Discovery of bioactive nitrated lipids and nitro-lipid-protein adducts using mass spectrometry-based approaches

Nitro-fatty acids (NO2-FA) undergo reversible Michael adduction reactions with cysteine and histidine residues leading to the post-translational modification (PTM) of proteins. This electrophilic character of NO2-FA is strictly related to their biological roles. The NO2-FA-induced PTM of signaling proteins can lead to modifications in protein structure, function, and subcellular localization. The nitro lipid-protein adducts trigger a series of downstream signaling events that culminates with anti-inflammatory, anti-hypertensive, and cytoprotective effects mediated by NO2-FA. These lipoxidation adducts have been detected and characterized both in model systems and in biological samples by using mass spectrometry (MS)-based approaches. These MS approaches allow to unequivocally identify the adduct together with the targeted residue of modification. The identification of the modified proteins allows inferring on the possible impact of the NO2-FA-induced modification. This review will focus on MS-based approaches as valuable tools to identify NO2-FA-protein adducts and to unveil the biological effect of this lipoxidation adducts.publishe

Liquid chromatography/tandem mass spectrometry characterization of nitroso, nitrated and nitroxidized cardiolipin products

Cardiolipins (CL) are anionic dimeric phospholipids bearing four fatty acids, found in inner mitochondrial membrane as structural components and are involved in several processes as oxidative phosphorylation or apoptotic signalling. As other phospholipids, CL can be modified by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can modulate various cellular functions. Modifications of CL by RNS remain largely unstudied although other nitrated lipids are emerging as bioactive molecules. In this work, we developed a C30-LC-HRMS/MS methodology to identify the nitrated and nitroxidized tetralinoleoyl-cardiolipin (TLCL), using a biomimetic model of nitration, and to disclose specific fragmentation pathways under HCD MS/MS. Using this lipidomics approach, we were able to separate and identify nitro, nitroso, nitronitroso, and nitroxidized TLCL derivatives, comprising 11 different nitrated compounds. These products were identified using accurate mass measurements and the fragmentation pattern acquired in higher-energy collision dissociation (HCD)-tandem MS/MS experiments. These spectra showed classifying fragmentation pathways, yielding phosphatidic acid (PA-), lysophosphatidic acid (LPA-), and carboxylate fragment ions with the modifying moiety. Remarkably, the typical neutral losses associated with the added moieties were not observed. In conclusion, this work has developed a new method for the identification of nitroso, nitrated and nitroxidized cardiolipin products by using a C30LC-MS platform method, potentially allowing their detection in biological samples.publishe

