Tracking lipid synthesis using 2H2O and 2H-NMR spectroscopy in black soldier fly (Hermetia illucens) larvae fed with macroalgae.

Black soldier fly (Hermetia illucens) larvae are used to upcycle biowaste into insect biomass for animal feed. Previous research on black soldier fly has explored the assimilation of dietary fatty acids (FAs), but endogenous FA synthesis and modification remain comparatively unexplored. This study presents a 1H/2H-NMR methodology for measuring lipid synthesis in black soldier fly larvae using diluted deuterated water (2H2O) as a stable isotopic tracer delivered through the feeding media. This approach was validated by measuring 2H incorporation into the larvae's body water and consequent labelling of FA esterified into triacylglycerols. A 5% 2H enrichment in the body water, adequate to label the FA, is achieved after 24 h in a substrate with 10% 2H2O. A standard feeding trial using an invasive macroalgae was designed to test this method, revealing de novo lipogenesis was lower in larvae fed with macroalgae, probably related to the poor nutritional value of the diet.

Tracing the Impact of Domestic Storage Conditions on Antioxidant Activity and Lipid Profiles in the Edible Microalgae Chlorella vulgaris and Tetraselmis chui.

The microalgae Chlorella vulgaris and Tetraselmis chui are valued for their nutrient-rich content, including lipids and polyunsaturated fatty acids (PUFA). However, little is known about how storage and processing affect their lipid quality. This study aimed to assess the impact of domestic storage and cooking practices in dried biomass of C. vulgaris and T. chui. Four conditions were tested: control (newly opened package), light (storage at room temperature and daily light regimen for three weeks), frozen (storage in the freezer at -20 °C for three weeks), and heated (three cycles of 90 min at 100 °C). Lipid extracts were analyzed by GC-MS and LC-MS, and antioxidant activity through DPPH and ABTS radical scavenging assays. Tested storage conditions promoted a decrease in fatty acid content and in diacyl/lyso lipid species ratios of phospholipid (PC/LPC, PE/LPE) and betaine lipids (DGTS/MGTS). Lipid extracts from light treatment showed the lowest antioxidant activity in C. vulgaris (ABTS, IC40: 104.9; DPPH, IC20: 187.9 ± 15.0), while heat affected the antioxidant activity of T. chui (ABTS, IC40: 88.5 ± 2.8; DPPH, IC20 209.4 ± 10.9). These findings underscore the impact of managing storage and processing conditions to optimize the nutritional and functional benefits of C. vulgaris and T. chui in food and feed applications.

Fatty Acid Profile and Escherichia coli and Salmonella sp. Load of Wild-Caught Seaweed Fly Fucellia maritima (Haliday, 1838) (Diptera: Anthomyiidae).

World aquaculture is expected to continue to grow over the next few decades, which amplifies the need for a higher production of sustainable feed ingredients for aquatic animals. Insects are considered good candidates for aquafeed ingredients because of their ability to convert food waste into highly nutritional biomass. However, commercially available terrestrial insect species lack n-3 long-chain polyunsaturated fatty acids (LC-PUFAs), which are essential biomolecules for marine cultured species. Nevertheless, several coastal insect species feature LC-PUFAs in their natural fatty acid (FA) profile. Here, we analysed the lipidic profile of wild-caught seaweed fly Fucellia maritima, with a focus on their FA profile, to evaluate its potential to be used as an aquafeed ingredient, as well as to screen for the presence of pathogenic bacteria. Results showed that the flies had a total lipid content of 13.2% of their total dry weight. The main classes of phospholipids (PLs) recorded were phosphatidylethanolamines (PEs) (60.8%), followed by phosphatidylcholine (PC) (17.1%). The most abundant FA was palmitoleic acid (C16:0) with 34.9% ± 4.3 of total FAs, followed by oleic acid (C18:1) with 30.4% ± 2.3. The FA composition of the flies included essential fatty acids (EFAs) for both freshwater fish, namely linoleic acid (C18:2 n-6) with 3.4% ± 1.3 and alpha-linoleic acid (C18:3 n-3) with 3.4% ± 1.9, and marine fish, namely arachidonic acid (C20:4 n-6) with 1.1% ± 0.3 and eicosapentaenoic acid (C20:5 n-3) with 6.1% ± 1.2. The microbiological analysis found 9.1 colony-forming units per gram (CFU/g) of Enterobacteriaceae and no presence of Salmonella sp. was detected in a sample of 25 g of fresh weight. These findings indicate that Fucellia maritima biomass holds the potential to be used as an additional aquafeed ingredient due to its FA profile and the low count of pathogenic bacteria, which can contribute to the optimal growth of fish and shrimp with a low risk of pathogen transfer during the feed production chain.

