Polar Lipids of Marine Microalgae Nannochloropsis oceanica and Chlorococcum amblystomatis Mitigate the LPS-Induced Pro-Inflammatory Response in Macrophages.

Microalgae are recognized as a relevant source of bioactive compounds. Among these bioactive products, lipids, mainly glycolipids, have been shown to present immunomodulatory properties with the potential to mitigate chronic inflammation. This study aimed to evaluate the anti-inflammatory effect of polar lipids isolated from Nannochloropsis oceanica and Chlorococcum amblystomatis. Three fractions enriched in (1) digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG), (2) monogalactosyldiacylglycerol (MGDG), and (3) diacylglyceryl-trimethylhomoserine (DGTS) and phospholipids (PL) were obtained from the total lipid extracts (TE) of N. oceanica and C. amblystomatis, and their anti-inflammatory effect was assessed by analyzing their capacity to counteract nitric oxide (NO) production and transcription of pro-inflammatory genes Nos2, Ptgs2, Tnfa, and Il1b in lipopolysaccharide (LPS)-activated macrophages. For both microalgae, TE and Fractions 1 and 3 strongly inhibited NO production, although to different extents. A strong reduction in the LPS-induced transcription of Nos2, Ptgs2, Tnfa, and Il1b was observed for N. oceanica and C. amblystomatis lipids. The most active fractions were the DGTS-and-PL-enriched fraction from N. oceanica and the DGDG-and-SQDG-enriched fraction from C. amblystomatis. Our results reveal that microalgae lipids have strong anti-inflammatory capacity and may be explored as functional ingredients or nutraceuticals, offering a natural solution to tackle chronic inflammation-associated diseases.

Maternal high-fat diet programs white and brown adipose tissue lipidome and transcriptome in offspring in a sex- and tissue-dependent manner in mice.

OBJECTIVE: The prevalence of overweight and obesity among children has drastically increased during the last decades and maternal obesity has been demonstrated as one of the ultimate factors. Nutrition-stimulated transgenerational regulation of key metabolic genes is fundamental to the developmental origins of the metabolic syndrome. Fetal nutrition may differently influence female and male offspring. METHODS: Mice dam were fed either a control diet or a high-fat diet (HFD) for 6-week prior mating and continued their respective diet during gestation and lactation. At weaning, female and male offspring were fed the HFD until sacrifice. White (WAT) and brown (BAT) adipose tissues were investigated in vivo by nuclear magnetic resonance at two different timepoints in life (midterm and endterm) and tissues were collected at endterm for lipidomic analysis and RNA sequencing. We explored the sex-dependent metabolic adaptation and gene programming changes by maternal HFD in visceral AT (VAT), subcutaneous AT (SAT) and BAT of offspring. RESULTS: We show that the triglyceride profile varies between adipose depots, sexes and maternal diet. In female offspring, maternal HFD remodels the triglycerides profile in SAT and BAT, and increases thermogenesis and cell differentiation in BAT, which may prevent metabolic complication later in life. Male offspring exhibit whitening of BAT and hyperplasia in VAT when born from high-fat mothers, with impaired metabolic profile. Maternal HFD differentially programs gene expression in WAT and BAT of female and male offspring. CONCLUSION: Maternal HFD modulates metabolic profile in offspring in a sex-dependent manner. A sex- and maternal diet-dependent gene programming exists in VAT, SAT, and BAT which may be key player in the sexual dimorphism in the metabolic adaptation later in life.

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.

Ethanol Extraction of Polar Lipids from Nannochloropsis oceanica for Food, Feed, and Biotechnology Applications Evaluated Using Lipidomic Approaches.

Nannochloropsis oceanica can accumulate lipids and is a good source of polar lipids, which are emerging as new value-added compounds with high commercial value for the food, nutraceutical, and pharmaceutical industries. Some applications may limit the extraction solvents, such as food applications that require safe food-grade solvents, such as ethanol. However, the effect of using ethanol as an extraction solvent on the quality of the extracted polar lipidome, compared to other more traditional methods, is not yet well established. In this study, the polar lipid profile of N. oceanica extracts was obtained using different solvents, including chloroform/methanol (CM), dichloromethane/methanol (DM), dichloromethane/ethanol (DE), and ethanol (E), and evaluated by modern lipidomic methods using LC-MS/MS. Ultrasonic bath (E + USB)- and ultrasonic probe (E + USP)-assisted methodologies were implemented to increase the lipid extraction yields using ethanol. The polar lipid signature and antioxidant activity of DM, E + USB, and E + USP resemble conventional CM, demonstrating a similar extraction efficiency, while the DE and ethanol extracts were significantly different. Our results showed the impact of different extraction solvents in the polar lipid composition of the final extracts and demonstrated the feasibility of E + USB and E + USP as safe and food-grade sources of polar lipids, with the potential for high-added-value biotechnological applications.

