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Vitrified shoots of strawberry tree propagated in vitro showed poor lignification when analysed by FTIR-ATR and by the acetyl bromide method, as well as marked disturbances on leaf tissues organization. Arbutus unedo (Ericaceae), commonly known as strawberry tree, is a bush or small tree grown mainly for the production of edible fruits that can be eaten fresh, processed or used to make liquors. In vitro propagation of A. unedo in liquid media is an advantageous procedure that reduces the time for multiplication. However, tissue culture in liquid media often leads to excessive accumulation of water in the apoplast that results in a physiological mechanism characterized by low lignification, impaired stomatal function and reduced mechanical strength of tissue culture-generated plants. Fourier-transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) was employed to assess the chemical-derived differences between vitrified and non-vitrified cell walls. FTIR-based spectral assignments suggested higher contents of aromatic compounds in leaves than in stems. Lignin measurements by the acetyl bromide procedure showed that there are no significant differences in the percentage of lignin by dry weight between the different types of leaves. Anatomical studies using light microscopy and scanning electron microscopy showed changes on epidermis organization of vitrified leaves as well as the presence of abnormal stomata in the abaxial surface. These observations may explain the difficulty of vitrified plants to balance absorption and evapotranspiration, and cope with hydric stress during the acclimatization phase. Moreover, cross-sectioned leaves revealed broad lacunar spaces and cross-sectioned stems had spaced-hypertrophic cortical parenchyma cells, where the water vapor may accumulate. Finally, the characterization of vitrified tissues was considered an important contribution to uncover the mechanisms that cause plant vitrification, not just by improving the methods developed for the micropropagation of this species, but also contributing for its agroeconomic valorization.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are key events in the initiation and/or progression of several diseases, are correlated with alterations at ER-mitochondria contact sites, the so-called "Mitochondria-Associated Membranes" (MAMs). These intracellular structures are also implicated in NLRP3 inflammasome activation which is an important driver of sterile inflammation, however, the underlying molecular basis remains unclear. This work aimed to investigate the role of ER-mitochondria communication during ER stress-induced NLRP3 inflammasome activation in both peripheral and central innate immune systems, by using THP-1 human monocytes and BV2 microglia cells, respectively, as in vitro models. Markers of ER stress, mitochondrial dynamics and mass, as well as NLRP3 inflammasome activation were evaluated by Western Blot, IL-1β secretion was measured by ELISA, and ER-mitochondria contacts were quantified by transmission electron microscopy. Mitochondrial Ca

Exosomes are extracellular vesicles of endosomal origin released by virtually all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs and proteins between different cells, tissues or organs. However, the mechanisms that regulate the selective loading of cytosolic proteins into these vesicles are still largely unknow. Here we describe a mechanism whereby proteins containing a pentapeptide sequence, biochemically related to the KFERQ-motif, are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, this mechanism is independent of the ESCRT machinery components TSG101 and VPS4b and dependent on HSC70, CD63, Alix, Syntenin-1, Rab31 and ceramides. The transcription factor and master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. Additionally, by tagging fluorescent proteins with KFERQ-like sequences we were able to follow inter-organ transfer of exosomes in zebrafish larvae. Our findings identify LAMP2A as a key component in exosome biogenesis while opening new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

Background: Cardiovascular diseases are the leading cause of global mortality with a tendency to increase due to population ageing as well as an increase in associated risk factors. Although current therapies improve survival rates, they are associated with several side effects, thus justifying the development of novel preventive and/or therapeutic approaches. In this way, plant metabolites such as essential oils have emerged as promising agents due to their biological effects. Objective: Bearing in mind that several essential oils are characterized by high amounts of phenylpropanoids, which may play a crucial role in the activity of these volatile extracts, a comprehensive and systematic review focusing on the cardiovascular effects of phenylpropanoid-rich essential oils is presented. Methods: Popular search engines including PubMed, Science Direct, Scopus and Google Scholar were consulted and papers from 2000 onwards were selected. Non-volatile phenylpropanoids were not considered in this review. Results: A compilation of the current knowledge on this thematic pointed out beneficial effects for volatile phenylpropanoids namely hypotensive, vasorelaxant, antiplatelet aggregation, antidyslipidaemic and antidiabetic, as well as protective properties against ischemia/reperfusion injury and heart hypertrophy. Conclusion: A better understanding of the protective effects of phenylpropanoids on the cardiovascular system is presented, thus paving the way towards future research on plant-based therapies for cardiovascular diseases.