Elaboração de elementos finitos para estudos biomecânicos em prostodontia

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas MonizO método dos elementos finitos tornou-se na técnica mais utilizada na análise de fenómenos físicos no campo da estrutura, mecanismos sólidos e fluidos. Na biomecânica, este método sofreu avanços consideráveis quer na investigação quer como instrumento de ensino e pode ser usado para encontrar factos ou processos de estudo que com outro instrumento não seriam possíveis (3). A análise por elementos finitos é um método numérico e tem vantagens consideráveis na fotoelasticidade. É muito útil na análise de geometrias complexas e permite a determinação das tensões e distorções sofridas por um componente tridimensional. Estes elementos finitos são formados pela divisão da estrutura original numa série de secções de forma adequada, onde cada uma delas conserva as mesmas propriedades dos materiais reais (4,5). Muitos destes métodos têm sido aplicados em estudos de medicina dentária restauradora, que consegue preservar e restaurar dentes cariados, restaurações defeituosas, dentes perdidos ou traumatizados. Em geral, o método dos elementos finitos é especialmente indicado para prever tendências e realizar análises paramétricas (2). Obviamente, é muito importante conhecer em detalhe as propriedades dos materiais anisotrópicos e as suas relações constituintes para desenvolver um modelo válido de elementos finitos (1). Portanto, é uma ferramenta útil que permite a realização de um grande número de análises em componentes e estruturas complexas, difíceis de obter pelos métodos analíticos clássicos. Um programa de elementos finitos consiste num software complexo onde convergem inúmeras operações. A preparação dos dados e a análise dos resultados numéricos que aparecem como produto do cálculo são tarefas que tendem a ser integradas no próprio software. Os cálculos de elementos finitos são efetuados num processador no qual estão incluídas todas as ajudas para a preparação dos dados e produção dos resultados, e num pós-processador que facilita a análise e interpretação dos mesmos, geralmente sob a forma de gráficos (traçado de curvas, gráficos tridimensionais, tabelas, etc.) O objetivo desta tese é rever a literatura de forma a compreender como é feito um elemento finito que permita o estudo biomecânico dos diferentes tipos de próteses feitas pelo médico dentista. No âmbito da investigação, serão revistos os diferentes tipos de materiais utilizados atualmente e o seu comportamento com as forças interpretadas na boca, bem como os movimentos de mastigação e os contactos dentários na oclusão. Isto dará ao médico dentista uma ideia geral sobre a seleção de materiais de acordo com o tipo de reabilitação (10).The finite element method has become the most widely used technique for the analysis of physical phenomena in the field of structure, solid and fluid mechanisms. For biomechanics, this method has received considerable advances in research and as a teaching tool, it can be used to find facts or study processes that with another tool are not possible (3). Finite element analysis is a numerical method and has important advantages over photoelasticity. It is very useful in the analysis of complex geometries and allows the determination of the stresses and distortions experienced by a three-dimensional component. These finite elements are formed by dividing the original structure into a series of suitably shaped sections, each of which retains the same properties as real materials (4,5). Many of these methods have been applied in studies of restorative dentistry, which can pre-serve and restore teeth that have decay, defective restorations, lost and traumatized teeth. In general, the finite element method is especially suitable for predicting trends and performing parametric analysis (2). Obviously, it is very important to know in detail the properties of anisotropic materials and their constituent relationships to develop a valid finite element model (1). Therefore, it is a useful tool that allows performing a large number of analyses on complex components and structures, which are difficult to obtain by classical analytical methods. A finite element program is a complex piece of software in which numerous operations converge. The preparation of the data and the analysis of the numerical results that appear as a product of the calculation are tasks that tend to be integrated into the software itself. Finite element calculations consist of a processor which includes all the aids for the preparation of the data and generation of the results, and a post-processor which facilitates the analysis and interpretation of the results, usually in the form of graphs (plotting curves, three dimensional graphs, tables, etc.). As part of the research, the different types of materials currently used and their behavior with the forces interpreted in the mouth, as well as chewing movements and tooth contacts in occlusion will be reviewed. This will give the dentist a general idea about the selection of materials according to the type of rehabilitation

Unravelling the fatty acid profiles of different polychaete species cultured under integrated multi-trophic aquaculture (IMTA)

[Abstract] Polychaetes can be successfully employed to recover otherwise wasted nutrients present in particulate organic matter (POM) of aquaculture effluents. The present study describes the fatty acid (FA) profile of four different polychaete species cultured in sand filters supplied with effluent water from a marine fish farm. The FA profile of cultured and wild Hediste diversicolor was compared and revealed a ≈ 24.2% dissimilarity, with cultured biomass displaying a higher content in two essential n-3 highly unsaturated FA (HUFA) (EPA [20:5 n-3] and DHA [22:6 n-3]—eicosapentaenoic and docosahexaenoic acid, respectively). The comparison of the FA profile of cultured H. diversicolor with that of other polychaete species whose larvae successfully settled on the sand filters (Diopatra neapolitana, Sabella cf. pavonina and Terebella lapidaria) revealed that their FA profile, which is here described for the first time, displayed high levels of EPA and DHA (≈ 1.5–4.8 and 1.0–1.1 µg mg−1 DW, respectively). The highest concentration of total FA per biomass of polychaete was recorded in H. diversicolor and T. lapidaria, with both species being the ones whose FA profiles revealed a lowest level of dissimilarity and more closely resembled that of the aquafeed used in the fish farm. In the present work it was demonstrated that it is possible to produce polychaetes biomass with high nutritional value through an eco-design concept such as integrated multi-trophic aquaculture (IMTA). Indeed, this framework promotes a cleaner production and, in this specific case, allowed to recover essential fatty acids that are commonly wasted in aquaculture effluents.European Commission; MAR-02.01.01-FEAMP-0038European Commission; Centro-01-0145-FEDER-000018Portugal. Fundação para a Ciência e a Tecnologia; PD/BD/127989/2016Portugal. Centro de Estudos do Ambiente e do Mar; UIDB/50017/2020Portugal. Centro de Estudos do Ambiente e do Mar; UIDP/50017/202

