Epigenetic reprogramming as a key to reverse ageing and increase longevity.

The pursuit for the fountain of youth has long been a fascination amongst scientists and humanity. Ageing is broadly characterized by a cellular decline with increased susceptibility to age-related diseases, being intimately associated with epigenetic modifications. Recently, reprogramming-induced rejuvenation strategies have begun to greatly alter longevity research not only to tackle age-related defects but also to possibly reverse the cellular ageing process. Hence, in this review, we highlight the major epigenetic changes during ageing and the state-of-art of the current emerging epigenetic reprogramming strategies leveraging on transcription factors. Notably, partial reprogramming enables the resetting of the ageing clock without erasing cellular identity. Promising chemical-based rejuvenation strategies harnessing small molecules, including DNA methyltransferase and histone deacetylase inhibitors are also discussed. Moreover, in parallel to longevity interventions, the foundations of epigenetic clocks for accurate ageing assessment and evaluation of reprogramming approaches are briefly presented. Going further, with such scientific breakthroughs, we are witnessing a rise in the longevity biotech industry aiming to extend the health span and ideally achieve human rejuvenation one day. In this context, we overview the main scenarios proposed for the future of the socio-economic and ethical challenges associated with such an emerging field. Ultimately, this review aims to inspire future research on interventions that promote healthy ageing for all.

Exploiting Potential Probiotic Lactic Acid Bacteria Isolated from Chlorella vulgaris Photobioreactors as Promising Vitamin B12 Producers.

Lactic acid bacteria (LAB) have been documented as potential vitamin B12 producers and may constitute an exogenous source of cobalamin for the microalga Chlorella vulgaris, which has been described as being able to perform vitamin uptake. Hence, there is an interest in discovering novel B12-producing probiotic LAB. Therefore, the purpose of the current work was to perform a phenotype-genotype analysis of the vitamin B12 biosynthesis capacity of LAB isolated from C. vulgaris bioreactors, and investigate their probiotic potential. Among the selected strains, Lactococcus lactis E32, Levilactobacillus brevis G31, and Pediococcus pentosaceus L51 demonstrated vitamin B12 biosynthesis capacity, with the latter producing the highest (28.19 ± 2.27 pg mL-1). The genomic analysis confirmed the presence of pivotal genes involved in different steps of the biosynthetic pathway (hemL, cbiT, cobC, and cobD). Notably, P. pentosaceus L51 was the only strain harboring cobA, pduU, and pduV genes, which may provide evidence for the presence of the cobalamin operon. All strains demonstrated the capability to withstand harsh gastrointestinal conditions, although P. pentosaceus L51 was more resilient. The potential for de novo cobalamin biosynthesis and remarkable probiotic features highlighted that P. pentosaceus L51 may be considered the most promising candidate strain for developing high-content vitamin B12 formulations.

Scenedesmus rubescens Heterotrophic Production Strategies for Added Value Biomass.

Microalgae attract interest worldwide due to their potential for several applications. Scenedesmus is one of the first in vitro cultured algae due to their rapid growth and handling easiness. Within this genus, cells exhibit a highly resistant wall and propagate both auto- and heterotrophically. The main goal of the present work is to find scalable ways to produce a highly concentrated biomass of Scenedesmus rubescens in heterotrophic conditions. Scenedesmus rubescens growth was improved at the lab-scale by 3.2-fold (from 4.1 to 13 g/L of dry weight) through medium optimization by response surface methodology. Afterwards, scale-up was evaluated in 7 L stirred-tank reactor under fed-batch operation. Then, the optimized medium resulted in an overall productivity of 8.63 g/L/day and a maximum biomass concentration of 69.5 g/L. S. rubescens protein content achieved approximately 31% of dry weight, similar to the protein content of Chlorella vulgaris in heterotrophy.

Untangling the role of biotic and abiotic ageing of various environmental plastics toward the sorption of metals.