Dermatophytosis is an important health concern with an increasing negative impact due to unsuccessful therapeutic approaches. Natural products have emerged as promising alternative/complementary agents, due to long traditional uses and increasing scientific recognition. Current knowledge on the antidermatophytic activity of natural products, mainly plant extracts with an up-to-date status on their activity, mechanism of action, developed pharmaceutical formulations, and effectiveness in animal models of infection and patients with dermatophytosis is presented. Although the information available on this matter points out an undeniable potential of these compounds, their usage by the pharmaceutical industry and medical community remains a challenge.

Thymus albicans is an endemic species of the Iberian Peninsula with a vulnerable conservation status. In an attempt to contribute to the valorization of this species, the present study brings new insights on the antifungal and anti-inflammatory mechanism of action of T. albicans essential oil. The antifungal activity of the oil and its major compounds was assessed for the first time against standard and clinically isolated strains of yeasts and filamentous fungi. The effect on the two major virulence factors of Candida albicans (germ tube formation and biofilm disruption) was considered in more detail. At 0.08 μL/mL, the oil inhibited C. albicans germ tube formation by more than 40% and decreased biofilm biomass at MIC values, thus pointing out its antivirulent potential. The anti-inflammatory activity of the essential oil was investigated on LPS-stimulated mouse macrophages (RAW 264.7) by evaluating the levels of several pro-inflammatory mediators, namely nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). T. albicans oil reduced the production of nitrites, a NO derived sub-product, at non-cytotoxic concentrations of 0.32 and 0.64 μL/mL, by 27 and 41%, respectively. In addition, the iNOS protein levels of essential oil pre-treated cells were reduced by 14%. Overall, the high essential oil yield of T. albicans as well as its bioactive effects at concentrations without cytotoxicity, encourage further studies on the potential pharmacological applications of this species. Furthermore, these results raise awareness for the need to preserve endangered species that may hold relevant medicinal value.

PurposeThis study aimed to endow the cell-penetrating peptide (CPP) S4(13)-PV with adequate features towards a safe and effective application in cancer gene therapy.MethodsPeptide/siRNA complexes were prepared with two new derivatives of the CPP S4(13)-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S4(13)-PV peptide, being named C12-H-5-S4(13)-PV and H-5-S4(13)-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum.ResultsPeptide/siRNA complexes prepared with the C12-H-5-S4(13)-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H-5-S4(13)-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes.ConclusionsThese encouraging results pave the way for a potential application of the C12-H-5-S4(13)-PV peptide as a promising tool in cancer gene therapy. European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme [CENTRO-010145-FEDER-000008] European Regional Development Fund (ERDF), through the COMPETE 2020 -Operational Programme for Competitiveness and Internationalisation Portuguese national funds via FCT - Fundacao para a Ciencia e a Tecnologia [POCI-01-0145-FEDER-016390: CANCEL STEM, POCI-01-0145-FEDER-007440, UIDB/04539/2020] Fundacao para a Ciencia e Tecnologia (FCT, Portugal)Portuguese Foundation for Science and Technology [UID/QUI/50006/2013, UID/MULTI/04378/2013, IF/00092/2014, SFRH/BD/79077/2011, PD/BD/106035/2015] info:eu-repo/semantics/publishedVersion

Ubiquitin signals the sorting of Cx43 into EVs, which is impaired during ischemia, resulting in decreased levels of Cx43 in cardiomyocyte-derived and circulating EVs from myocardial infarcted patients., Ischemic heart disease has been associated with an impairment on intercellular communication mediated by both gap junctions and extracellular vesicles. We have previously shown that connexin 43 (Cx43), the main ventricular gap junction protein, assembles into channels at the extracellular vesicle surface, mediating the release of vesicle content into target cells. Here, using a comprehensive strategy that included cell-based approaches, animal models and human patients, we demonstrate that myocardial ischemia impairs the secretion of Cx43 into circulating, intracardiac and cardiomyocyte-derived vesicles. In addition, we show that ubiquitin signals Cx43 release in basal conditions but appears to be dispensable during ischemia, suggesting an interplay between ischemia-induced Cx43 degradation and secretion. Overall, this study constitutes a step forward for the characterization of the signals and molecular players underlying vesicle protein sorting, with strong implications on long-range intercellular communication, paving the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders.

