Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cell homeostasis.

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, which is characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMC homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam-cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMC proliferation and migration, impacting atherosclerosis. In summary, this work shows that ChA is sufficient to induce lysosomal dysfunction in VSMCs, that, in ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered, and, finally, that recombinant LAL can be a therapeutic approach for lysosomal dysfunction.

2,3-Diphosphoglycerate and the Protective Effect of Pyruvate Kinase Deficiency against Malaria Infection-Exploring the Role of the Red Blood Cell Membrane.

Malaria remains a major world public health problem, contributing to poverty and inequality. It is urgent to find new efficacious tools with few adverse effects. Malaria has selected red blood cell (RBC) alterations linked to resistance against infection, and understanding the protective mechanisms involved may be useful for developing host-directed tools to control Plasmodium infection. Pyruvate kinase deficiency has been associated with resistance to malaria. Pyruvate kinase-deficient RBCs display an increased concentration of 2,3-diphosphoglycerate (2,3-DPG). We recently showed that 2,3-DPG impacts in vitro intraerythrocytic parasite growth, induces a shift of the metabolic profile of infected cells (iRBCs), making it closer to that of noninfected ones (niRBCs), and decreases the number of parasite progenies that invade new RBCs. As an increase of 2,3-DPG content may also have an adverse effect on RBC membrane and, consequently, on the parasite invasion, in this study, we explored modifications of the RBC morphology, biomechanical properties, and RBC membrane on Plasmodium falciparum in vitro cultures treated with 2,3-DPG, using atomic force microscopy (AFM)-based force spectroscopy and other experimental approaches. The presence of infection by P. falciparum significantly increased the rigidity of parasitized cells and influenced the morphology of RBCs, as parasitized cells showed a decrease of the area-to-volume ratio. The extracellular addition of 2,3-DPG also slightly affected the stiffness of niRBCs, making it more similar to that of infected cells. It also changed the niRBC height, making the cells appear more elongated. Moreover, 2,3-DPG treatment influenced the cell surface charge, becoming more negative in treated RBCs than in untreated ones. The results indicate that treatment with 2,3-DPG has only a mild effect on RBCs in comparison with the effect of the presence of the parasite on the host cell. 2,3-DPG is an endogenous host metabolite, which may, in the future, originate a new antimalarial tool with few adverse effects on noninfected cells.

Erythrocytes' surface properties and stiffness predict survival and functional decline in ALS patients.

Erythrocytes play a fundamental role in oxygen delivery to tissues and binding to inflammatory mediators. Evidences suggest that dysregulated erythrocyte function could contribute to the pathophysiology of several neurodegenerative diseases. We aimed to evaluate changes in morphological, biomechanical, and biophysical properties of erythrocytes from amyotrophic lateral sclerosis (ALS) patients, as new areas of study in this disease. Blood samples were collected from ALS patients, comparing with healthy volunteers. Erythrocytes were assessed using atomic force microscopy (AFM) and zeta potential analysis. The patients' motor and respiratory functions were evaluated using the revised ALS Functional Rating Scale (ALSFRS-R) and percentage of forced vital capacity (%FVC). Patient survival was also assessed. Erythrocyte surface roughness was significantly smoother in ALS patients, and this parameter was a predictor of faster decline in ALSFRS-R scores. ALS patients exhibited higher erythrocyte stiffness, as indicated by reduced AFM tip penetration depth, which predicted a faster ALSFRS-R score and respiratory subscore decay. A lower negative charge on the erythrocyte membrane was predictor of a faster ALSFRS-R and FVC decline. Additionally, a larger erythrocyte surface area was an independent predictor of lower survival. These changes in morphological and biophysical membrane properties of ALS patients' erythrocytes, lead to increased cell stiffness and morphological variations. We speculate that these changes might precipitate motoneurons dysfunction and accelerate disease progression. Further studies should explore the molecular alterations related to these observations. Our findings may contribute to dissect the complex interplay between respiratory function, tissue hypoxia, progression rate, and survival in ALS.

A mathematical model of fibrinogen-mediated erythrocyte-erythrocyte adhesion.

