Analysis of sensory effects of chitosan-based coatings applied on frozen salmon over six-months storage

The 19th Gums & Stabilisers for the Food Industry Conference: Hydrocolloid MultifunctionalityDue to an increase in fish consumption over the past years, in combination with the perishable nature of fish, the fish industry has given an added focus on the improvement of the currently used fish preservation techniques. Chitosan coatings may add improvements to the traditional water glazing, namely in physical and anti-microbial protection, allowing for shelf life extension. This work was meant to determine the influence of chitosan coatings in the organoleptic characteristics of salmon. The effects of chitosan coatings on microbiological (Total Viable Count – TVC) and chemical (Total Volatile Base Nitrogen – TVB-N) parameters were also assessed. A 15 g/L solution of chitosan was used to coat frozen salmon samples, and its effect on the sensory properties of Atlantic salmon (Salmo salar) was compared with uncoated and water glazed samples, and was studied over six months of storage at -18 °C. Samples were dipped in the chitosan solution at 8 °C during 10 s; water glazing was applied at 0.5 °C, with a dipping time of 40 s. Textural properties were evaluated through Texture Profile Analysis (TPA), while sensory properties of frozen, frozen+thawed and frozen+thawed+cooked samples were assessed by a trained panel of judges. Microbiological stability was assessed through TVC (ISO 4833-1:2013 standard), and chemical stability was determined as TVB-N (NP 2930:2009 standard). TVC analysis showed an anti-microbial effect of chitosan in the coated samples (reduction of the number of microorganisms), while TVB-N results showed to remain stable during the experiment. Textural results from the TPA analysis showed no significant differences between different coatings. Results of the trained panel indicated that for frozen samples chitosan was the preferred choice, while no significant differences existed between chitosan-coated and glazed samples in thawed and cooked samples. Flavor diffusion from the chitosan coating to the samples was assessed by Principal Component Analysis and no correlation between coating type and sample flavor could be established, meaning that no chitosan flavor was detected by the panellists.info:eu-repo/semantics/publishedVersio

Hyaluronic acid-amphotericin B nanocomplexesa: a promising anti-leishmanial targeted drug delivery system

Leishmaniasis has been classified as one of the most neglected tropical diseases, causing 50 thousand deaths and 1.5 to 2 million new cases every year, according to the World Health Organization. This disease, promoted by protozoan parasites of the genus Leishmania, has a high incidence affecting 89 countries worldwide. Nowadays, current treatment strategies still rely on the antifungal agent amphotericin B (AmB) but are rather inadequate due to the high prevalence of the disease within low-income population of sub-developed regions, the intracellular location of the parasite and the emergence of parasite resistance. Thus, other strategies have been pursued to improve the therapeutic efficacy and to reduce the toxicity of AmB such as the use of biocompatible polysaccharides as carriers. In this work, a simple and inexpensive production process using hyaluronic acid (HA, 50 kDa) was used in order to develop water-soluble hyaluronic acid-amphotericin B nanocomplex (HA-AmB). HA is the main ligand of CD44 receptor, thus being favorably internalized by macrophages that overexpress this receptor upon infection. Therefore, HA arises as a suitable polysaccharide to target the AmB delivery to the leishmania-infected macrophages. The nanocomplex, obtained by simply processing the mixture of the polysaccharide with the drug in a nanospray dryer (HA-AmB SD), was characterized in terms of size/zeta potential (DLS) and morphology (SEM and Cryo-SEM). Furthermore, an HPLC-MS detection method was optimized and used to determine the AmB content in the nanocomplex. Also, to ascertain the interaction between AmB and the HA, FTIR, DSC and PXRD analysis were performed. Cytotoxic and hemolytic effects were assessed on different cell lines through the resazurin test and in dogs blood, respectively. Anti-leishmanial activity was assessed in vitro in axenic cultures of Leishmania by resazurin and in infected bone marrow-derived macrophages (BMM) stained with different fluorescent probes using high-content microscopy. Our results shown that the produced material has a spherical morphology in aqueous solution with a mean hydrodynamic diameter of 318.4 ± 34.7 nm and low polydispersity (0.239 ± 0.02). Moreover, this material that presents an AmB content of 13.56 ± 3.49 %, has a good colloidal stability due to the highly negative surface charge (-39.45 ± 1.12 mV). DSC and PXRD analysis strongly suggested the formation of an amorphous inclusion complex between AmB and the complex polysaccharide chain networks, explaining the high solubility of the drug in water. The in vitro assays showed that compared to free-AmB, the nanocomplex had significantly less cytotoxicity against BMM and HEK293T cell lines, significant less hemolytic effect and inhibited the infection in the Leishmania-infected BMM. Exploratory in vivo assays are being conducted in mice. In conclusion, this work has shown that the hyaluronic acid-AmB nanocomplex is a promising system for the treatment of Leishmaniasis, possessing similar effects to the free-AmB against Leishmania-infected macrophages and Leishmania axenic cultures, with reduced cytotoxicity. Given the affordability, simplicity, low-toxicity and facile scale up of the developed formulation, the hyaluronic acid-AmB nanocomplex may represent an alternative to the expensive nanoformulations available.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for supporting this study under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ricardo Silva-Carvalho also acknowledges FCT for the PhD scholarship SFRH/BD/118880/2016.info:eu-repo/semantics/publishedVersio