This study aimed to endow the cell-penetrating peptide (CPP) S4Peptide/siRNA complexes were prepared with two new derivatives of the CPP S4Peptide/siRNA complexes prepared with the C12-HThese encouraging results pave the way for a potential application of the C12-H

Ethnopharmacological relevance Thymus camphoratus and T. carnosus are widely used in Portugal for the treatment of inflammatory-related conditions, such as inflammation of the respiratory tract, being the later also used as an antitussive. Aim of the study Bearing in mind the lack of scientific studies focused on the pharmacological activity of Thymus camphoratus and T. carnosus , this work was designed to validate the anti-inflammatory properties ascribed to these traditional species and concomitantly to unveil both the putative molecular mechanisms behind their bioactivity as well as the safety profile of their essential oils and major compounds. Materials and methods The chemical composition of the essential oils was assessed by gas chromatography (GC) and gas chromatography – mass spectroscopy (GC/MS). The nitric oxide (NO) scavenging potential of the oils was tested using S-nitroso-N-acetyl-D,L-penicillamine (SNAP) as NO donor. The anti-inflammatory potential of the essential oils and their major compounds was evaluated by measuring the nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages as well as the expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Importantly, and in an attempt to assess the safety profile of the oils and respective major compounds, their effect on macrophages and hepatocytes viability was also determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results T. carnosus essential oil was characterized by high amounts of borneol and camphene whereas T. camphoratus oil was rich in 1,8-cineole and borneol. The later presented higher pharmacological activity showing inhibitory effects towards NO production at lower concentrations (0.16 µL/mL) and concomitantly inhibiting the expression of two crucial pro-inflammatory proteins, iNOS and COX-2 (at 0.32 µL/mL). Since no NO scavenging activity was achieved, it is reasonable to conclude that the anti-inflammatory activity of the essential oils occurs upstream of iNOS expression, probably through inhibition of relevant pro-inflammatory signal transduction pathways. Importantly, at bioactive concentrations, the essential oils were devoid of toxicity towards macrophages and hepatocytes. The activity of the isolated compounds was far from that observed for the essential oils, thus suggesting that the anti-inflammatory activity is due to a synergic effect between several compounds in the mixture. Conclusion Overall, the results herein presented sustain and strengthen the anti-inflammatory properties traditionally ascribed to T. carnosus and T. camphoratus. Additionally, the molecular mechanisms associated to their pharmacological activity were highlighted, opening new avenues for the development of effective anti-inflammatory herbal medicinal products.

Airborne environmental particles (EP) more commonly referred as particulate matter (PM) are an illustrative marker of air pollution that is associated with adverse effects on human health. Considering, PM is a complex mixture, not only in terms of its chemical composition, but also in the range of particle size, it is difficult to identify which attribute contributes more for the toxicity. Currently, there is no report about the immunotoxicological effects caused by PM with reduced content of heavy metals. This study intends to address this gap and provides a detailed characterization and immunotoxicity evaluation of PM collected in an urban area with heavy traffic congestion. Environmental particles were separated by different sizes though a sucrose gradient. This method allowed to achieve 4 sized fractions: EP f 15 % with a mean diameter of 284 nm ± 1.86 nm, EP f 25 % with a mean diameter of 461 nm ± 1.72 nm, EP f 35 % with a mean diameter of 1845 nm ± 251 nm and EP f 45 % with a mean diameter of 2204 nm ± 310 nm. Only the fractions with the smallest sizes (EP f 15 % and EP f 25 %) were subsequently studied. The chemical composition of both fractions was not substantially different, and the dominant elements were C, O, Ca and K. Only EP f 25 % showed to have a small amount of Fe. Therefore, the heavy metal elements were eliminated through centrifugation. Essentially, we found that the EP f 15 % was more cytotoxic in RAW 264.7 cells than EP f 25 %, which indicates the smaller size as the motive for the higher toxicity. In addition, both fractions of EP presented a good internalization in macrophages after 2 h exposure and induced the production of reactive oxygen species in a concentration-dependent manner. Moreover, EP f 15 % and EP f 25 % led to a strong secretion of proinflammatory cytokines (TNF-α and IL-6) in human peripheral blood mononuclear cells (hPBMCs) in the 3 concentrations tested. The inflammatory response observed was independent of the presence of heavy metals and endotoxins, since these last were suppressed by using polymyxin B sulfate. This report emphasizes the importance of an adequate physicochemical characterization and adequate controls in the experiments to achieve a right interpretation of the biological effects caused by PM.