Potential Anti-Obesity, Anti-Steatosis, and Anti-Inflammatory Properties of Extracts from the Microalgae Chlorella vulgaris and Chlorococcum amblystomatis under Different Growth Conditions.

Microalgae are known as a producer of proteins and lipids, but also of valuable compounds for human health benefits (e.g., polyunsaturated fatty acids (PUFAs); minerals, vitamins, or other compounds). The overall objective of this research was to prospect novel products, such as nutraceuticals from microalgae, for application in human health, particularly for metabolic diseases. Chlorella vulgaris and Chlorococcum amblystomatis were grown autotrophically, and C. vulgaris was additionally grown heterotrophically. Microalgae biomass was extracted using organic solvents (dichloromethane, ethanol, ethanol with ultrasound-assisted extraction). Those extracts were evaluated for their bioactivities, toxicity, and metabolite profile. Some of the extracts reduced the neutral lipid content using the zebrafish larvae fat metabolism assay, reduced lipid accumulation in fatty-acid-overloaded HepG2 liver cells, or decreased the LPS-induced inflammation reaction in RAW264.7 macrophages. Toxicity was not observed in the MTT assay in vitro or by the appearance of lethality or malformations in zebrafish larvae in vivo. Differences in metabolite profiles of microalgae extracts obtained by UPLC-LC-MS/MS and GNPS analyses revealed unique compounds in the active extracts, whose majority did not have a match in mass spectrometry databases and could be potentially novel compounds. In conclusion, microalgae extracts demonstrated anti-obesity, anti-steatosis, and anti-inflammatory activities and could be valuable resources for developing future nutraceuticals. In particular, the ultrasound-assisted ethanolic extract of the heterotrophic C. vulgaris significantly enhanced the anti-obesity activity and demonstrated that the alteration of culture conditions is a valuable approach to increase the production of high-value compounds.

Fertility preservation with successful pregnancy outcome in a patient with transplanted heart and non-Hodgkin's lymphoma - a case report.

BACKGROUND: Fertility preservation must be discussed with reproductive age women before cancer treatment. Heart transplantation raises complex issues in pregnancy. Pregnancy in a heart transplant woman after pelvic irradiation involves close multidisciplinary follow-up to avoid complications in the mother and the foetus. We report the first live birth in a heart transplant woman after pelvic irradiation, chemotherapy and fertility preservation. CASE PRESENTATION: A 36-year-old heart transplant woman with pelvic non-Hodgkin lymphoma spared her fertility, with cryopreservation of oocytes and embryos, before chemotherapy and pelvic irradiation. After multidisciplinary discussion and pre-conception evaluation, pregnancy was achieved. A close follow-up by a multidisciplinary team allowed a normal pregnancy without maternal or foetal complications and the delivery of a healthy infant. CONCLUSIONS: Achieving pregnancy in heart transplant women with iatrogenic ovarian failure after oncologic treatment including pelvic irradiation is possible and can be successful. Careful and close surveillance by a multidisciplinary team is mandatory due to increased risk of maternal and foetal complications.

A Rare Cause of Vitamin B12 Deficiency in the Pediatric Age.

Vitamin B12 plays an important role in DNA synthesis, hematopoiesis, and neuronal mechanisms, and its deficiency can be associated with insufficient intake or poor absorption and autoimmune and genetic diseases. Although rare, excessive, and chronic alcohol consumption may also justify a deficiency in this vitamin. We present a case of a 17-year-old Iraqi adolescent refugee in Portugal with a deficiency of vitamin B12 due to excessive and chronic alcohol consumption. Although intramuscular (IM) supplementation with cyanocobalamin is the most used, it was decided to initiate oral supplementation. After four months, vitamin B12 levels were replenished. With this clinical case, we aim to raise awareness of the cause of cyanocobalamin deficiency, which although rare in pediatric age should be considered in adolescents, given the possibility of risky behaviors in this age group. Additionally, given the therapeutic options, we have found that oral administration of vitamin B12 has been effective, as reported in the literature.