Influence of different types of bacterial nanocellulose on development of oil-in-water Pickering emulsions

Bacterial nanocelluloses have been studied to stabilize oil-in-water emulsions due to its ability to adsorb on the oil-water interface, promoting highly stable and surfactant-free systems. However, several features of the bacterial nanocellulose may influence the resultant emulsion, such as the cellulose nature, size, surface charge, shape and chemical surface. Thus, this work aims to produce sunflower oil-in-water emulsions using bacterial nanocelluloses produced by fermentation of Komagataeibacter xylinus in Hestrin e Schramm (HS) medium and processed by two different treatments: 2,2,6,6-tetramethyl-1-piperidinoxyl (TEMPO) mediated oxidation for the production of cellulose nanofibrils (CNF) and acid hydrolysis with sulfuric acid for the production of cellulose nanocrystals (CNC). The oxidized bacterial cellulose suspension was further nanofibrillated by high-speed homogenizer that produced the CNF (82 nm diameter and -46.5 mV surface charge). Nanocrystals had an average length of 491 nm, mean diameter of 70 nm and -50.3 mV of surface charge. For each nanocellulose, different o/w ratios were tested, in order to produce stable emulsions. The emulsions were formulated with an oil phase of 10-1% and an aqueous phase of 90-99%, with a nanocellulose concentration 0.5-1%. Different salt concentrations (NaCl) were tested in the aqueous phase (0-50 mM). The emulsion stability was evaluated by visual inspection considering that the absence of oil in the emulsion surface represents emulsion stability. Emulsions stabilized by CNC exhibited a mean droplet diameter varying between 1.2 and 2.0 m, with a white color and fluid texture. On the other hand, the emulsions stabilized by CNF formed droplets above 2.0 m with a less fluid texture, which confirmed the influence of the bacterial nanocellulose features on the characteristics of the emulsions formed. The microscopy analysis performed with polarized light demonstrated the presence of fibrils and crystals on the oil-water interface of the droplets. The oil droplets were also analysed by fluorescence microscopy after nile red staining. These results indicated that pickering emulsions composed by CNC and CNF, can be used as a carrier to encapsulate active compounds. Beta-carotene will be incorporated in the oil-in-water emulsions, as an hydrophobic model molecule, and digestibility studies will be performed to assess the beta-carotene bioaccessibility in different formulations.info:eu-repo/semantics/publishedVersio

Portuguese study of familial dilated cardiomyopathy: the FATIMA study

Dilated cardiomyopathy (DCM) is a myocardial disease, characterized by ventricular dilatation and impaired systolic function, that in more than 30% of cases has a familial or genetic origin. Given its age-dependent penetrance, DCM frequently manifests in adults by signs or symptoms of heart failure, arrhythmias or sudden death. The predominant mode of inheritance is autosomal dominant, and in these cases mutations are identified in genes coding for cytoskeletal, sarcomeric or nuclear envelope proteins. To date, most studies aimed at molecular diagnosis of DCM have been in selected families, or in larger groups of patients, but screening for mutations in a limited number of genes. Consequently, the epidemiology of mutations in familial DCM remains unknown. There is thus a need for multicenter studies, involving screening for a wide range of mutations in several families and in cases of idiopathic DCM. The present article describes the methodology of a multicenter study, aimed at clinical and molecular characterization of familial DCM patients in the Portuguese population.A miocardiopatia dilatada (MCD) é uma doença do músculo cardíaco caracterizada pela dilatação ventricular e compromisso da função sistólica, sendo possível identificar, numa percentagem superior a 30% dos casos, uma origem familiar ou genética. Dada a penetrância dependente da idade, manifesta-se muitas vezes em adultos por sinais ou sintomas de insuficiência cardíaca, arritmias ou morte súbita. O padrão autossómico dominante predomina, sendo possível identificar, nestes casos, mutações em genes de proteínas do citoesqueleto celular, sarcómero ou membrana nuclear. Até ao momento, a maioria dos trabalhos visando o diagnóstico molecular nos casos de MCD foi realizada em famílias seleccionadas, ou em grupos mais abrangentes de doentes, mas rastreando mutações num número restrito de genes. Consequentemente a epidemiologia das mutações nos casos familiares de MCD continua por esclarecer. É neste contexto que se coloca a necessidade de efectuar estudos multicêntricos, envolvendo uma pesquisa mutacional diversificada em várias familias e nos casos idiopáticos de MCD. O presente artigo descreve a metodologia de um estudo multicêntrico que tem como objectivo a caracterização clínica e molecular de casos familiares de MCD na população portuguesa

Behavior of lactoferrin nanohydrogels incorporating curcumin as model compound into food simulants