Ana Raquel Madureira,1 Sara Nunes,2 Débora ACampos,1 João C Fernandes,2 Cláudia Marques,3 Monica Zuzarte,2 Beatriz Gullón,1 Luís M Rodríguez-Alcalá,1 Conceição Calhau,3,4 Bruno Sarmento,5–7 Ana Maria Gomes,1 Maria Manuela Pintado,1 Flávio Reis2 1Catholic University of Portugal, CBQF– Center for Biotechnology and Fine Chemistry – Associate Laboratory, Faculty of Biotechnology, Porto, Portugal; 2Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, and CNC.IBILI Consortium, University of Coimbra, Coimbra, Portugal; 3Department of Biochemistry, Faculty of Medicine, University of Porto, Porto, Portugal; 4Center for Health Technology and Services Research (CINTESIS), Porto, Portugal; 5Department of Pharmaceutical Sciences, Institute of Health Sciences-North, CESPU, Gandra, Portugal; 6“I3S” Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal; 7INEB, Institute of Biomedical Engineering, University of Porto, Porto, Portugal Abstract: Rosmarinic acid (RA) possesses several protective bioactivities that have attracted increasing interest by nutraceutical/pharmaceutical industries. Considering the reduced bioavailability after oral use, effective (and safe) delivery systems are crucial to protect RA from gastrointestinal degradation. This study aims to characterize the safety profile of solid lipid nanoparticles produced with Witepsol and Carnauba waxes and loaded with RA, using in vitro and in vivo approaches, focused on genotoxicity and cytotoxicity assays, redox status markers, hematological and biochemical profile, liver and kidney function, gut bacterial microbiota, and fecal fatty acids composition. Free RA and sage extract, empty nanoparticles, or nanoparticles loaded with RA or sage extract (0.15 and 1.5 mg/mL) were evaluated for cell (lymphocytes) viability, necrosis and apoptosis, and antioxidant/prooxidant effects upon DNA. Wistar rats were orally treated for 14 days with vehicle (control) and with Witepsol or Carnauba nanoparticles loaded with RA at 1 and 10 mg/kg body weight/d. Blood, urine, feces, and several tissues were collected for analysis. Free and loaded RA, at 0.15 mg/mL, presented a safe profile, while genotoxic potential was found for the higher dose (1.5 mg/mL), mainly by necrosis. Our data suggest that both types of nanoparticles are safe when loaded with moderate concentrations of RA, without in vitro genotoxicity and cytotoxicity and with an in vivo safety profile in rats orally treated, thus opening new avenues for use in nutraceutical applications. Keywords: solid lipid nanoparticles, rosmarinic acid, Witepsol and Carnauba waxes, safety profile, in vitro and animal toxicity

We thank Dr Nuno Alves (Cardiology Department, CHUC‐HG) who performed the collection of human blood samples and Doctor Francisco Caramelo (iCBR/FMUC) for helping with the statistical analysis. This work was supported by the European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) [under the projects PAC “NETDIAMOND” POCI‐01‐0145‐FEDER‐016385; HealthyAging2020 CENTRO‐01‐0145‐ FEDER‐000012‐N2323; POCI‐01‐0145‐FEDER‐007440, CENTRO‐01‐ 0145‐FEDER‐032179, CENTRO‐01‐0145‐FEDER‐032414 and FCT‐ UID/NEU/04539/2013 to CNC.IBILI]. TMM was supported by PD/ BD/106043/2015 and TRR by PD/BD/52294/2013 from Fundação para a Ciência e a Tecnologia (FCT). JS was supported by Horizon2020 ERC‐2016‐COG EVICARE (725229). Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell-cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte-derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro-inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL-1β and IL-6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL-1β, IL-6, IL-10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum-circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte-derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post-infarction remodelling. publishersversion published

Fungal infections remain a burden worldwide, thus underpinning the need for effective new therapeutic approaches. In the present study, the antifungal effect of the essential oils of two thyme species, Thymus camphoratus and Thymus carnosus, used in traditional medicine in Portugal, as well as their major compounds was assessed. A special focus was placed on their effect on Candida albicans virulence factors. Also, the safety profile of the essential oils was assessed on keratinocytes. The essential oils were analyzed by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The minimal inhibitory and minimal fungicidal concentrations of the essential oils and their main compounds were assessed on reference and clinical strains. Also, their effect on C. albicans germ tube formation, metabolism, and biofilm disruption were considered. T. camphoratus oil was rich in 1,8-cineole and α-pinene whereas T. carnosus oil showed high amounts of borneol and camphene. Regarding the antifungal effect, both oils were more active against Cryptococcus neoformans and dermatophytes and very effective in inhibiting C. albicans germ tube formation, at doses well below their MIC and in a higher extend than the isolated compounds and fluconazole, an antifungal drug widely used in the clinic. The oils also disrupted preformed C. albicans biofilms. Furthermore, no toxicity was observed at pharmacological relevant concentrations towards keratinocytes. Our study validates the traditional uses ascribed to these Iberian species. Furthermore, it brings new insights on the antifungal potential and mechanism of action of these thyme species, thus paving the way for the development of novel effective antifungal drugs.