From stigma to increased social acceptance? Living with Machado-Joseph disease in São Miguel, Azores, Portugal.

This study describes the experiences with the stigma attached to Machado-Joseph disease (MJD) in São Miguel Island, the Azores (Portugal). We draw on semi-structured interviews with persons with MJD, family members, healthcare professionals, and direct care providers recruited through the local patient's association (n = 28). Qualitative thematic analysis revealed three main themes: (i) the intense stigma associated with MJD in the past; (ii) the current tendency towards increased openness; and (iii) increased availability of information about MJD and support. The findings suggest that stigmatization was more frequent and intense in the past. Still, there is currently a decrease in the intensity of perceived stigma, accompanied by an increasing awareness about MJD within the community. The local patient's association is noted for playing a pivotal role in raising awareness about MJD in the community and fostering the confidence of individuals with MJD and their families to engage socially, which may help to reduce or mitigate feelings of stigma. This raises questions about whether the diminished stigma towards MJD in São Miguel results from heightened awareness about the condition, a decrease in the social acceptability of stigma, or a gradual internalization and normalization of stigma among individuals with MJD as a coping mechanism.

Nonalcoholic Fatty Liver Disease and Continuous Metabolic Syndrome in Adolescents with Overweight/Obesity.

INTRODUCTION: Nonalcoholic fatty liver disease is the leading cause of pediatric chronic liver disease. Although nonalcoholic fatty liver disease is closely associated with obesity, its relationship with metabolic syndrome in children is not fully understood. The main aim of this study was to evaluate the association between nonalcoholic fatty liver disease and a combination of cardiometabolic risk factors in adolescents with overweight/obesity, using a pediatric metabolic syndrome score (PsiMS) to predict metabolic syndrome. METHODS: A retrospective cohort study was conducted. Subjects with overweight/obesity aged 10 to 17 followed at two clinical centers in Portugal (2018 - 2021) were enrolled. The independent association of nonalcoholic fatty liver disease with PsiMS, and of other potential predictors, was tested through multiple regression analyses. Receiver operator characteristic curve analysis was performed to estimate the optimal cutoff of PsiMS to discriminate metabolic syndrome. RESULTS: Eighty-four subjects were included (median age at baseline 11.5 years). The prevalence rate of nonalcoholic fatty liver disease was 51% and the prevalence rate of metabolic syndrome was 7%. The mean PsiMS was 2.05 ± 0.48 at the first evaluation, and 2.11 ± 0.52 at the last evaluation (mean follow-up time was 15 months). The nonalcoholic fatty liver disease group had significantly (p < 0.05) higher weight and body mass index z-scores, higher rate of severe obesity and higher waist circumference percentile. PsiMS was highly accurate in predicting metabolic syndrome (area under the curve = 0.96), with an optimal cutoff of 2.46 (sensitivity 100%, specificity 89%). In the univariate analysis, no statistically significant association was observed between nonalcoholic fatty liver disease and PsiMS. In the multiple regression analysis, female sex had a negative association with PsiMS (first and last evaluation). Independent predictors of a higher PsiMS at first evaluation were: ≥ 2 metabolic syndrome criteria, body mass index z-score, insulin resistance and dyslipidemia. At the last evaluation, independent predictors of a higher PsiMS were: nonalcoholic fatty liver disease, baseline PsiMS and body mass index increase from baseline. CONCLUSION: The results suggest a good performance of the PsiMS to assess metabolic syndrome and that nonalcoholic fatty liver disease is associated with PsiMS at follow-up.

Adapting the Nominal Group Technique to a virtual version: an experience report.