Erythrocytes are deformable cells that undergo progressive biophysical and biochemical changes affecting the normal blood flow. Fibrinogen, one of the most abundant plasma proteins, is a primary determinant for changes in haemorheological properties, and a major independent risk factor for cardiovascular diseases. In this study, the adhesion between human erythrocytes is measured by atomic force microscopy (AFM) and its effect observed by micropipette aspiration technique, in the absence and presence of fibrinogen. These experimental data are then used in the development of a mathematical model to examine the biomedical relevant interaction between two erythrocytes. Our designed mathematical model is able to explore the erythrocyte-erythrocyte adhesion forces and changes in erythrocyte morphology. AFM erythrocyte-erythrocyte adhesion data show that the work and detachment force necessary to overcome the adhesion between two erythrocytes increase in the presence of fibrinogen. The changes in erythrocyte morphology, the strong cell-cell adhesion and the slow separation of the two cells are successfully followed in the mathematical simulation. Erythrocyte-erythrocyte adhesion forces and energies are quantified and matched with experimental data. The changes observed on erythrocyte-erythrocyte interactions may give important insights about the pathophysiological relevance of fibrinogen and erythrocyte aggregation in hindering microcirculatory blood flow.

Fatty acids composition in yellow-legged (Larus michahellis) and lesser black-backed (Larus fuscus) gulls from natural and urban habitats in relation to the ingestion of anthropogenic materials.

Urban habitats offer spatially and temporally predictable anthropogenic food sources for opportunistic species, such as several species of gulls that are known to exploit urban areas and take advantage of accessible and diverse food sources, reducing foraging time and energy expenditure. However, human-derived food may have a poorer nutritional quality than the typical natural food resources and foraging in urban habitats may increase birds' susceptibility of ingesting anthropogenic debris materials, with unknown physiological consequences for urban dwellers. Here we compare the fatty acids (FA) composition of two opportunistic gull species (the yellow-legged gull, Larus michahellis, and the lesser black-backed gull, Larus fuscus) from areas with different levels of urbanization, to assess differences in birds' diet quality among foraging habitats, and we investigate the effects of ingesting anthropogenic materials, a toxicological stressor, on gulls' FA composition. Using GC-MS, 23 FAs were identified in the adipose tissue of both gull species. Significant differences in gulls' FA composition were detected among the three urbanization levels, mainly due to physiologically important highly unsaturated FAs that had lower percentages in gulls from the most urbanized habitats, consistent with a diet based on anthropogenic food resources. The deficiency in omega (ω)-3 FAs and the higher ω-6:ω-3 FAs ratio in gulls from the most urbanized location may indicate a diet-induced susceptibility to inflammation. No significant differences in overall FA composition were detected between gull species. While we were unable to detect any effect of ingested anthropogenic materials on gulls' FA composition, these data constitute a valuable contribution to the limited FA literature in gulls. We encourage studies to explore the long-term physiological effects of the lower nutritional quality diet for urban dwellers, and to detect the sub-lethal impacts of the ingestion of anthropogenic materials.

Corrigendum: γ' Fibrinogen as a Predictor of Survival in Amyotrophic Lateral Sclerosis.

[This corrects the article DOI: 10.3389/fcvm.2021.715842.].

Ingestion of anthropogenic materials by yellow-legged gulls (Larus michahellis) in natural, urban, and landfill sites along Portugal in relation to diet composition.

Pollution is a global concern, increasing rapidly throughout marine and terrestrial ecosystems, and affecting many species. Urbanization enhances waste production, leading to the opening of landfills that constitute a spatially and temporally predictable food source for opportunistic species. Several species of gulls are known to exploit and breed in urban areas, taking advantage of accessible and diverse food resources. The exploitation of anthropogenic food subsidies at sea (e.g. fishery discards), urban sites, and landfills leads to debris ingestion by gulls with potential negative effects. Here we characterize anthropogenic debris ingested by yellow-legged gulls (Larus michahellis) along Portugal, by analysing the content of pellets collected in (1) natural and urban breeding locations, and in (2) urban and landfill resting sites, to assess seasonal patterns in the ingestion of anthropogenic debris. We also relate diet with the presence of anthropogenic debris. Debris materials were found in 28.8% of pellets from breeding locations (natural and urban) and in 89.7% of pellets from resting sites (urban and landfill). Gulls from the most urbanized breeding location exhibited higher levels of ingested materials during the entire breeding cycle, however, gulls from a natural breeding site also ingested high levels of debris during the pre-breeding season. At resting sites, small seasonal differences were detected in the number and mass of debris items ingested, which were both higher during spring and summer. Gulls that typically fed on pelagic fish had significantly less sheet and fragment plastics in their pellets. The presence of certain debris categories in gull pellets was positively related to the presence of some prey items, suggesting that gulls may accidentally ingest debris while foraging at multiple habitats. The quantity of anthropogenic materials ingested by gulls from urban locations and landfills indicates a need for improved waste management.