Plastic particles can impact the environmental fate and bioavailability of essential inorganic micronutrients and non-essential (toxic) metals. The sorption of metals to environmental plastic has been demonstrated to be facilitated by plastic ageing, a phenomenon encompassing an array of physical, chemical, and biological processes. This study deploys a factorial experiment to untangle the role of different ageing processes in determining the sorption of metals. Plastics made of three different polymer types were aged both through abiotic (ultraviolet irradiation, UV) and biotic (through the incubation with a multispecies algal inoculum forming a biofilm) processes under controlled laboratory conditions. Pristine and aged plastic samples were characterized for their physiochemical properties through Fourier-transformed infrared spectroscopy, scanning electron microscopy and water contact angle measurements. Their sorption affinity toward aluminum (Al) and copper (Cu) in aqueous solutions was then assessed as a response variable. All ageing processes (alone or combined) influenced plastic surface properties resulting in reduced hydrophobicity, changes in surface functional groups (i.e., increase of oxygen containing functional groups after UV ageing and the appearance of marked bands as amides and polysaccharides after biofouling), as well as in nanomorphology. The sorption of Al and Cu was instead statistically dependent (p < 0.01) on the degree of biofouling covering the specimens. Biofouled plastic displayed in fact substantial affinity for metal sorption causing the depletion of up to tenfold Cu and Al compared to pristine polymers, regardless of the polymer type and presence or absence of other ageing treatments. These results confirm the hypothesis that the accumulation of metals on plastic is substantially driven by the biofilm present on environmental plastics. These findings also highlight the importance of investigating the implications of environmental plastic for metal and inorganic nutrients availability in environments impacted by this pollution.

Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signaling mediators of cellular responses. However, despite representing a promising alternative to cell-based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred-tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred-tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC-derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential.

Nisin Mutant Prevention Concentration and the Role of Subinhibitory Concentrations on Resistance Development by Diabetic Foot Staphylococci.

The most prevalent microorganism in diabetic foot infections (DFI) is Staphylococcus aureus, an important multidrug-resistant pathogen. The antimicrobial peptide nisin is a promising compound for DFI treatment, being effective against S. aureus. However, to avoid the selection of resistant mutants, correct drug therapeutic doses must be established, being also important to understand if nisin subinhibitory concentrations (subMIC) can potentiate resistant genes transfer between clinical isolates or mutations in genes associated with nisin resistance. The mutant selection window (MSW) of nisin was determined for 23 DFI S. aureus isolates; a protocol aiming to prompt vanA horizontal transfer between enterococci to clinical S. aureus was performed; and nisin subMIC effect on resistance evolution was assessed through whole-genome sequencing (WGS) applied to isolates subjected to a MEGA-plate assay. MSW ranged from 5-360 µg/mL for two isolates, from 5-540 µg/mL for three isolates, and from 5-720 µg/mL for one isolate. In the presence of nisin subMIC values, no transconjugants were obtained, indicating that nisin does not seem to promote vanA transfer. Finally, WGS analysis showed that incubation in the presence of nisin subMIC did not promote the occurrence of significant mutations in genes related to nisin resistance, supporting nisin application to DFI treatment.

Food grade extraction of Chlorella vulgaris polar lipids: A comparative lipidomic study.

Glycolipids and phospholipids are the main reservoirs of omega polyunsaturated fatty acids in microalgae. Their extraction for the food industry requires food grade solvents, however, the use of these solvents is generally associated with low extraction yields. In this study, we evaluated the lipid extraction efficiency of food-grade ethanol, ultrasound-assisted ethanol (UAE) and dichloromethane/methanol (DCM) from Chlorella vulgaris cultivated under autotrophic and heterotrophic conditions. Yields of lipids, fatty acids (FA), and complex lipid profiles were determined by gravimetry, GC-MS, and LC-MS/MS, respectively. UAE and DCM showed the highest lipid yields with similar purity. The FA profiles were identical for all extracts. The polar lipidome of the DCM and UAE extracts was comparable, while the EtOH extracts were significantly different. These results demonstrated the effectiveness of UAE extraction to obtain high yields of polar lipids and omega-3 and -6-rich extracts from C. vulgaris that can be used for food applications.