OBJECTIVE: To report on the adaptations made to the original Nominal Group Technique (NGT), allowing it to be applied to the virtual format, preserving all its key elements. METHOD: An experience report on the adaptations and adjustments made to the original NGT to the virtual format using Information and Communication Technologies (ICT), using digital tools that are available free of charge or are low cost and easy to use. RESULTS: The NGT was carried out entirely virtually and underwent adaptations in each of its four stages through the incorporation of specific digital resources. It was possible to present the most voted ideas and obtain final approval from the participants. The participants had no difficulty in using the virtual resources provided and, based on the reaction evaluation, they were satisfied with the tools provided. CONCLUSION: The adapted NGT proved to be an effective method when used in a virtual setting, capable of producing a significant number of ideas and developing consensus. The adapted tool can be used by other researchers in countries with similar resources or dimensions to Brazil.

Epidemiological and Clinical Aspects of Cutaneous and Mucosal Leishmaniases in Portugal: Retrospective Analysis of Cases Diagnosed in Public Hospitals and Reported in the Literature between 2010 and 2020.

Leishmania infantum, a zoonotic vector-born parasite, is endemic in the Mediterranean region, presenting mostly as visceral (VL), but also as cutaneous (CL) and mucosal leishmaniasis (ML). This study aimed to describe the epidemiological and clinical aspects of the CL and ML cases diagnosed in mainland Portugal between 2010 and 2020. Collaboration was requested from every hospital of the Portuguese National Health System. Cases were screened through a search of diagnostic discharge codes or positive laboratory results for Leishmania infection. Simultaneously, a comprehensive literature search was performed. Descriptive statistics and hypothesis testing were performed using IBM® SPSS® Statistics. A total of 43 CL and 7 ML cases were identified, with a predominance of autochthonous cases (86%). In CL, immunosuppressed individuals constituted a significant proportion of patients (48%), and in this group, disseminated CL (22%) and simultaneous VL (54%) were common. In autochthonous cases, lesions, mostly papules/nodules (62%), were frequently observed on the head (48%). The approach to treatment was very heterogeneous. ML cases were all autochthonous, were diagnosed primarily in older immunosuppressed individuals, and were generally treated with liposomal amphotericin B. The findings suggest a need for enhanced surveillance and reporting, clinical awareness, and diagnostic capacity of these forms of leishmaniasis to mitigate underdiagnosis and improve patient outcomes. A holistic One Health approach is advocated to address the multifaceted challenges posed by leishmaniases in Portugal and beyond.

Differences and Similarities in Lipid Composition, Nutritional Value, and Bioactive Potential of Four Edible Chlorella vulgaris Strains.

The microalga Chlorella vulgaris is a popular food ingredient widely used in the industry, with an increasing market size and value. Currently, several edible strains of C. vulgaris with different organoleptic characteristics are commercialized to meet consumer needs. This study aimed to compare the fatty acid (FA) and lipid profile of four commercialized strains of C. vulgaris (C-Auto, C-Hetero, C-Honey, and C-White) using gas- and liquid-chromatography coupled to mass-spectrometry approaches, and to evaluate their antioxidant and anti-inflammatory properties. Results showed that C-Auto had a higher lipid content compared to the other strains and higher levels of omega-3 polyunsaturated FAs (PUFAs). However, the C-Hetero, C-Honey, and C-White strains had higher levels of omega-6 PUFAs. The lipidome signature was also different between strains, as C-Auto had a higher content of polar lipids esterified to omega-3 PUFAs, while C-White had a higher content of phospholipids with omega-6 PUFAs. C-Hetero and C-Honey showed a higher content of triacylglycerols. All extracts showed antioxidant and anti-inflammatory activity, highlighting C-Auto with greater potential. Overall, the four strains of C. vulgaris can be selectively chosen as a source of added-value lipids to be used as ingredients in food and nutraceutical applications for different market needs and nutritional requirements.

Short Communication: Restrictions in care following the COVID-19 pandemic severely impacted Machado-Joseph disease patients: a study in the Azores Islands, Portugal.

This qualitative study describes how the restrictions imposed by the COVID-19 pandemic impacted on Machado-Joseph disease (MJD) patients and their care, in the island of São Miguel (the Azores, Portugal). In-person semi-structured interviews were conducted with 11 participants, including patients, family members, healthcare professionals, and care providers. Main findings highlighted the key role played by the local association in psychosocial and healthcare for MJD patients and families, and the adverse effects on their care following the onset of the COVID-19 pandemic. In particular, hindered access to the day-care centre increased isolation and had a negative impact on mental health and disease progression. For persons with a progressive and severe neurological disease, there is no "back to normal." Future restrictive measures ensuing need to be accompanied by a careful definition of daily care routines for patients.