γ' Fibrinogen as a Predictor of Survival in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disorder related to neuroinflammation that is associated with increased risk of thrombosis. We aimed to evaluate γ' fibrinogen plasma level (an in vivo variant of fibrinogen) as a biomarker in ALS, and to test its role as a predictor of disease progression and survival. Sixty-seven consecutive patients with ALS were followed and the results were compared with those from 82 healthy blood donors. Patients were clinically evaluated at the time of blood sampling and on follow-up (every 3 months for the beginning of the follow-up until death) by applying the revised ALS Functional Rating Scale. Human plasma γ' fibrinogen concentration was quantified using a specific two-site sandwich kit enzyme-linked immunosorbent assay. We found, for the first time, a positive association between γ' fibrinogen concentration and survival in ALS patients: patients with higher γ' fibrinogen plasma levels survived longer, and this finding was not influenced by confounders such as age, gender, respiratory impairment, or functionality (ALSFRS-R score). Since increased levels have a positive impact on outcome, this novel biomarker should be further investigated in ALS.

Characterization of anthropogenic materials on yellow-legged gull (Larus michahellis) nests breeding in natural and urban sites along the coast of Portugal.

Anthropogenic materials are a persistent pressure on ecosystems, affecting many species. Seabirds can collect these materials to construct their nests, which may modify nest characteristics and cause entanglement of chicks and adults, with possible consequences on breeding success. The incorporation of anthropogenic materials in nests of seabird species that breed in both natural and urban environments, such as gulls, is poorly known. Here, we characterize and compare anthropogenic materials incorporated in yellow-legged gull (Larus michahellis) nests from two natural and two urban breeding sites across their Portuguese breeding range and during 2 consecutive years. Anthropogenic materials were found in 2.6% and 15.4% of gull nests from natural locations and in 47.6% and 95.7% of nests from urban breeding sites. No differences were found on hatching success between urban and natural breeding colonies. A significantly higher number of anthropogenic materials were found in the largest and more populated urban breeding colony, which on average included items of a greater mass but smaller size than items from the other three colonies. The higher incorporation of anthropogenic materials in urban locations could be a consequence of a lower access to natural nest construction materials and higher availability of anthropogenic debris. The quantity and diversity of anthropogenic materials incorporated in gull nests from urban locations indicate a need for improved debris management in urban settlements.

Long-term results after concomitant mitral and aortic valve surgery: repair or replacement?

OBJECTIVES: The reported superiority of mitral valve (MV) repair for isolated MV regurgitation has not been confirmed in mitroaortic valve surgery. Our goals were to evaluate the feasibility of repair in patients undergoing mitral and aortic valve surgery and to identify factors predisposing to MV replacement, to compare long-term outcomes (survival and MV reoperation) of repair and replacement and to perform a subgroup analysis in patients with rheumatic MV disease. METHODS: From January 1992 through December 2016, 1122 consecutive patients were submitted to concomitant aortic and MV surgery in 2 different centres (Coimbra and Santiago). Of these, 837 patients underwent MV repair (74.6%) and 285 patients had MV replacement (25.4%). Rheumatic aetiology was predominant (666 patients; 59.4%). Cumulative follow-up was 9522.6 patient-years (25th-75th percentile 2.6-13.2 years) and was complete for 95.6% of patients. Propensity score matching (1:1) was performed in 232 patients for comparing each treatment option (MV repair and MV replacement). RESULTS: Previous MV intervention, rheumatic aetiology, chronic obstructive pulmonary disease, higher degrees of tricuspid and mitral regurgitation and pulmonary hypertension were independently correlated with MV replacement. The 30-day mortality rate was higher in patients with MV replacement (4.2% vs 1.8%, P = 0.021) and was confirmed in the propensity score matching (4.7% vs 1.7%, P = 0.06). Late survival was lower in the MV replacement group (53.3 ± 4.5% vs 61.7 ± 2.0% at 12 years; P = 0.026) and was confirmed in the propensity score matching (54.6 ± 4.9% vs 63.2 ± 3.8%, P = 0.062) and rheumatic subgroup (57.9 ± 4.8% vs 68.0 ± 2.5%, P = 0.018). Freedom from MV reoperation at 12 years was higher in the MV repair group (94.7 ± 1.1% vs 89.0 ± 3.1%, P = 0.004) but similar in patients with rheumatic MV disease. CONCLUSIONS: MV repair can be performed in most patients undergoing aortic valve replacement. It should be the procedure of choice whenever feasible, because it is associated with lower early and late mortality rates and with freedom from reoperation in non-rheumatic patients.