Formation of lipofuscin-like autofluorescent granules in the retinal pigment epithelium requires lysosome dysfunction

Funding Information: Supported by Funda??o para a Ciência e Tecnologia (FCT) ? Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union?s Horizon 2020 programme under grant agreement No. 811087. Funding Information: Supported by Fundação para a Ciência e Tecnologia (FCT) – Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union’s Horizon 2020 programme under grant agreement No. 811087. Publisher Copyright: Copyright 2021 The AuthorsPURPOSE. We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS. We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS. AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS. We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.publishersversionpublishe

Using High-Pressure Technology to Develop Antioxidant-Rich Extracts from Bravo de Esmolfe Apple Residues

Bravo de Esmolfe (BE) is a traditional Portuguese apple highly appreciated by consumers due to its peculiar flavor and aroma. This apple contains higher concentration of phenolic compounds than other cultivars and is thus considered a rich source of antioxidants. Its sensorial and functional properties have attracted farmers' associations to increase BE production. However, a large quantity of apples is wasted due to storage/transportation procedures that impact BE's quality attributes. In this work, we applied high-pressure extraction methodologies to generate antioxidant-rich fractions from BE residues aiming at adding high value to these agro-food by-products. We performed a first extraction step using supercritical CO2, followed by a second extraction step where different CO2 + ethanol mixtures (10-100% v/v) were tested. All experiments were carried out at 25 MPa and 50 °C. Extracts were characterized in terms of global yield, phenolic content and antioxidant activity using chemical (ORAC, HOSC, HORAC) and cell-based assays (CAA). We demonstrated that, although the pressurized 100% ethanol condition promoted the highest recovery of phenolic compounds (509 ± 8 mg GAE/100 g BE residues), the extract obtained with 40% ethanol presented the highest CAA (1.50 ± 0.24 µmol QE/g dw) and ORAC (285 ± 16 µmol TEAC/g dw), as well as HOSC and HORAC values, which correlated with its content of epicatechin and procyanidin B2. Noteworthy, this fraction inhibited free radical production in human neurospheroids derived from NT2 cells, a robust 3D cell model for neuroprotective testing

Single-Step Self-Assembly of Zein–Honey–Chitosan Nanoparticles for Hydrophilic Drug Incorporation by Flash Nanoprecipitation

Zein- and chitosan-based nanoparticles have been described as promising carrier systems for food, biomedical and pharmaceutical applications. However, the manufacture of size-controlled zein and chitosan particles is challenging. In this study, an adapted anti-solvent nanoprecipitation method was developed. The effects of the concentration of zein and chitosan and the pH of the collection solution on the properties of the zein–honey–chitosan nanoparticles were investigated. Flash nanoprecipitation was demonstrated as a rapid, scalable, single-step method to achieve the self-assembly of zein–honey–chitosan nanoparticles. The nanoparticles size was tuned by varying certain formulation parameters, including the total concentration and ratio of the polymers. The zein–honey–chitosan nanoparticles’ hydrodynamic diameter was below 200 nm and the particles were stable for 30 days. Vitamin C was used as a hydrophilic model substance and efficiently encapsulated into these nanoparticles. This study opens a promising pathway for one-step producing zein–honey–chitosan nanoparticles by flash nanoprecipitation for hydrophilic compounds’ encapsulation

Environmentally-safe scCO2 P. pinaster branches extracts: Composition and properties

Considering the vast array of compounds that plants manufacture that help protect them against insects’ attacks, plant raw materials are a potential source of future insecticides/larvicides. In this work, volatiles from maritime pine (Pinus pinaster) branches, a pruning residue, were extracted by supercritical carbon dioxide (scCO2), and also by hydrodistillation for comparison purposes. The volatiles naturally emitted from the pine branches were identified as well. Experimental design results showed higher extraction yields (up to 6%), when scCO2 density and temperature increased, while the inverse occurred for total phenolic contents (1.2–3.3% GAE, gallic acid equivalents). Emitted volatiles from the branches were mainly α-pinene, β-pinene, β-myrcene, limonene, and β-caryophyllene. In general, the volatile profiles of the scCO2 and hydrodistillation extracts were quite similar, revealing abietadiene, the precursor of abietic acid, as the main compound. Oxidation inhibitions of ∼42 – 59% were obtained for the scCO2 extracts in the β-carotene assay. The acetylcholinesterase inhibitory activity assay revealed the potential of these extracts to have insecticidal/larvicidal activity, with IC50 values of 2.8–10.7 mg/mL. Overall, these extracts may be applied in environmentally-safe insect/larvae control strategies, in formulations that benefit from the presence of antioxidants