Relocation Facilitates the Acquisition of Short Cis-Regulatory Regions that Drive the Expression of Retrogenes during Spermatogenesis in Drosophila

Retrogenes are functional processed copies of genes that originate via the retrotranscription of an mRNA intermediate and often exhibit testis-specific expression. Although this expression pattern appears to be favored by selection, the origin of such expression bias remains unexplained. Here, we study the regulation of two young testis-specific Drosophila retrogenes, Dntf-2r and Pros28.1A, using genetic transformation and the enhanced green fluorescent protein reporter gene in Drosophila melanogaster. We show that two different short (< 24 bp) regions upstream of the transcription start sites (TSSs) act as testis-specific regulatory motifs in these genes. The Dntf-2r regulatory region is similar to the known beta 2 tubulin 14-bp testis motif (beta 2-tubulin gene upstream element 1 [beta 2-UE1]). Comparative sequence analyses reveal that this motif was already present before the Dntf-2r insertion and was likely driving the transcription of a noncoding RNA. We also show that the beta 2-UE1 occurs in the regulatory regions of other testis-specific retrogenes, and is functional in either orientation. In contrast, the Pros28.1A testes regulatory region in D. melanogaster appears to be novel. Only Pros28.1B, an older paralog of the Pros28.1 gene family, seems to carry a similar regulatory sequence. It is unclear how the Pros28.1A regulatory region was acquired in D. melanogaster, but it might have evolved de novo from within a region that may have been preprimed for testes expression. We conclude that relocation is critical for the evolutionary origin of male germline-specific cis-regulatory regions of retrogenes because expression depends on either the site of the retrogene insertion or the sequence changes close to the TSS thereafter. As a consequence we infer that positive selection will play a role in the evolution of these regulatory regions and can often act from the moment of the retrocopy insertion.National Institute of General Medical Sciences of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R01GM071813]; ARRA; UT; NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R01GM071813] Funding Source: NIH RePORTERThe authors thank J. Coyne, P. Gibert, F. Lemeunier, M. Long, M.-L. Wu, C.-I. Wu, and the UC San Diego Drosophila Stock Center for providing wild-type Drosophila strains used in this work and Genetic Services, Inc. and the Bloomington Drosophila Stock Center at Indiana University for providing the mutant stocks used in this study. They also thank H. White-Cooper for providing them with her in situ hybridization protocol before publication and for all her advice on its implementation in their lab. They want to thank four anonymous reviewers for comments on this work. M. G. wants to thank Arndt von Haeseler for his support when finalizing the manuscript. This work was supported by, the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM071813, an ARRA Supplement and UT Arlington startup funds to E. B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels

Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human platelet lysate reinforced with cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications

Influence of heartwood on wood density and pulp properties explained by machine learning techniques

The aim of this work is to develop a tool to predict some pulp properties e.g., pulp yield, Kappa number, ISO brightness (ISO 2470:2008), fiber length and fiber width, using the sapwood and heartwood proportion in the raw-material. For this purpose, Acacia melanoxylon trees were collected from four sites in Portugal. Percentage of sapwood and heartwood, area and the stem eccentricity (in N-S and E-W directions) were measured on transversal stem sections of A. melanoxylon R. Br. The relative position of the samples with respect to the total tree height was also considered as an input variable. Different configurations were tested until the maximum correlation coefficient was achieved. A classical mathematical technique (multiple linear regression) and machine learning methods (classification and regression trees, multi-layer perceptron and support vector machines) were tested. Classification and regression trees (CART) was the most accurate model for the prediction of pulp ISO brightness (R = 0.85). The other parameters could be predicted with fair results (R = 0.64–0.75) by CART. Hence, the proportion of heartwood and sapwood is a relevant parameter for pulping and pulp properties, and should be taken as a quality trait when assessing a pulpwood resourceinfo:eu-repo/semantics/publishedVersio