The present study was conducted to elucidate the composition, antifungal and anti-inflammatory activities and cytotoxicity of Daucus carota subsp. gummifer essential oil. Aerial parts of the plants were hydrodistilled and the oil analyzed by GC and GC–MS. The oil was characterized by high contents of monoterpenes (83.9%), the major compounds being geranyl acetate (37.0%) and α-pinene (30.9%). The daucane sesquiterpene, carotol, was also found in relatively high amounts (11.0%). For the antifungal activity, minimal inhibitory and minimal lethal concentrations (MIC and MLC, respectively) were determined against several pathogenic fungi strains. The oil was particularly active against dermatophytes and Cryptococcus neoformans, with MIC values ranging from 0.32 to 0.64 μL/mL. Concerning the anti-inflammatory potential, the oil demonstrated a strong anti-inflammatory activity by inhibiting nitric oxide (NO) production in both lipopolysaccharide (LPS)-triggered macrophages and microglia cells. NO scavenging activity was also assessed using the NO donor S-nitroso-N-acetyl- d , l -penicillamine and a significant effect was disclosed. To assure that the bioactivity of the oil was achieved without detrimental effects to cells, the toxicity of D. carota subsp. gummifer oil was investigated using the MTT assay in several mammalian cell lines: macrophages (Raw 264.7), keratinocytes (HaCat), hepatocytes (HepG2) and microglia (N9). Interestingly, we detected a cytotoxic effect only for the highest concentrations of the oil, thus assuring a safe toxicological profile at bioactive concentrations. These results advise that D. carota subsp. gummifer essential oil should be explored as a natural source of antifungal and anti-inflammatory drugs with potential application both at the peripheral and central nervous system levels, thus supporting in vivo studies focused in the management of dermatophytosis and/or inflammatory-related diseases.

Ethnopharmacological relevance Santolina species are widely used in traditional medicine in the Mediterranean region for their anti-inflammatory, antimicrobial, antispasmodic, digestive, and analgesic properties. S. impressa, a Portuguese endemism, is traditionally recognized for its beneficial anti-inflammatory properties in several gastrointestinal affections and is also used in oropharyngeal infections. Aim of the study The present study aims to characterize the essential oil of S. impressa growing in Portugal and validate its traditional uses by assessing the anti-inflammatory potential of its essential oil at concentrations without toxicity. The antifungal properties of the oil are also addressed, as well as, the putative mechanism of action underlying these effects. Material and methods The essential oil was obtained in accordance with the European Pharmacopoeia and characterized by GC and GC-MS. The anti-inflammatory potential of the oil was assessed on LPS-stimulated macrophages, through the production of nitric oxide (NO) using the Griess reaction. Putative mechanisms of action included the role of the oil as a NO scavenger, as well as its effect on the expression of two key pro-inflammatory enzymes, iNOS and COX-2 by Western blot analysis. The antifungal effect of the oil was evaluated according to the CLSI guidelines on several yeast and filamentous strains and on two major virulence factors in Candida albicans, namely germ tubes and biofilms. Ultrastructural modifications on dermatophytes were also unveiled by transmission electron microscopy. Results S. impressa essential oil was primarily characterized by the presence of monoterpene hydrocarbons and oxygenated monoterpenes, being the main compounds β-pinene (22.5%), 1,8-cineole (10.0%), limonene (9.1%), camphor (8.1%) and β-phellandrene (8.0%). A significant decrease (ca 60.0%) in nitrite levels was observed in LPS-stimulated macrophages treated with the oil without affecting cell viability. This effect could be explained by a great reduction on iNOS expression (85.0% inhibition), thus underpinning the anti-inflammatory potential of the oil. The oil also showed a fungicidal effect, being more active against Cryptococcus neoformans, Epidermophyton floccosum and Trichophytum rubrum. For these dermatophytes, significant ultrastructural modifications in cell wall structure were detected. Strikingly, for C. albicans, the oil showed a significant anti-infective potential (at 0.07 mg/mL for germ tube inhibition and 0.02 mg/mL for biofilm disruption) before fungal growth inhibition occurred. Conclusions Our results validate the main traditional use ascribed to S. impressa, namely its anti-inflammatory effect. In addition, an antifungal potential is pointed out, thus corroborating the antimicrobial uses and adding new value to an endemic species poorly recognized by the industry.

