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Background Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles. Methods Based on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles. Results We hereby present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals’ exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer’s disease. Our findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology. Conclusions Overall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology.

Blueberries (Vaccinium corymbosum L.) are cultivated worldwide and represent an important asset for the Portuguese economy. Pathogen infection and water deficiency are known to affect crops productivity worldwide, thus limiting plant yield or fruit quality. Diaporthe is a cosmopolitan genus comprising plant pathogens and endophytes, that may switch their behavior to a pathogenic phase when the host is under environmental stress (e.g., drought, precipitation). Given the scarce physiological studies on blueberry plant- pathogen interactions, in this study we aimed to evaluate the effect of Diaporthe amygdali and D. eres on 9-month-old clonal blueberries under two different water scenarios: well-watered (WW) and water deficit (WD). Morphological (lesion length) and physiological parameters (water status, leaf gas exchange, photosynthetic pigments, proline, phenolic compounds, flavonoids, starch, total soluble sugars, and lipid peroxidation) were assessed. Our results suggest that the irrigation regime applied was not sufficient to cause severe stress to plants. Under WW conditions, plants inoculated with D. eres may have used malondialdehyde content (MDA) as a signaling molecule. Although D. amygdali has caused plant mortality, this study shows that under WW conditions, plants manage to deal with pathogen attack, maintaining their physiological performance. This study also demonstrates that the interaction between fungal pathogens and water limitation seems to stimulate plant defense, through the accumulation of proline. Our findings offer crucial insights to understand how blueberry plants cope with infection by species of Diaporthe, and how plants can adapt to climate changes in the Mediterranean area (e.g., water scarcity).

Agave bagasse is a waste material generated during agave sap collection for “pulque” production. This residue is a potential feedstock for steroidal saponins and other phytochemicals. Ultrasonically assisted extraction (UAE) allows the use of green solvents such as water or ethanol for phytochemicals recovery. The influence of solvent composition and its effect on the intensity of the ultrasonic field during the UAE of agave bagasse saponins were evaluated. The best extraction conditions were a temperature of 60 °C (T) and a solvent-to-mass ratio of 20 (S/M), for conventional and UAE. In contrast to conventional extraction, where mixtures of ethanol-water were more efficient, water was the best extraction solvent in UAE. The amount of saponins obtained without ultrasound in 58% ethanol (22.48 ± 1.34 mg PE/g dw) was similar to the amount obtained by UAE in water (24.41 ± 0.84 mg PE/g dw). When only water was used, the ultrasound intensity was higher (271.40 ± 11.91 W/L) than in ethanol mixtures (144.81–202.30 W/L) due to cavitation intensity, demonstrated in a test on aluminum foil and SEM images of exhausted bagasse. Despite lower chemical affinity of water for saponins, its lower vapor pressure and viscosity, as well as higher surface tension, compared with ethanol, provided a greater cavitation intensity. Therefore, UAE using water is an excellent alternative to recover bioactive compounds from agave bagasse.

Silvetia compressa is a brown seaweed native to the coast of Baja California, Mexico. It is a rich source of phlorotannins and polysaccharides, two compound families with important nutraceutical applications. Optimal conditions for obtaining highly concentrated phlorotannin and polysaccharide extracts from S. compressa were determined using Box-Behnken experimental design combined with response surface methodology (RSM). The effect of extraction temperature (X1: 50–65 °C), ultrasound power density (X2: 1.2–3.8 W cL−1), solvent/seaweed ratio (X3: 10–30 mL g−1 seaweed meal), and ethanol concentration (X4: 25–100% ethanol in water) on phlorotannin and polysaccharide yield was explored. Experimental results were fitted to a second-degree polynomial model, while model fitness was assessed using analysis of variance (ANOVA). From this analysis, optimal phlorotannin and polysaccharide extraction conditions were determined for the evaluated parameters (X1 = 50 °C, X2 = 3.8 W cL−1, X3 = 30 mL g−1 seaweed meal, and X4 = 32.33%). Under these conditions, experimental phlorotannin and polysaccharide yields were 0.73% and 23% (w/w), respectively. Phlorotannin extraction was significantly enhanced by ultrasound power density, while polysaccharide extraction improved when using low ethanol concentration in the solvent, therefore both families of compounds were obtained combining those parameters. Experimental data agreed with predictions from the RSM model, indicating suitability of the obtained model and the success of RSM in optimizing the extraction conditions. In addition, the identification of the main compounds in the phlorotannin extract was carried out by HPLC-MS-TOF; 8 of the 12 identified phenolic compounds belonged to the phlorotannin family, 4 from the fuhalol group, 3 being eckol derivatives, and 1 being a phloroglucinol subunit.