Attitudes and behaviors towards vaccination in Portuguese nursing students

Knowing the attitudes and behaviors of nursing students in relation to vaccination is important because they will soon be determinant for the health literacy of the population. Vaccination remains the most effective response in the fight against communicable diseases, including COVID-19 and influenza. The objective of this study is to analyze the attitudes and behaviors of Portuguese nursing students with regard to vaccination. A cross-sectional study was carried out, with data collection from nursing students at a university in Lisbon, Portugal. A sample of 216 nursing students was obtained, representing 67.1% of the students enrolled in this university. What stands out from the results of the questionnaire “Attitudes and Behaviors in Relation to Vaccination among Students of Health Sciences” is that for the majority of students the answers were positive; in addition, 84.7% had a completed vaccination schedule for COVID-19. Being a nursing student, being in the final years of the course and being a woman are the factors that most influence the positive attitude of the students. The results obtained are motivating, because these students will be the future health professionals most likely to integrate health promotion programs through vaccination.info:eu-repo/semantics/publishedVersio

Perspectives on the development of CLA/CLNA-enriched milk through in situ microbial production

Over the past decade several food-derived lipids with potential bioactive properties to be used in the development of innovative functional foods have been identified. These include conjugated linoleic (CLA) and conjugated linolenic (CLNA) acids with well described anti- inflammatory, anti-obesity and anticarcinogenic properties. Limited availability in their natural sources (e.g. ruminants’ milk and meat or vegetable oils) has driven studies on in situ microbial production in dairy products to improve CLA/CLNA daily intake. Several probiotic strains have been reported to produce CLA/CLNA isomers using linoleic (LA) and alpha-linolenic (α-LNA) acids as precursor substrates, respectively. This research work aimed to evaluate the viability of developing a CLA/CLNA-enriched milk through in situ microbial production at a laboratory scale. A combination of genetic screening, substrate tolerance and production assays, identified three CLA/CLNA-producing candidates from a pool of 85 probiotic bacteria. Since Bifidobacterium breve DSM 20091 stood out from the others with CLA/CLNA production yields around 0.3 mg/mL, it was selected to proceed with milk CLA/CLNA-enrichment. Seeking to explore industrial viability, edible vegetable oils were applied as substrate sources instead, namely soybean (SBO; rich in LA) and flaxseed (FSO; rich in α-LNA) oils, which had been previously hydrolyzed with Candida rugosa lipase. Microbial CLA/CLNA-enrichment of pasteurized bovine milk was tested with each hydrolyzed oil, individually or in combination, to provide 0.5 mg/mL of LA and/or α-LNA. Viable cell numbers of B. breve achieved 9 log cycles upon 24 h fermentation. Highest CLA/CLNA-enrichment was achieved using FSO alone (~0.4 mg/g), where CLNA isomers were those mainly produced. Surprisingly, only traces of CLA were found, either with SBO alone or in combination. Milk enrichment was further assayed with FSO alone to concentrations up to 10 mg/mL α-LNA, where at 2 mg/mL the maximum yield of ~1 mg/g CLA+CLNA was obtained. In conclusion, the development of a CLA/CLNA-enriched bovine milk through in situ microbial production remains challenging, given the limited number of strains able to produce CLA/CLNA at considerable levels, and because it is difficult to develop a dairy product simultaneously enriched in CLA and CLNA. An in situ strategy has its limitations; nevertheless, this study demonstrates that B. breve DSM 20091 has potential for the development of CLNA-enriched dairy products.info:eu-repo/semantics/publishedVersio