Microalgal Lipid Extracts Have Potential to Modulate the Inflammatory Response: A Critical Review.

Noncommunicable diseases (NCD) and age-associated diseases (AAD) are some of the gravest health concerns worldwide, accounting for up to 70% of total deaths globally. NCD and AAD, such as diabetes, obesity, cardiovascular disease, and cancer, are associated with low-grade chronic inflammation and poor dietary habits. Modulation of the inflammatory status through dietary components is a very appellative approach to fight these diseases and is supported by increasing evidence of natural and dietary components with strong anti-inflammatory activities. The consumption of bioactive lipids has a positive impact on preventing chronic inflammation and consequently NCD and AAD. Thus, new sources of bioactive lipids have been sought out. Microalgae are rich sources of bioactive lipids such as omega-6 and -3 polyunsaturated fatty acids (PUFA) and polar lipids with associated anti-inflammatory activity. PUFAs are enzymatically and non-enzymatically catalyzed to oxylipins and have a significant role in anti and pro-resolving inflammatory responses. Therefore, a large and rapidly growing body of research has been conducted in vivo and in vitro, investigating the potential anti-inflammatory activities of microalgae lipids. This review sought to summarize and critically analyze recent evidence of the anti-inflammatory potential of microalgae lipids and their possible use to prevent or mitigate chronic inflammation.

Microalgae as Sustainable Bio-Factories of Healthy Lipids: Evaluating Fatty Acid Content and Antioxidant Activity.

The demand for sustainable and environmentally friendly food sources and food ingredients is increasing, and microalgae are promoted as a sustainable source of essential and bioactive lipids, with high levels of omega-3 fatty acids (ω-3 FA), comparable to those of fish. However, most FA screening studies on algae are scattered or use different methodologies, preventing a true comparison of its content between microalgae. In this work, we used gas-chromatography mass-spectrometry (GC-MS) to characterize the FA profile of seven different commercial microalgae with biotechnological applications (Chlorella vulgaris, Chlorococcum amblystomatis, Scenedesmus obliquus, Tetraselmis chui, Phaeodactylum tricornutum, Spirulina sp., and Nannochloropsis oceanica). Screening for antioxidant activity was also performed to understand the relationship between FA profile and bioactivity. Microalgae exhibited specific FA profiles with a different composition, namely in the ω-3 FA profile, but with species of the same phylum showing similar tendencies. The different lipid extracts showed similar antioxidant activities, but with a low activity of the extracts of Nannochloropsis oceanica. Overall, this study provides a direct comparison of FA profiles between microalgae species, supporting the role of these species as alternative, sustainable, and healthy sources of essential lipids.

Polar lipidomic profile shows Chlorococcum amblystomatis as a promising source of value-added lipids.

There is a growing trend to explore microalgae as an alternative resource for the food, feed, pharmaceutical, cosmetic and fuel industry. Moreover, the polar lipidome of microalgae is interesting because of the reports of bioactive polar lipids which could foster new applications for microalgae. In this work, we identified for the first time the Chlorococcum amblystomatis lipidome using hydrophilic interaction liquid chromatography-high resolution electrospray ionization- tandem mass spectrometry (HILIC-HR-ESI-MS/MS). The Chlorococcum amblystomatis strain had a lipid content of 20.77% and the fatty acid profile, determined by gas chromatography-mass spectrometry, has shown that this microalga contains high amounts of omega-3 polyunsaturated fatty acids (PUFAs). The lipidome identified included 245 molecular ions and 350 lipid species comprising 15 different classes of glycolipids (6), phospholipids (7) and betaine lipids (2). Of these, 157 lipid species and the main lipid species of each class were esterified with omega-3 PUFAs. The lipid extract has shown antioxidant activity and anti-inflammatory potential. Lipid extracts also had low values of atherogenic (0.54) and thrombogenic index (0.27). In conclusion, the lipid extracts of Chlorococcum amblystomatis have been found to be a source of lipids rich in omega-3 PUFAs for of great value for the food, feed, cosmetic, nutraceutical and pharmaceutical industries.