Short Communication: Restrictions in care following the COVID-19 pandemic severely impacted Machado-Joseph disease patients: a study in the Azores Islands, Portugal.

This qualitative study describes how the restrictions imposed by the COVID-19 pandemic impacted on Machado-Joseph disease (MJD) patients and their care, in the island of São Miguel (the Azores, Portugal). In-person semi-structured interviews were conducted with 11 participants, including patients, family members, healthcare professionals, and care providers. Main findings highlighted the key role played by the local association in psychosocial and healthcare for MJD patients and families, and the adverse effects on their care following the onset of the COVID-19 pandemic. In particular, hindered access to the day-care centre increased isolation and had a negative impact on mental health and disease progression. For persons with a progressive and severe neurological disease, there is no "back to normal." Future restrictive measures ensuing need to be accompanied by a careful definition of daily care routines for patients.

Applications of lipidomics in marine organisms: progress, challenges and future perspectives.

Marine ecosystems comprise a high diversity of life forms, such as algae, invertebrates, and vertebrates. These organisms have adapted their physiology according to the conditions of the environments in which they inhabit. In the last few years, the study of lipids of marine origin has received increasing attention due to the diversity of their composition. The progress of analytical techniques such as LC-MS and MS/MS has allowed researchers to improve accurate processing of samples and lipid characterization. Lipidomics is a useful research field to understand marine ecosystems and the physiology of their organisms. The screening of biological systems in marine environments has demonstrated a significant diversity of lipids in their living resources. In this review, we provide the state-of-the-art marine of lipidomics studies. We describe in detail the lipidomics approach for the analysis of marine lipidomes, including sample collection and preparation, interpretation of MS/MS fragmentation fingerprinting, data analysis and processing. Special attention is also given to illustrate the latest applications and advances of applied LC-MS lipidomic analyses in diversified studies of different marine organisms, as well as the challenges and future perspectives of marine lipidomics.

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.

Bioprospecting antibiotic properties in photodynamic therapy of lipids from Codium tomemtosum and Chlorella vulgaris.

The growing resistance from pathogens against antibiotics has increased the research for new compounds and strategies with antibacterial potential. Lipids from algae are emerging as natural and potent bioactive molecules with interesting results regarding the inactivation of bacteria, viruses, and fungi. The combination of algae lipids with innovative strategies, such as antibacterial photodynamic therapy (aPDT) can enhance their antimicrobial potential. In this work, we aimed to evaluate the antibacterial potential in aPDT of total lipid extracts and polar lipid fractions from the green macroalga, Codium tomentosum, and the green microalga, Chlorella vulgaris on a Gram-positive bacteria Staphylococcus aureus. Total lipid extracts and polar lipid fractions were characterized by LC-MS. The results revealed that the total extracts of algae promote S. aureus inhibition after light irradiation, with a decrease of ca. 6 log10 (CFU/mL) after 15 min of treatment with both extracts of algae. The polar lipid fractions, composed by phospholipids, glycolipids and betaine lipids, from C. tomentosum and C. vulgaris also revealed antibacterial potential in combination with aPDT, but a decrease of ca. 6 log10 (CFU/mL) was reached at 60 min of treatment, later than with the total extracts. These results unveil algae lipids as antibacterial compounds in combination with aPDT displaying an alternative from natural origin to tackle pathogen resistance.

Molecular programming modulates hepatic lipid metabolism and adult metabolic risk in the offspring of obese mothers in a sex-specific manner.

Male and female offspring of obese mothers are known to differ extensively in their metabolic adaptation and later development of complications. We investigate the sex-dependent responses in obese offspring mice with maternal obesity, focusing on changes in liver glucose and lipid metabolism. Here we show that maternal obesity prior to and during gestation leads to hepatic steatosis and inflammation in male offspring, while female offspring are protected. Females from obese mothers display important changes in hepatic transcriptional activity and triglycerides profile which may prevent the damaging effects of maternal obesity compared to males. These differences are sustained later in life, resulting in a better metabolic balance in female offspring. In conclusion, sex and maternal obesity drive differently transcriptional and posttranscriptional regulation of major metabolic processes in offspring liver, explaining the sexual dimorphism in obesity-associated metabolic risk.

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.