Plasma lipidomics analysis reveals altered profile of triglycerides and phospholipids in children with Medium-Chain Acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most prevalent mitochondrial fatty acid ß-oxidation disorder. In this study, we assessed the variability of the lipid profile in MCADD by analysing plasma samples obtained from 25 children with metabolically controlled MCADD (following a normal diet with frequent feeding and under l-carnitine supplementation) and 21 paediatric control subjects (CT). Gas chromatography-mass spectrometry was employed for the analysis of esterified fatty acids, while high-resolution C18-liquid chromatography-mass spectrometry was used to analyse lipid species. We identified a total of 251 lipid species belonging to 15 distinct lipid classes. Principal component analysis revealed a clear distinction between the MCADD and CT groups. Univariate analysis demonstrated that 126 lipid species exhibited significant differences between the two groups. The lipid species that displayed the most pronounced variations included triacylglycerols and phosphatidylcholines containing saturated and monounsaturated fatty acids, specifically C14:0 and C16:0, which were found to be more abundant in MCADD. The observed changes in the plasma lipidome of children with non-decompensated MCADD suggest an underlying alteration in lipid metabolism. Therefore, longitudinal monitoring and further in-depth investigations are warranted to better understand whether such alterations are specific to MCADD children and their potential long-term impacts.

Bioconversion of olive oil pomace by black soldier fly increases eco-efficiency in solid waste stream reduction producing tailored value-added insect meals.

Olive oil is one of the most important agricultural products in Mediterranean areas, and currently the European Union is the largest producer. Due to technological innovations, Portugal has become one of the main olive oil producing countries over the last few years, accompanied by large amounts of olive oil pomace (OOP), the most representative residue of the olive oil extraction process. This is causing serious waste management problems since current management solutions also present environmental impacts. Here we explored the black soldier fly (Hermetia illucens) potential to biotransform OOP into valuable insect meals by feeding them OOP-based diets as substrates. Results show that despite survival rates not being affected by higher replacement (75% and 50%) levels of OOP, there was an increase in larval instar duration. Substrate reduction was significantly lower for higher replacement levels but was not affected up to the 50% replacement level. Feed conversion rate differed among all the treatments, increasing as the replacement level increased, while bioconversion rate, which also differed among all the treatments, decreased as replacement level increased. Differences in larval protein content were only seen at higher replacement levels (75%), with an increase in protein content for replacements of up to 25%. One of the most striking results was the change in fatty acid profile, which became more abundant in monounsaturated fatty acids (mostly oleic acid) as the olive pomace replacement levels increased in comparison with the control substrate, rich in saturated fatty acids (palmitic acid). These results show that BSF can be an effective OOP bioconversion agent, and resulting insect meals can be used as alternatives to currently available saturated fatty acid insect meals.

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.

Bioconversion and performance of Black Soldier Fly (Hermetia illucens) in the recovery of nutrients from expired fish feeds.

In modern aquaculture systems, feed is the main source of the waste being produced, including expired aquafeeds. There is a link between the expiration date of aquafeeds enriched with fish oil for marine fish and the observation of several physical and microbiological changes. Among these, lipid oxidation is worth highlighting, as this process is responsible for the loss of palatability of aquafeeds, which can lead to feeding rejection by the species being farmed. In this study, we used an expired fish aquafeed, which otherwise would be discarded as waste, as a substrate to feed Black Soldier Fly (BSF) larvae. Different replacement levels of expired aquafeed were used which unravelled the amount of n-3 fatty acids added to larval tissues of BSF larvae after 2, 7, and 10 days of feeding. Our results also showed that shorter trials and higher diet replacement levels induced a deleterious effect on final larval weight. Furthermore, amino acid and fatty acid larval contents were shaped by the supplied diet, with results supporting the inclusion of BSF meal in aquafeeds, due to the levels of lysine (5.6-8.9%), methionine (1.9-3.2%), and omega-3 fatty acids (14.5%) recorded. These results demonstrate that the re-introduction of an expired resource aiming to diversify the source of aquafeeds raw materials can be safely achieved through BSF biotransformation. Overall, BSF larvae can successfully recover important nutrients for aquafeeds targeting marine species and foster the production of value-added insects under a circular bioeconomy framework.

Plasma Phospholipidomic Profile Differs between Children with Phenylketonuria and Healthy Children.