The present study reports the chemical composition, antifungal, antioxidant and anti-inflammatory properties as well as the cytotoxicity of Oenanthe crocata essential oil and one of its main compounds. The essential oil was obtained from the aerial parts of the plant by hydrodistillation and analysed by GC and GC/MS. The oil was predominantly composed of monoterpene hydrocarbons (85.8%), being the main compounds trans-β-ocimene (31.3%), sabinene (29.0%) and cis-β-ocimene (12.3%). For the antifungal activity, the minimal inhibitory and minimal lethal concentrations (MICs and MLCs) were determined. The oil was particularly active against dermatophytes and Cryptococcus neoformans, with MIC values ranging from 0.08 to 0.16 μL/mL. Regarding the anti-inflammatory activity, both the oil and sabinene demonstrated strong anti-inflammatory activity through nitric oxide (NO) production inhibition in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-triggered macrophages. Furthermore, the essential oil showed a potent NO scavenging effect and inhibited inducible NO synthase expression. Interestingly, and although we detected a cytotoxic effect in macrophages and keratinocytes for the highest concentrations tested of the oil and sabinene, we also disclosed bioactive and safe concentrations to be further explored for therapeutic proposes. Taking together, these results support the use of the oil and sabinene for the management of dermatophytosis and/or inflammatory-related diseases.

As part of our ongoing study on the valorization of aromatic plants, the present study was designed to elucidate the composition, scavenging potential, anti-inflammatory activity and cytotoxicity of Margotia gummifera essential oils. Umbels were submitted to hydrodistillation in a Clevenger-type apparatus and the oils were analyzed by GC and GC–MS. For the anti-inflammatory activity, an in vitro model of lipopolysaccharide-stimulated macrophages was used and the inhibition of nitric oxide (NO) production was quantified through the Griess reagent, in the presence of the essential oil or its main compounds. NO scavenging potential was assessed using an NO donor and the cytotoxicity was evaluated on macrophages, keratinocytes and alveolar epithelial cells. The oils were characterized by high contents of monoterpene hydrocarbons, being the major compounds myrcene (20.4–23.0%) and sabinene (21.0–23.5%). The oil, myrcene and sabinene significantly inhibited NO production without affecting cell viability and showed a very effective NO scavenging potential, sabinene being the most active compound. These results suggest that M. gummifera essential oil, sabinene and myrcene should be explored as a natural source of new antioxidant and anti-inflammatory drugs for the development of food supplements, nutraceuticals or plant-based medicines.

Copyright © 2017, Oxford University Press, Aims: Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results: Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions: This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply., This work was supported by European Regional Development Fund (FEDER) through the Operational Program for Competitiveness Factors (COMPETE) [HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, POCI-01-0145-FEDER-016385, POCI-01-0145-FEDER-007440 to CNC.IBILI, POCI-01-0145-FEDER-007274 to i3S/INEB and NORTE-01-0145- FEDER-000012 to T.L.L.]; national funds through the Portuguese Foundation for Science and Technology (FCT) [PTDC/SAU-ORG/119296/2010, PTDC/NEU-OSD/0312/2012, PESTC/ SAU/UI3282/2013-2014, MITP-TB/ECE/0013/2013, FCT-UID/NEU/04539/2013], PD/BD/52294/2013 to T.M.R.R., SFRH/BD/85556/2012 (co-financed by QREN) to V.C.S]; Lisboa Portugal Regional Operational Programme (LISBOA 2020) and Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement; and by INFARMED Autoridade Nacional do Medicamento e Produtos de Saúde, I.P. [FIS-FIS-2015-01_CCV_20150630-157].