Nisin Influence on the Antimicrobial Resistance Ability of Canine Oral Enterococci.

Periodontal disease (PD) is one of the most common diseases in dogs. Although previous studies have shown the potential of the antimicrobial peptide nisin for PD control, there is no information regarding its influence in the development of antimicrobial resistance or horizontal gene transfer (HGT). Nisin's mutant prevention concentration (MPC) and selection window (MSW) were determined for a collection of canine oral enterococci. Isolates recovered after the determination of the MPC values were characterized for their antimicrobial profile and its nisin minimum inhibitory and bactericidal concentrations. The potential of vanA HGT between Enterococcus faecium CCGU36804 and nine clinical canine staphylococci and enterococci was evaluated. Nisin MPC values ranged from 400 to more than 600 µg/mL. In comparison with the original enterococci collection, the isolates recovered after the determination of the nisin MPC showed increased resistance towards amoxicillin/clavulanate (5%), vancomycin (5%), enrofloxacin (10%), gentamicin (10%) and imipenem (15%). The HGT of vanA gene was not observed. This work showed that nisin selective pressure may induce changes in the bacteria's antimicrobial resistance profile but does not influence horizontal transfer of vanA gene. To our knowledge, this is the first report of nisin's MPC and MSW determination regarding canine enterococci.

Lipidomic study of cell lines reveals differences between breast cancer subtypes.

Breast cancer (BC) is the most prevalent type of cancer in women in western countries. BC mortality has not declined despite early detection by screening, indicating the need for better informed treatment decisions. Therefore, a novel noninvasive diagnostic tool for BC would give the opportunity of subtype-specific treatment and improved prospects for the patients. Heterogeneity of BC tumor subtypes is reflected in the expression levels of enzymes in lipid metabolism. The aim of the study was to investigate whether the subtype defined by the transcriptome is reflected in the lipidome of BC cell lines. A liquid chromatography mass spectrometry (LC-MS) platform was applied to analyze the lipidome of six cell lines derived from human BC cell lines representing different BC subtypes. We identified an increased abundance of triacylglycerols (TG) ≥ C-48 with moderate or multiple unsaturation in fatty acyl chains and down-regulated ether-phosphatidylethanolamines (PE) (C-34 to C-38) in cell lines representing estrogen receptor and progesterone receptor positive tumor subtypes. In a cell line representing HER2-overexpressing tumor subtype an elevated expression of TG (≤ C-46), phosphatidylcholines (PC) and PE containing short-chained (≤ C-16) saturated or monounsaturated fatty acids were observed. Increased abundance of PC ≥ C-40 was found in cell lines of triple negative BC subtype. In addition, differences were detected in lipidomes within these previously defined subtypes. We conclude that subtypes defined by the transcriptome are indeed reflected in differences in the lipidome and, furthermore, potentially biologically relevant differences may exist within these defined subtypes.

Two new spongian diterpene analogues isolated from the marine sponge Acanthodendrilla sp.

Two new marine natural compounds, 3ß-acetoxy-15-hydroxyspongia-12-en (1) and 3-methylspongia-3,12-dien-16-one (2), were isolated from the marine sponge Acanthodendrilla sp., collected in Pulau-Pulau. Compounds 1 and 2 represent new chemical entities of the known spongian diterpene family. Compound 1 is a new 3-acetoxyspongia and compound 2 presents an unreported rearranged 3-methylspongia-3-en. Their structures, including relative configurations, were fully elucidated based on 1D and 2D NMR analyses, as well as HRESTOFMS experiments. No significant bioactivities were found for these compounds. This work reports two new chemical structures, compounds 1 and 2, together with the first isolation of spongian diterpenes from Acanthodendrilla genus.

Monitoring Work Ability Index During a Two-Year Period Among Portuguese Municipality Workers.