Oscar L. Ramos acknowledge his Post-Doctoral grant (SFRH/BPD/80766/2011) to the Fundação para a Ciência e Tecnologia (FCT, Portugal). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. João F. Araújo acknowledge the Escola de Ciências and Centro de Engenharia Biológica from Universidade do Minho (Braga, Portugal) for their support, under the scope of the Biophysics and Bionanosystems Master program.info:eu-repo/semantics/publishedVersio

In vitro digestion and stability assessment of b-lactoglobulin/riboflavin nanostructures

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period.Oscar L. Ramos, Ricardo N. Pereira and Ana C. Pinheiro gratefully acknowledge their Post-Doctoral grants (SFRH/BPD/80766/2011, SFRH/BPD/81887/2011 and SFRH/BPD/101181/2014, respectively) and Ana I. Bourbon gratefully acknowledge her Doctoral grant (SFRH/BD/73178/2010) to Fundacao para a Ciencia e a Tecnologia (FCT). The authors would like to acknowledge to Francisco Figueiredo from the Institute for Molecular and Cell Biology (IBMC), University of Porto, for assistance in taking the TEM pictures. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project "BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER

Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice

<p>Abstract</p> <p>Background</p> <p>Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned <it>Lgl1</it>, a developmentally regulated secreted glycoprotein in the lung. In rat, O₂toxicity caused reduced levels of <it>Lgl1</it>, which normalized during recovery. We report here on the generation of an <it>Lgl1 </it>knockout mouse in order to determine whether deficiency of <it>Lgl1 </it>is associated with arrested alveolarization and contributes to neonatal lung injury.</p> <p>Methods</p> <p>An <it>Lgl1 </it>knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the <it>Lgl1 </it>gene. To evaluate the pulmonary phenotype of <it>Lgl1</it>^+/-mice, we assessed lung morphology, <it>Lgl1 </it>RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.</p> <p>Results</p> <p>Absence of <it>Lgl1 </it>was lethal prior to lung formation. Postnatal <it>Lgl1</it>^+/-lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of T_H2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of <it>Lgl1 </it>was associated with elevated tropoelastin expression and altered pulmonary mechanics.</p> <p>Conclusion</p> <p>Our findings confirm that <it>Lgl1 </it>is essential for viability and is required for developmental processes that precede lung formation. <it>Lgl1</it>^+/-mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. <it>Lgl1 </it>haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.</p

Tracing exogenous surfactant in vivo in rabbits by the natural variation of 13C

BACKGROUND: Respiratory Distress Syndrome (RDS) is a prematurity-related breathing disorder caused by a quantitative deficiency of pulmonary surfactant. Surfactant replacement therapy is effective for RDS newborns, although treatment failure has been reported. The aim of this study is to trace exogenous surfactant by 13C variation and estimate the amount reaching the lungs at different doses of the drug. METHODS: Forty-four surfactant-depleted rabbits were obtained by serial bronchoalveolar lavages (BALs), that were merged into a pool (BAL pool) for each animal. Rabbits were in nasal continuous positive airway pressure and treated with 0, 25, 50, 100 or 200 mg/kg of poractant alfa by InSurE. After 90 min, rabbits were depleted again and a new pool (BAL end experiment) was collected. Disaturated-phosphatidylcholine (DSPC) was measured by gas chromatography. DSPC-Palmitic acid (PA) 13C/12C was analyzed by isotope ratio mass spectrometry. One-way non-parametric ANOVA and post-hoc Dunn's multiple comparison were used to assess differences among experimental groups. RESULTS: Based on DSPC-PA 13C/12C in BAL pool and BAL end experiment, the estimated amount of exogenous surfactant ranged from 61 to 87% in dose-dependent way (p < 0.0001) in animals treated with 25 up to 200 mg/kg. Surfactant administration stimulated endogenous surfactant secretion. The percentage of drug recovered from lungs did not depend on the administered dose and accounted for 31% [24-40] of dose. CONCLUSIONS: We reported a risk-free method to trace exogenous surfactant in vivo. It could be a valuable tool for assessing, alongside the physiological response, the delivery efficiency of surfactant administration techniques

MC EMiNEM Maps the Interaction Landscape of the Mediator

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors

Cohesive versus Flexible Evolution of Functional Modules in Eukaryotes

Although functionally related proteins can be reliably predicted from phylogenetic profiles, many functional modules do not seem to evolve cohesively according to case studies and systematic analyses in prokaryotes. In this study we quantify the extent of evolutionary cohesiveness of functional modules in eukaryotes and probe the biological and methodological factors influencing our estimates. We have collected various datasets of protein complexes and pathways in Saccheromyces cerevisiae. We define orthologous groups on 34 eukaryotic genomes and measure the extent of cohesive evolution of sets of orthologous groups of which members constitute a known complex or pathway. Within this framework it appears that most functional modules evolve flexibly rather than cohesively. Even after correcting for uncertain module definitions and potentially problematic orthologous groups, only 46% of pathways and complexes evolve more cohesively than random modules. This flexibility seems partly coupled to the nature of the functional module because biochemical pathways are generally more cohesively evolving than complexes