Lavandula L. and Thymus L. comprise several relevant species for the food, cosmetic, perfumery and pharmaceutical industries. Considering the traditional medicinal use of L. stoechas and T. herba-barona and the lack of scientific studies on their biological activities, the present study was designed to elucidate the composition and antifungal activity of their essential oils against fungi responsible for human infections as well as the anti-inflammatory potential and their cytotoxicity on a macrophage cell line. Moreover, the antifungal activity against fungi responsible for food contamination is also reported. Flowering parts of the plants were submitted to hydrodistillation in a Clevenger-type apparatus and the oils were analysed by GC and GC–MS. The minimal inhibitory and minimal lethal concentrations of the oils against fungi strains were determined using a macrodilution broth method. For the anti-inflammatory activity, an in vitro model of lipopolysaccharide-stimulated macrophages was used and the inhibition of nitric oxide production quantified. Assessment of the oils cytotoxicity was performed using the MTT reduction assay. L. stoechas essential oil was rich in fenchone (37.0%) and camphor (27.3%) while T. herba-barona oil showed high amounts of two phenols, carvacrol (54.0%) and thymol (30.2%). The latter was the most active oil against the tested fungi but evidenced high cytotoxicity on macrophages. L. stoechas was active against dermatophyte strains and showed potential anti-inflammatory activity at concentrations without affecting cell viability. These results support the use of L. stoechas in the development of phytopharmaceuticals or food supplements/nutraceuticals for the management of dermatophytosis and/or inflammatory-related diseases. Regarding T. herba-barona, it can be used as a preservative in storage products, due to its ability to inhibit Aspergilllus growth.

Extracellular vesicles (EVs) are major conveyors of biological information, mediating local and systemic cell-to-cell communication under physiological and pathological conditions. These endogenous vesicles have been recognized as prominent drug delivery vehicles of several therapeutic cargoes, including doxorubicin (dox), presenting major advantages over the classical approaches. Although dox is one of the most effective anti-tumour agents in the clinical practice, its use is very often hindered by its consequent dramatic cardiotoxicity. Despite significant advances witnessed in the past few years, more comprehensive studies, supporting the therapeutic efficacy of EVs, with decreased side effects, are still scarce. The main objective of this study was to evaluate the role of the gap junction protein connexin43 (Cx43) in mediating the release of EV content into tumour cells. Moreover, we investigated whether Cx43 improves the efficiency of dox-based anti-tumour treatment, with a concomitant decrease of cardiotoxicity. In the present report, we demonstrate that the presence of Cx43 in EVs increases the release of luciferin from EVs into tumour cells in vitro and in vivo . In addition, using cell-based approaches and a subcutaneous mouse tumour model, we show that the anti-tumour effect of dox incorporated into EVs is similar to the administration of the free drug, regardless the presence of Cx43. Strikingly, we demonstrate that the presence of Cx43 in dox-loaded EVs reduces the cardiotoxicity of the drug. Altogether, these results bring new insights into the concrete potential of EVs as therapeutic vehicles and open new avenues toward the development of strategies that help to reduce unwanted side effects. Keywords: extracellular vesicles; drug delivery; cancer; cardioprotection; intercellular communication (Published: 29 September 2016)

This work reports the antifungal activity of Lavandula luisieri essential oils against yeast, dermatophyte and Aspergillus strains responsible for human infections and food contamination. The oil's cytotoxicity and its effect on the yeast-mycelium transition in Candida albicans, an important virulence factor, were also evaluated. Analyses by GC and GC/MS showed a peculiar composition of irregular monoterpenes. Significant differences between the samples occurred in the amounts of 1,8-cineole, fenchone and trans-α-necrodyl acetate. The oil with higher amounts of irregular monoterpenes was the most effective. The influence of the oils on the dimorphic transition in C. albicans was also studied through the germ tube inhibition assay. Filamentation was completely inhibited at concentrations sixteen times lower than the minimal inhibitory concentration. The results support the use of L. luiseiri essential oils in the development of new phytopharmaceuticals and food preservatives and emphasise its antifungal properties at concentrations not cytotoxic or with very low detrimental effects on mammalian cells.