Phenylketonuria (PKU) is a disease of the catabolism of phenylalanine (Phe), caused by an impaired function of the enzyme phenylalanine hydroxylase. Therapeutics is based on the restriction of Phe intake, which mostly requires a modification of the diet. Dietary restrictions can lead to imbalances in specific nutrients, including lipids. In the present study, the plasma phospholipidome of PKU and healthy children (CT) was analyzed by hydrophilic interaction liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Using this approach, 187 lipid species belonging to nine different phospholipid classes and three ceramides were identified. Principal component analysis of the lipid species data set showed a distinction between PKU and CT groups. Univariate analysis revealed that 146 species of phospholipids were significantly different between both groups. Lipid species showing significant variation included phosphatidylcholines, containing polyunsaturated fatty acids (PUFA), which were more abundant in PKU. The high level of PUFA-containing lipid species in children with PKU may be related to a diet supplemented with PUFA. This study was the first report comparing the plasma polar lipidome of PKU and healthy children, highlighting that the phospholipidome of PKU children is significantly altered compared to CT. However, further studies with larger cohorts are needed to clarify whether these changes are specific to phenylketonuric children.

Evaluation of MicroRNA Delivery In Vivo.

MicroRNAs (miRNAs) are a family of short noncoding RNA molecules that fine-tune expression of mRNAs. Often their altered expression is associated with a number of diseases, including cancer. Given that miRNAs target multiple genes and "difficult to drug" oncogenes, they present attractive candidates to manipulate as an anti-cancer strategy. MicroRNA-7 (miR-7) is a tumor suppressor miRNA that has been shown to target oncogenes overexpressed in cancers, such as the epidermal growth factor receptor (EGFR) and the nuclear factor-κ B subunit, RelA. Here, we describe methods for evaluating systemic delivery of miR-7 using a lipid nanoparticle formulation in an animal model. The microRNA is delivered three times, over 1 week and tissues collected 24 h after the last injection. RNA and protein are extracted from snap frozen tissues and processed to detect miRNA distribution and subsequent assessment of downstream targets and signaling mediators, respectively. Importantly, variability in efficiency of miRNA delivery will be observed between organs of the same animal and also between animals. Additionally, delivering the microRNA to organs other than the liver, particularly the brain, remains challenging. Furthermore, large variation in miRNA targets is seen both within tissues and across tissues depending on the lysis buffer used for protein extraction. Therefore, analyzing protein expression is dependent upon the method used for isolation and requires optimization for each individual application. Together, these methods will provide a foundation for those planning on assessing the efficacy of delivery of a miRNA in vivo.

Characterization of a mouse model of hyperglycemia and retinal neovascularization.

One of the limitations of research into diabetic retinopathy is the lack of suitable animal models. To study how the two important factors--hyperglycemia and vascular endothelial growth factor--interact in diabetic retinopathy, the Akimba mouse (Ins2AkitaVEGF+/-) was generated by crossing the Akita mouse (Ins2Akita) with the Kimba mouse (VEGF+/+). C57Bl/6 and the parental and Akimba mouse lines were characterized by biometric measurements, histology, immunohistochemistry, and Spectralis Heidelberg retinal angiography and optical coherence tomography. The Akimba line not only retained the characteristics of the parental strains, such as developing hyperglycemia and retinal neovascularization, but developed higher blood glucose levels at a younger age and had worse kidney-body weight ratios than the Akita line. With aging, the Akimba line demonstrated enhanced photoreceptor cell loss, thinning of the retina, and more severe retinal vascular pathology, including more severe capillary nonperfusion, vessel constriction, beading, neovascularization, fibroses, and edema, compared with the Kimba line. The vascular changes were associated with major histocompatibility complex class II+ cellular staining throughout the retina. Together, these observations suggest that hyperglycemia resulted in higher prevalences of edema and exacerbated the vascular endothelial growth factor-driven neovascular and retinal changes in the Akimba line. Thus, the Akimba line could become a useful model for studying the interplay between hyperglycemia and vascular endothelial growth factor and for testing treatment strategies for potentially blinding complications, such as edema.

Análise das variaçöes dos tempos de exposiçäo e de reveleçäo dos filmes periapicais Kodak (Ektaspeed) e da agfa-gevaert (dentus M-2) / Analysis of the time variations of exposure and revelations of periapical Kodak (Ektaspeed) e Agfa-Gevaert (Dentus M-2)

Avaliaçäo dos efeitos das variaçöes nos tempos de exposiçäo e de revelaçäo nos filmes radiográficos periapicais Kodak EP-21 e Agfa-Gevaert, quando processados no Revelador Kodak para Raios X Dental na temperatura de 20-C. A metodologia utilizada foi a da análise subjetiva, executada por 30 examinadores (cirurgiöes-dentistas e técnicos em Radiologia). Verificou-se, pelos resultados apresentados, que o maior número de radiografias consideradas satisfatórias foi do filme Agfa-Pevaert. Os dois tipos de filmes, quando expostos corretamente, produzem radiografias boas para diagnóstico, na opiniäo da maioria dos examinadores. Os filmes superexpostos e sub-revelados produzem imagem radiográfica aceitável, o que também é observado, mas em menor intensidade, pelos examinadores, nos filmes subexpostos e super-revelados