In Portugal, little is known about the work ability profiles of municipal workers and their changes during working life. In order to characterize and understand the changes in work ability among municipal workers, a prospective study was designed to begin in 2015 in the municipality of Sintra, in the surroundings of Lisbon, and to collect data every two years. The present paper aims at characterizing the changes in the work ability of those workers between 2015 and 2017 and to identify the main predictors. Data collection was based on a questionnaire that encompassed socio-demographic data, the Copenhagen Psychosocial Questionnaire II (COPSOQ II), the Nordic questionnaire adapted, and the Work Ability Index (WAI). In this two-year period, the work ability of municipal workers decreased and the main predictive factors were age, lower-back pain, negative health perception, the presence of burnout, and making manual efforts. Still, there were factors that act as positive predictors of an excellent work ability, such as having training in the previous two years, a good sense of community at work, and a favorable meaning of work. In summary, the intervention strategies in the work field should take into consideration the main predictors of work ability that are relevant for each organization.

New Aromatic Bisabolane Derivatives with Lipid-Reducing Activity from the Marine Sponge Myrmekioderma sp.

The previously reported 1-(2,4-dihydroxy-5-methylphenyl)ethan-1-one (1), (1'Z)-2-(1',5'-dimethylhexa-1',4'-dieny1)-5-methylbenzene-1,4-diol (2), and 1,8-epoxy-1(6),2,4,7,10-bisaborapentaen-4-ol (5) together with four new structures of aromatic bisabolane-related compounds (3, 4, 6, 7) were isolated from the marine sponge Myrmekioderma sp. Compounds 1, 2, and 5 were identified based on spectral data available in the literature. The structures of the four new compounds were experimentally established by 1D and 2D-NMR and (-)-HRESIMS spectral analysis. Cytotoxic and lipid-reducing activities of the isolated compounds were evaluated. None of the isolated compounds were active against the tested cancer cell lines; however, lipid-reducing activity was found for compounds 2-5 and 7 in the zebrafish Nile red fat metabolism assay. This class of compounds should be further explored for their suitability as possible agents for the treatment of lipid metabolic disorders and obesity.

Identification of Cyanobacterial Strains with Potential for the Treatment of Obesity-Related Co-Morbidities by Bioactivity, Toxicity Evaluation and Metabolite Profiling.

Obesity is a complex disease resulting in several metabolic co-morbidities and is increasing at epidemic rates. The marine environment is an interesting resource of novel compounds and in particular cyanobacteria are well known for their capacity to produce novel secondary metabolites. In this work, we explored the potential of cyanobacteria for the production of compounds with relevant activities towards metabolic diseases using a blend of target-based, phenotypic and zebrafish assays as whole small animal models. A total of 46 cyanobacterial strains were grown and biomass fractionated, yielding in total 263 fractions. Bioactivities related to metabolic function were tested in different in vitro and in vivo models. Studying adipogenic and thermogenic gene expression in brown adipocytes, lipid metabolism and glucose uptake in hepatocytes, as well as lipid metabolism in zebrafish larvae, we identified 66 (25%) active fractions. This together with metabolite profiling and the evaluation of toxicity allowed the identification of 18 (7%) fractions with promising bioactivity towards different aspects of metabolic disease. Among those, we identified several known compounds, such as eryloside T, leptosin F, pheophorbide A, phaeophytin A, chlorophyll A, present as minor peaks. Those compounds were previously not described to have bioactivities in metabolic regulation, and both known or unknown compounds could be responsible for such effects. In summary, we find that cyanobacteria hold a huge repertoire of molecules with specific bioactivities towards metabolic diseases, which needs to be explored in the future.

Epidemiology and forensic aspects of carbon monoxide intoxication in Portugal: A three years' analysis.

