región fronteriza entre Melilla-Nador se ha convertido un espacio de diversidad y variabilidad multicultural, como espacio de ensayo de carácter interdisciplinar. Concluimos que el discurso transfronterizo de las personas que practican el movimiento entre ambas orillas y el examen de las narrativas definen a una población sin miedo al cambio donde la identidad nacional cede terreno ante la identidad fronteriza y/o transfronterizo.Abstract: This article aims to study and analyze the border region of Melilla (Spain) - Nador (Morocco) highlighting the conflict of cross-border mobility and emphasizing peaceful relations between individuals and/or citizens. However, the definition of border diluted from neighboring areas when we approach the centers of power. This border is a multicultural geological fault defined both a colonial border between Spain and Morocco in economic terms between Europe and Africa, a geopolitical division between the north and south of the Mediterranean and, primarily, a religious conflict between Christianity and Islam. The border region between Melilla-Nador has become a multicultural diversity and variability space, as a rehearsal interdisciplinary space. Concluding that the transboundary speech of people who practice the movement between both sides and examining narratives define a population without fear of exchange where national identity is giving way to the border and/or transborder identity

Assessment of Enterococcus durans F21 Isolated from Lben, a Moroccan Fermented Milk as a Biopreservative Agent

Abstract  Background and Objective: Lactic acid bacteria used in food processing for a long time are known for their benefits to consumers and their ability to produce natural antimicrobial compounds used as bio-preservatives in foods. The aim of the present study was the characterization and assessment of F21 strain, isolated from Lben (a traditional Moroccan fermented milk), as food biopreservative. Material and Methods: Isolate F21 was isolated from Lben, subjected to screening of inhibitory activity production, and identified based on morphological, biochemical and molecular identification. Then, the production and physicochemical characterization of the antagonistic substance were determined. Also, the safety profiling and biotechnological properties of isolate were evaluated. Finally, a biopreservative powder with antimicrobial activity was produced and assessed in various food systems (milk, ground beef and fresh cheese). Results and Conclusion: Of the isolated lactic acid bacteria, Enterococcus durans F21, isolated from Lben (a traditional Moroccan fermented milk), was remarkably endowed with interesting enterocin-like substance (heat stable and pH resistant) active against potentially pathogens and food spoilages (Listeria monocytogenes, Listeria innocua, Enterococcus faecalis, Brochothrix thermosphacta, and Mycobacterium smegmatis). Concerning the safety properties, Enterococcus durans F21 was not hemolytic, sensible to antibiotics tested, unable to produce biogenic amines and other virulence enzymes (gelatinase, DNase and urease). In addition, Enterococcus durans F21 showed satisfactory biotechnological characteristics such as acidification power, exopoly-saccharides production and antioxidant activity. The biopreservative powder containing enterocin-like substance F21 that was achieved via freeze-drying showed a minimum inhibition concentration of 60 AU ml-1 against Listeria monocytogenes in culture media. In addition, this biopreservative powder (at 665 AU ml-1) was able to improve safety and shelf-life of numerous foods (milk, Jben and ground beef). Thus, these results provided foundations for further uses of Enterococcus durans F21 as producer of potential food biopreservative agent. Conflict of interest: The authors declare no conflict of interest

A Molecular Investigation of Malaria Infections From High-Transmission Areas of Southern Togo Reveals Different Species of Plasmodium Parasites

Malaria particularly burdens people in poor and neglected settings across the tropics of Africa. Meanwhile, a large proportion of the Togo population have poor understanding of malaria epidemiology and parasites. This study carried out a molecular survey of malaria cases in southern Togo during 2017–2019. We estimated Plasmodium species infection rates and microscopic examination compliance with nested PCR results. Sensitivity and specificity analyses were performed in conjunction with predictive values. Also, phylogenetic characterization of species of malaria parasites was assessed. Plasmodium genus-specific nested PCR identified 565 positive cases including 536/611 (87.8%) confirmed cases from the microscopy-positive group and 29/199 (14.6%) diagnosed malaria cases from the microscopy-negative group. Our findings revealed a disease prevalence (69.8%) higher than that reported (25.5–55.1%) for the country. The diagnostic test had 94.9% sensitivity and 69.4% specificity, i.e., it missed 120 of the people who had malaria and about one-third of the people tested positive for the disease, which they did not have, respectively. In conjunction, the test showed 87.7% positive predictive value and 85.4% negative predictive value, which, from a clinical perspective, indicates the chance that a person with a positive diagnostic test truly has the disease and the probability that a person with a negative test does not have the disease, respectively. Further species-specific nested PCR followed by analysis of gene sequences confirmed species of malaria parasites and indicated infection rates for Plasmodium falciparum (Pf), 95.5% (540/565); P. ovale (Po), 0.5% (3/565); and P. malariae (Pm), 0.4% (2/565). In addition, 20 cases were coinfection cases of Pf-Po (15/565) and Pf-Pm (5/565). This study publicly reports, for the first time, a molecular survey of malaria cases in Togo and reveals the presence of other malaria parasites (Po and Pm) other than Pf. These findings might provide answers to some basic questions on the malaria scenario and, knowledge gained could help with intervention deployment for effective malaria control in Togo