In the present work we report for what we believe to be the first time the antifungal activity and mechanism of action of the essential oils of Lavandula viridis from Portugal. The essential oils were isolated by hydrodistillation and analysed by GC and GC/MS. The MIC and the minimal lethal concentration (MLC) of the essential oil and its major compounds were determined against several pathogenic fungi. The influence of subinhibitory concentrations of the essential oil on the dimorphic transition in Candida albicans was also studied, as well as propidium iodide and FUN-1 staining of Candida albicans cells by flow cytometry following short treatments with the essential oil. The oils were characterized by a high content of oxygen-containing monoterpenes, with 1,8-cineole being the main constituent. Monoterpene hydrocarbons were present at lower concentrations. According to the determined MIC and MLC values, the dermatophytes and Cryptococcus neoformans were the most sensitive fungi (MIC and MLC values ranging from 0.32 to 0.64 µl ml⁻¹), followed by Candida species (at 0.64-2.5 µl ml⁻¹). For most of these strains, MICs were equivalent to MLCs, indicating a fungicidal effect of the essential oil. The oil was further shown to completely inhibit filamentation in Candida albicans at concentrations well below the respective MICs (as low as MIC/16). Flow cytometry results suggested a mechanism of action ultimately leading to cytoplasmic membrane disruption and cell death. Our results show that L. viridis essential oils may be useful in the clinical treatment of fungal diseases, particularly dermatophytosis and candidosis, although clinical trials are required to evaluate the practical relevance of our in vitro research.

Lavanders (Lavandula spp.) are aromatic plants with high industrial and commercial value, used in food industry, perfumery and pharmaceutical preparations. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Lavandula pedunculata and to evaluate the potential of these plants for essential oil production with industrial application. In vitro cultures were established on media with different concentrations of benzyladenine. Trichome morphology was examined by SEM and their secretion analyzed by histochemical tests. Essential oils were isolated by hydrodistillation and analyzed by GC and GC-MS. The essential oils and trichomes of in vitro plants were compared with those of field-growing plants. Best propagation rates were achieved with 0.25 mg/l BA. Rooting occurred without treatment with auxins thus preventing callus growth. Trichomes and essential oils of the in vitro propagated plants were similar to those of the parent plants. Two chemotypes were characterized: 1,8-cineole/camphor type and fenchone type. Since trichomes and essential oils of L. pedunculata micropropagated plantlets are identical to those produced by field-growing plants, in vitro cultures can be used for essential oil production without affecting natural resources. Moreover, the quality of the essential oils is assured and this protocol can be further applied to the propagation of selected chemotypes for industrial purposes.

The chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav., harvested in North and Central Portugal, were investigated. The essential oils were isolated by hydrodistillation and analyzed by GC and GC/MS. The minimal-inhibitory concentration (MIC) and the minimal-lethal concentration (MLC) of the essential oils and of their major constituents were used to evaluate the antifungal activity against different strains of fungi involved in candidosis, dematophytosis, and aspergillosis. The oils were characterized by a high percentage of oxygenated monoterpenes, the main compounds being 1,8-cineole (2.4-55.5%), fenchone (1.3-59.7%), and camphor (3.6-48.0%). Statistical analysis differentiated the essential oils into two main types, one characterized by the predominance of fenchone and the other one by the predominance of 1,8-cineole. Within the 1,8-cineole chemotype, two subgroups were well-defined taking into account the percentages of camphor. A significant antifungal activity of the oils was found against dermatophyte strains. The essential oil with the highest content of camphor was the most active with MIC and MLC values ranging from 0.32-0.64 microl/ml.

The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria , which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria , we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.

The essential oil ofDaucus carotasubsp.carotafrom Portugal, with high amounts of geranyl acetate (29.0%),α-pinene (27.2%), and 11αH-himachal-4-en-1β-ol (9.2%), was assessed for its biological potential. The antimicrobial activity was evaluated against several Gram-positive and Gram-negative bacteria, yeasts, dermatophytes, andAspergillusstrains. The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) were evaluated showing a significant activity towards Gram-positive bacteria (MIC = 0.32–0.64 μL/mL),Cryptococcus neoformans(0.16 μL/mL), and dermatophytes (0.32–0.64 μL/mL). The inhibition of the germ tube formation and the effect of the oil onCandida albicansbiofilms were also unveiled. The oil inhibited more than 50% of filamentation at concentrations as low as 0.04 μL/mL (MIC/128) and decreased both biofilm mass and cell viability. The antioxidant capacity of the oil, as assessed by twoin chemicomethods, was not relevant. Still, it seems to exhibit some anti-inflammatory potential by decreasing nitric oxide production around 20% in LPS-stimulated macrophages, without decreasing macrophages viability. Moreover, the oils safety profile was assessed on keratinocytes, alveolar epithelial cells, macrophages, and hepatocytes. Overall, the oil demonstrated a safety profile at concentrations below 0.64 μL/mL. The present work highlights the bioactive potential ofD. carotasubsp.carotasuggesting its industrial exploitation.

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.