Browsing by Author "Santos, N. C."
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- Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18Publication . Pinto, M. L.; Rios, E.; Silva, A. C.; Neves, S. C.; Caires, H. R.; Pinto, A. T.; Durães, C.; Carvalho, F. A.; Cardoso, A. P.; Santos, N. C.; Barrias, C. C.; Nascimento, D. S.; Pinto-do-Ó, P.; Barbosa, M. A.; Carneiro, F.; Oliveira, M. J.Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-β and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.
- Differential mesenteric fat deposition in bovines fed on silage or concentrate is independent of glycerol membrane permeabilityPublication . Martins, A. P.; Lopes, P. A.; Costa, A. S. H.; Martins, S. V.; Santos, N. C.; Prates, J. A. M.; Moura, T. F.; Soveral, G.In the meat industry, the manipulation of fat deposition in cattle is of pivotal importance to improve production efficiency, carcass composition and ultimately meat quality. There is an increasing interest in the identification of key factors and molecular mechanisms responsible for the development of specific fat depots. This study aimed at elucidating the influence of breed and diet on adipose tissue membrane permeability and fluidity and their interplay on fat deposition in bovines. Two Portuguese autochthonous breeds, Alentejana and Barrosã, recognized as late- and early-maturing breeds, respectively, were chosen to examine the effects of breed and diet on fat deposition and on adipose membrane composition and permeability. Twenty-four male bovines from these breeds were fed on silage-based or concentrate-based diets for 11 months. Animals were slaughtered to determine their live slaughter and hot carcass weights, as well as weights of subcutaneous and visceral adipose depots. Mesenteric fat depots were excised and used to isolate adipocyte membrane vesicles where cholesterol content, fatty acid profile as well as permeability and fluidity were determined. Total accumulation of neither subcutaneous nor visceral fat was influenced by breed. In contrast, mesenteric and omental fat depots weights were higher in concentrate-fed bulls relative to silage-fed animals. Membrane fluidity and permeability to water and glycerol in mesenteric adipose tissue were found to be independent of breed and diet. Moreover, the deposition of cholesterol and unsaturated fatty acids, which may influence membrane properties, were unchanged among experimental groups. Adipose membrane lipids from the mesenteric fat depot of ruminants were rich in saturated fatty acids, and unaffected by polyunsaturated fatty acids dietary levels. Our results provide evidence against the involvement of cellular membrane permeability to glycerol on fat accumulation in mesenteric fat tissue of concentrate-fed bovines, which is consistent with the unchanged membrane lipid profile found among experimental groups.
- Fluorescent probes DPH, TMA-DPH and C17-HC induce erythrocyte exovesiculationPublication . Saldanha, C.; Santos, N. C.; Martins-Silva, J.An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.
- Higher membrane fluidity mediates the increased subcutaneous fatty acid content in pigs fed reduced protein dietsPublication . Lopes, P. A.; Martins, A. P.; Martins, S. V.; Madeira, M. S.; Santos, N. C.; Moura, T. F.; Prates, J. A. M.; Soveral, G.The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing-finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain - a lean genotype) and purebred (Alentejana breed - a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig's subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane's fatty acid composition. Adipose membrane's cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.
- In vivo efficacy of measles virus fusion protein-derived peptides is modulated by the properties of self-assembly and membrane residencePublication . Figueira, T. N.; Palermo, L. M.; Veiga, A. S.; Huey, D.; Alabi, C. A.; Santos, N. C.; Welsch, J. C.; Mathieu, C.; Horvat, B.; Niewiesk, S.; Moscona, A.; Castanho, Miguel A. R. B.; Porottob, M.Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The selfassembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides.
- Lipid selectivity in novel antimicrobial peptides : implication on antimicrobial and hemolytic activityPublication . Maturana, P.; Martinez, M.; Noguera, M. E.; Santos, N. C.; Disalvo, E. A.; Semorile, L.; Maffia, P. C.; Hollmann, A.Antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against awide range of bacteria, fungi and viruses. For an AMP to be considered as a therapeutic option, it musthave not only potent antibacterial properties but also low hemolytic and cytotoxic activities [1]. Eventhough many studies have been conducted in order to correlate the antimicrobial activity with affinitytoward model lipid membranes, the use of these membranes to explain cytotoxic effects (especiallyhemolysis) has been less explored. In this context, we studied lipid selectivity in two related novel AMPs,peptide 6 (P6) and peptide 6.2 (P6.2). Each peptide was designed from a previously reported AMP, andspecific amino acid replacements were performed in an attempt to shift their hydrophobic momentor net charge. P6 showed no antimicrobial activity and high hemolytic activity, and P6.2 exhibited goodantibacterial and low hemolytic activity. Using both peptides as a model we correlated the affinity towardmembranes of different lipid composition and the antimicrobial and hemolytic activities. Our results fromsurface pressure and zeta potential assays showed that P6.2 exhibited a higher affinity and faster bindingkinetic toward PG-containing membranes, while P6 showed this behavior for pure PC membranes. Thefinal position and structure of P6.2 into the membrane showed an alpha-helix conversion, resulting in aparallel alignment with the Trps inserted into the membrane. On the other hand, the inability of P6 toadopt an amphipathic structure, plus its lower affinity toward PG-containing membranes seem to explainits poor antimicrobial activity. Regarding erythrocyte interactions, P6 showed the highest affinity towarderythrocyte membranes, resulting in an increased hemolytic activity. Overall, our data led us to concludethat affinity toward negatively charged lipids instead of zwitterionic ones seems to be a key factor thatdrives from hemolytic to antimicrobial activity.
- Study of surface damage on cell envelope assessed by AFM and flow cytometry of Lactobacillus plantarum exposed to ethanol and dehydrationPublication . Bravo-Ferrada, B. M.; Gonçalves, S.; Semorile, L.; Santos, N. C.; Tymczyszyn, E. E.; Hollmann, A.Aims: In this work, we evaluated freeze-drying damage at the surface level of oenological strain Lactobacillus plantarum UNQLp155, as well as its ability to grow in a synthetic wine with and without pre-acclimation. Methods and Results: Damage on cell surface was studied by flow cytometry, zeta potential and atomic force microscopy, and cell survival was analysed by plate count. Results showed that beside cells acclimated at lower ethanol concentration (6% v/v) became more susceptible to drying than nonacclimated ones, after rehydration they maintain their increased ability to grow in a synthetic wine. Acclimation at a higher ethanol concentration (10% v/v) produces several damages on the cell surface losing its ability to grow in a synthetic wine. Conclusions: In this work, we showed for the first time that sublethal alterations on bacterial surface induced by a pre-acclimation with a low ethanol concentration (6%), upon a freeze-drying process, result in a better bacterial adaptation to the stress conditions of wine-like medium, as well as to the preservation process. Significance and Impact of the Study: Understanding the adaptation to ethanol of oenological strains and their effects on the preservation process has a strong impact on winemaking process and allows to define the most appropriate conditions to obtain malolactic starters cultures.