Insights into Chagas treatment based on the potential of bacteriocin AS-48

Chagas disease caused by the protozoan parasite Trypanosoma cruzi represents a significant public health problem in Latin America, affecting around 8 million cases worldwide. Nowadays is urgent the identification of new antichagasic agents as the only therapeutic options available, Nifurtimox and Benznidazole, are in use for>40 years, and present high toxicity, limited efficacy and frequent treatment failures in the chronic phase of the disease. Recently, it has been described the antiparasitic effect of AS-48, a bacteriocin produced by Enterococcus faecalis, against Trypanosoma brucei and Leishmania spp. In this work, we have demonstrated the in vitro potential of the AS-48 bacteriocin against T. cruzi. Interesting, AS-48 was more effective against the three morphological forms of different T. cruzi strains, and displayed lower cytotoxicity than the reference drug Benznidazole. In addition, AS-48 combines the criteria established as a potential antichagasic agent, resulting in a promising therapeutic alternative. According to the action mechanism, AS-48 trypanocidal activity could be explained in a mitochondrion-dependent manner through a reactive oxygen species production and mitochondrial depolarization, causing a fast and severe bioenergetic collapse.This work was supported by the Spanish Ministry of Economy and Competitiveness [grant numbers SAF2013-48971-C2-1-R, CSD2010- 00065], both including funds from the European Regional Development Fundings (ERDF), and the Ministry of Education of Spain [RM-E, grant number FPU14/01537]

Analysis of the Promoters Involved in Enterocin AS-48 Expression

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.This work was supported in part by the Ministerio de Ciencia e Innovación project BIO2008-01708, the Plan Propio from the University of Granada (Spain) and by the Research Plan Group (BIO 160)

Control of Propionibacterium acnes by natural antimicrobial substances: Role of the bacteriocin AS-48 and lysozyme

We report the high susceptibility of several clinical isolates of Propionibacterium acnes from different sources (skin, bone, wound exudates, abscess or blood contamination) to the head-to-tail cyclized bacteriocin AS-48. This peptide is a feasible candidate for further pharmacological development against this bacterium, due to its physicochemical and biological characteristics, even when it is growing in a biofilm. Thus, the treatment of pre-formed biofilms with AS-48 resulted in a dose- and time-dependent disruption of the biofilm architecture beside the decrease of bacterial viability. Furthermore, we demonstrated the potential of lysozyme to bolster the inhibitory activity of AS-48 against P. acnes, rendering high reductions in the MIC values, even in matrix-growing cultures, according to the results obtained using a range of microscopy and bioassay techniques. The improvement of the activity of AS-48 through its co-formulation with lysozyme may be considered an alternative in the control of P. acnes, especially after proving the absence of cytotoxicity demonstrated by these natural compounds on relevant human skin cell lines. In summary, this study supports that compositions comprising the bacteriocin AS-48 plus lysozyme must be considered as promising candidates for topical applications with medical and pharmaceutical purposes against dermatological diseases such as acne vulgaris.This research was funded by a grant from the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R that included funds from European Regional Development, ERDF), and the Research Group General (BIO160, UGR)

Antibacterial activity and mode of action of selected glucosinolate hydrolysis products against bacterial pathogens

Plants contain numerous components that are important sources of new bioactive molecules with antimicrobial properties. Isothiocyanates (ITCs) are plant secondary metabolites found in cruciferous vegetables that are arising as promising antimicrobial agents in food industry. The aim of this study was to assess the antibacterial activity of two isothiocyanates (ITCs), allylisothiocyanate (AITC) and 2-phenylethylisothiocyanate (PEITC) against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. The antibacterial mode of action was also characterized by the assessment of different physiological indices: membrane integrity, intracellular potassium release, physicochemical surface properties and surface charge. The minimum inhibitory concentration (MIC) of AITC and PEITC was 100 g/mL for all bacteria. The minimum bactericidal concentration (MBC) of the ITCs was at least 10 times higher than the MIC. Both AITC and PEITC changed the membrane properties of the bacteria decreasing their surface charge and compromising the integrity of the cytoplasmatic membrane with consequent potassium leakage and propidium iodide uptake. The surface hydrophobicity was also non-specifically altered (E. coli and L. monocytogenes become less hydrophilic; P. aeruginosa and S. aureus become more hydrophilic). This study shows that AITC and PEITC have strong antimicrobial potential against the bacteria tested, through the disruption of the bacterial cell membranes. Moreover, phytochemicals are highlighted as a valuable sustainable source of new bioactive products.This work was supported by the Operational Programme for Competitiveness Factors - COMPETE and by the Portuguese Foundation for Science and Technology through Project Phytodisinfectants - PTDC/DTP-SAP/1078/2012 (COMPETE: FCOMP-01-0124-FEDER-028765), the PhD grant awarded to Ana Abreu (SFRH/BD/84393/2012), and the post-doctoral grants awarded to Anabela Borges (SFRH/BPD/98684/2013) and Lucia C. Simoes (SFRH/BPD/81982/2011). Also, this work was undertaken as part of the European Research Project SUSCLEAN (Contract no FP7-KBBE-2011-5, project number: 287514) and the COST Action FA1202. The authors are solely responsible for this work. It does not represent the opinion of the European Community, and the Community is not responsible for any use that might be made of data appearing herein