Blueberry (Vaccinium corymbosum) cultivation in Portugal has been growing considerably in the last decade, making it a highly profitable fruit crop. Species of Botryosphaeriaceae are relevant pathogens of blueberry causing stem blight and dieback. In this study a survey was carried out in blueberry orchards in the centre region of Portugal in order to assess which species of Botryosphaeriaceae are associated with stem blight and dieback symptoms. Their presence as endophytes was also evaluated in asymptomatic plants. Four species were identified, Botryosphaeria dothidea, Neofusicoccum parvum, N. australe and N. eucalyptorum. Of these, B. dothidea and N. parvum were the most commonly found. Pathogenicity trials showed that the four species tested were pathogenic to blueberry plants and revealed that N. parvum, was the most aggressive species, and most likely the main causal agent of blueberry stem blight and dieback in Portugal. Neofusicoccum eucalyptorum, a common Eucalyptus pathogen, is reported for the first time as a pathogen of blueberry. This represents a significant host jump whose potential future effects on blueberry plantations are not fully understood.

Separation of potentially bioactive components from foods and plant extracts is one of the main challenges for their study. Centrifugal partition chromatography has been a successful technique for the screening and identification of molecules with bioactive potential, such as steroidal saponins. Agave is a source of steroidal saponins with anticancer potential, though the activity of these compounds in concentrated agave sap has not been yet explored. In this study, fast centrifugal partition chromatography (FCPC) was used coupled with in vitro tests on HT-29 cells as a screening procedure to identify apoptotic saponins from an acetonic extract of concentrated agave sap. The three most bioactive fractions obtained by FCPC at partition coefficients between 0.23 and 0.4 contained steroidal saponins, predominantly magueyoside b. Flow cytometry analysis determined that the fraction rich in kammogenin and manogenin glycosides induced apoptosis, but when gentrogenin and hecogenin glycosides were also found in the fraction, a necrotic effect was observed. In conclusion, this study provides the evidence that steroidal saponins in concentrated agave sap were potential inductors of apoptosis and that it was possible to separate them using fast centrifugal partition chromatography.

Cardiovascular disease represents one of the most important public health problem in the Western countries, with sudden cardiac death (SCD) being the most common cause of death in adults under 65 years of age. The objective of the present study is to evaluate the leading causes of SCD in young adults who died suddenly in Northern Portugal between 2007 and 2012. This study included 288 cases of 20- to 45-year-old adults who died suddenly of a cardiac cause and whose forensic autopsy was performed in the North Branch of the National Institute of Legal Medicine and Forensic Sciences between 2007 and 2012. Data included the cause of death, forensic autopsy findings, previous medical history and cardiovascular risk factors. The mean age of the population was 37.36 years. Coronary artery disease (CAD) was the leading cause of death, representing 55.6 % of all cases (92.5 % of men and 7.5 % of women). Females died mostly from probable primary arrhythmia. From those who died of coronary disease, 72 (25 % of total cases) had histological evidence of a fatal acute infarction. Age, previous coronary disease, hypercholesterolemia, smoking habits and alcohol consumption seem to be associated with an increased risk of SCD by CAD. A total of 55.6 % of deaths were attributed to CAD. Prevention of cardiovascular risk factors should therefore be considered to prevent atherosclerosis in young adults.

To investigate the tensile biomechanical properties of round and uterosacral ligaments.Tissue samples were obtained from 15 female cadavers without pelvic organ prolapse. Uniaxial tensile tests were performed to obtain stiffness and maximum stress of round and uterosacral ligaments. Correlations were calculated using the Pearson correlation coefficient. Statistical differences between groups were tested using Student's paired and unpaired t test.There was a great variability in the measurements of stiffness and maximum stress in pelvic ligaments. The round ligaments demonstrated stiffness of 9.1 ± 1.6 MPa (mean ± SEM) (ranging from 2 to 25.6 MPa) and maximum stress of 4.3 ± 0.7 MPa (ranging from 1.2 to 11.5 MPa). The stiffness of the uterosacral ligaments was 14.1 ± 1.4 MPa (ranging from 5.7 to 26.1 MPa) with maximum stress of 6.3 ± 0.8 MPa (ranging from 2.2 to 11.9 MPa). There was a strong positive correlation between stiffness and maximum stress in female pelvic ligaments (ρ = 0.851; p0.001). The uterosacral ligaments demonstrated higher stiffness and maximum stress compared to the round ligaments (p = 0.006 and p = 0.034; respectively). Age, body mass index and menopausal status were not associated with the biomechanical proprieties of round and uterosacral ligaments. Nulliparous women had lower uterosacral stiffness (15.5 ± 1.3 vs. 10 ± 1.8 MPa; p = 0.033) and maximum stress (8.2 ± 0.9 vs. 4.2 ± 1.1 MPa; p = 0.028) compared to parous women.The uterosacral ligaments are significantly more resistant than round ligaments. Parturition seems to enhance the stiffness and maximum stress of the ligaments.

The objective of the present study was to investigate the tensile biomechanical properties of the human female bladder. Tissue samples were obtained from 13 cadavers without pelvic floor dysfunctions. We performed uniaxial tensile tests to measure the stiffness and maximum stress of the bladder tissue. Correlations were calculated using the Pearson correlation coefficient. The bladder tissue stiffness ranged from 1 to 4.1 MPa (mean stiffness, 1.9 ± 0.2 MPa) and the maximum stress ranged from 0.5 to 2.6 MPa (mean maximum stress, 0.9 ± 0.1 MPa). There was a strong positive correlation between stiffness and maximum stress in the bladder tissue (ρ = 0.829, p