Recovery and evaluation of cellulose from agroindustrial residues of corn, grape, pomegranate, strawberry-tree fruit and fava

Considering the expected increasing demand for cellulose fibers in the near future and that its major source is wood pulp, alternative sources such as vegetable wastes from agricultural activities and agro-food industries are currently being sought to prevent deforestation. In the present study, cellulose was successfully isolated from six agroindustrial residues: corncob, corn husk, grape stalk, pomegranate peel, marc of strawberry-tree fruit and fava pod. Cellulose fibers were characterized by Fourier-transform infrared spectroscopy, thermogravimetric analysis, stereomicroscopy and scanning electron microscopy (SEM). Despite the evident morphological differences among the extracted celluloses, results revealed similar compositional and thermal properties with the wood-derived commercial microcrystalline cellulose used as a control. Trace amounts of lignin or hemicellulose were detected in all cellulose samples, with the exception of corncob cellulose, that exhibited the greatest extraction yield (26%) and morphological similarities to wood-derived microcrystalline cellulose, visible through SEM. Furthermore, corncob cellulose was found to have thermal properties (TOnset of 307.17 °C, TD of 330.31 °C, and ΔH of 306.04 kJ/kg) suitable for biomedical applications. [Figure not available: see fulltext.

Fractioned High Pressure Extraction of Anthocyanins from Elderberry ( Sambucus nigra L.) Pomace

Fractionated high pressure extractions from dry and in natura elderberry pomace were performed in order to obtain anthocyanin rich extracts. Experiments were carried out using CO2 supercritical fluid extraction followed by enhanced solvent extraction (ESE) with CO2/EtOH–H2O mixtures (1–100%, v/v), to obtain anthocyanin rich fractions in the second step, at 313 K and ~20 MPa. Higher extract yields, anthocyanin contents and antioxidant activities occurred by the presence of water, both in the raw material and in the solvent mixture. The CO2 dissolved in the ESE solvent mixture favored either anthocyanin contents or antioxidant activities, which were not directly related. Comparing to the literature data for elderberries and grapes, these fractions had higher anthocyanins contents. From these results, an added economical value to this agroindustrial residue is proposed, using solvents and techniques “generally regarded as safe” in the food and pharmaceutical industries

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Management practices for postdural puncture headache in obstetrics: a prospective, international, cohort study

© 2020 British Journal of AnaesthesiaBackground: Accidental dural puncture is an uncommon complication of epidural analgesia and can cause postdural puncture headache (PDPH). We aimed to describe management practices and outcomes after PDPH treated by epidural blood patch (EBP) or no EBP. Methods: Following ethics committee approval, patients who developed PDPH after accidental dural puncture were recruited from participating countries and divided into two groups, those receiving EBP or no EBP. Data registered included patient and procedure characteristics, headache symptoms and intensity, management practices, and complications. Follow-up was at 3 months. Results: A total of 1001 patients from 24 countries were included, of which 647 (64.6%) received an EBP and 354 (35.4%) did not receive an EBP (no-EBP). Higher initial headache intensity was associated with greater use of EBP, odds ratio 1.29 (95% confidence interval 1.19–1.41) per pain intensity unit increase. Headache intensity declined sharply at 4 h after EBP and 127 (19.3%) patients received a second EBP. On average, no or mild headache (numeric rating score≤3) was observed 7 days after diagnosis. Intracranial bleeding was diagnosed in three patients (0.46%), and backache, headache, and analgesic use were more common at 3 months in the EBP group. Conclusions: Management practices vary between countries, but EBP was more often used in patients with greater initial headache intensity. EBP reduced headache intensity quickly, but about 20% of patients needed a second EBP. After 7 days, most patients had no or mild headache. Backache, headache, and analgesic use were more common at 3 months in patients receiving an EBP