The presented manuscript describes the carbon monoxide (CO) related deaths in Portugal over a period of 3 years, based on autopsies carried out at the National Institute of Legal Medicine and Forensic Sciences, from January 2012 to December 2014. Three hundred and forty-seven forensic autopsy reports with carboxyhaemoglobin (COHb) analysis requests were analysed and subdivided into three main groups: (1) improbable CO intoxication; (2) possible CO intoxication; (3) highly probable CO intoxication. In group 1, COHb analysis was negative, and the death circumstances, as well as the post mortem findings, didn't corroborate an exposition to CO. In group 2, with COHb positive in 1/3 of the cases, the death circumstances corroborated an exposition to CO, but the post mortem findings weren't enough to confirm an exposition to this substance. In group 3, the results of COHb were positive, and both circumstances of death and post mortem findings corroborated an exposition to CO. The first group (113 cases) had no specific suspicion of a CO intoxication and, thus, the request of a COHb analysis had no particular basis, reflected in the low COHb achieved percentage (between 0 and 12). In the second group (164 cases), 29% of the cases were directly or indirectly related to CO exposure (between 0% and 94%). In the third group (70 cases), 56 deaths were due to CO intoxication and 14 due to burns after CO inhalation (between 18% and 91%). This study intended to do, not only a 3-year assessment of CO poisoning, but also to enhance the fact that circumstantial information, as well as a correct evaluation at the forensic autopsy data are crucial, and allow an enhanced diagnosis of possible intoxication, as well as a better guidance for the consequent toxicological analysis requests.

Structure of Hierridin C, Synthesis of Hierridins B and C, and Evidence for Prevalent Alkylresorcinol Biosynthesis in Picocyanobacteria.

Small, single-celled planktonic cyanobacteria are ubiquitous in the world's oceans yet tend not to be perceived as secondary metabolite-rich organisms. Here we report the isolation and structure elucidation of hierridin C, a minor metabolite obtained from the cultured picocyanobacterium Cyanobium sp. LEGE 06113. We describe a simple, straightforward synthetic route to the scarcely produced hierridins that relies on a key regioselective halogenation step. In addition, we show that these compounds originate from a type III PKS pathway and that similar biosynthetic gene clusters are found in a variety of bacterial genomes, most notably those of the globally distributed picocyanobacteria genera Prochlorococcus, Cyanobium and Synechococcus.

Spectral and temporal auditory processing in the superior colliculus of aged rats.

Presbyacusis reflects dysfunctions present along the central auditory pathway. Given that the topographic representation of the auditory directional spatial map is deteriorated in the superior colliculus of aged animals, therefore, are spectral and temporal auditory processes altered with aging in the rat's superior colliculus? Extracellular single-unit recordings were conducted in the superior colliculus of anesthetized Sprague-Dawley adult (10 months) and aged (22 months) rats. In the spectral domain, level thresholds in aged rats were significantly increased when superior colliculus auditory neurons were stimulated with pure tones or Gaussian noise bursts. The sharpness of the frequency response tuning curve at 10 dB SPL above threshold was also significantly broader among the aged rats. Furthermore, in the temporal domain, the minimal silent gap thresholds to Gaussian noises were significantly longer in aged rats. Hence, these results highlight that spectral and temporal auditory processing in the superior colliculus are impaired during aging.

Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016-2017).

We report the detection of rabbit haemorrhagic disease virus 2 (RHDV2) in the Madeira archipelago, Portugal. Viral circulation was confirmed by RT-qPCR and vp60 sequencing. Epidemiological data revealed the outbreak initiated in October 2016 in Porto Santo affecting wild and domestic rabbits. It was then detected three months later on the island of Madeira. Five haplotypes were identified and a genetic overall similarity of 99.54 to 99.89% was observed between the two viral populations. Unique single nucleotide polymorphisms were recognised in the Madeira archipelago strains, two of which resulting in amino acid substitutions at positions 480 and 570 in the VP60 protein. Phylogenetic investigation by Maximum Likelihood showed all the vp60 sequences from the Madeira archipelago group together with high bootstraps. The analysis also showed that the Madeira archipelago strains are closely related to the strains detected in the south of mainland Portugal in 2016, suggesting a possible introduction from the mainland. The epidemiological data and high genetic similarity indicate a common source for the Porto Santo and Madeira RHDV2 outbreaks. Human activity related to hunting was most probably at the origin of the Madeira outbreak.