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In the present work, chemical and enzymatic assisted techniques were compared for protein extraction from lesser mealworm larvae (LM, Alphitobius diaperinus), recently approved as a novel food in the European Union. All extracts showed appreciable nutritional quality, with quantities of essential amino acids above the reference standard. Conventional alkali extraction allowed the isolation of only 73% of the protein, preserving the amino acid composition but potentially causing denaturation or racemisation. The “stepwise” method, following the Osborne fractionation, improved protein recovery to 91% by isolating four fractions with different solubility properties. Additionally, enzymatic hydrolysis using Bacillus licheniformis proteases was also tested, and it provided hydrolysates with an average degree of hydrolysis of 14%, making them a potential hypoallergenic solution. Overall, these findings indicate the ability to tailor the composition of LM protein to meet specific needs, offering promising prospects for the use of insect protein ingredients in various applications.

Despite the advent of “methylomic” analyses, bisulfite modification of genomic DNA followed by Sanger sequencing remains the most precise assay for investigating the methylation profile of specific sequences at single-cytosine resolution. We and others highlighted the possibility that cytosine methylation outside the canonical CpG dinucleotides—indicated as “non-CpG” or “CpH” methylation—can be significantly present, particularly in brain cells and tissues and that these non-CpG methyl-groups also display a functional role in modulating gene expression. The sequencing output obtained after bisulfite assay needs to be compared to the reference sequence to identify the modified cytosines. This task can be performed through software-assisted analysis but, so far, limitedly at the CpG moieties. To address this gap, the MethPy software has been developed to analyze non-CpG methylation profiles in an automated manner. MethPy was programmed in Python and enables the comparison of bisulfite-modified sequences with their corresponding genomic ones. The results are immediately showed with the option to show multiple output formats (text, tables, and graphs). To our knowledge, this is the first tool that allows the analysis of non-CpG methylation at single-base resolution and may contribute to streamlining and accelerating the analysis of Sanger sequencing of bisulfite PCR products.

Textbook and scientific papers addressing DNA methylation usually still cite “DNA methylation occurs at CpG cytosines”. Methylation at cytosines outside the CpG nucleotide, the so-called “non-CpG methylation”, is usually considered a minor and not biologically relevant process. However, the technical improvements and additional studies in epigenetics have demonstrated that non-CpG methylation is present with frequency higher than previously thought and retains biological activity, potentially relevant to the understanding and the treatment of human diseases.

Vitamin K is essential for many physiological processes, including coagulation, bone metabolism, tissue calcification, and antioxidant activity. Vitamin K vitamers are represented by lipophilic compounds with similar chemical structure (i.e., phylloquinone (vitamin K1) and menaquinone (vitamin K2)). Vitamin K deficiency can affect coagulation and vascular calcification, increasing the risk of hemorrhages, atherosclerosis, cerebrovascular diseases, and neurodegeneration. Recently, several studies have hypothesized a possible dual role of vitamin K vitamers in benefiting both vascular and cerebral health, e.g., by sphingolipids biosynthesis or ferroptosis inhibition. The aim of this narrative review is to deepen the understanding of biological activities of vitamin K and its possible dual protective/preventive actions in neurovascular and degenerative conditions, e.g., stroke and dementia. Given the difficulties related to hemorrhagic risk entailed in the prevention of strokes, the function of vitamin K antagonists is also investigated. Finally, we track the development of a clinical concept for a future preventive strategy and innovative use of vitamin K as a supplement to counteract neurovascular and pathological processes, focusing in particular on stroke and dementia.

Insects are becoming increasingly relevant as protein sources in food and feed. The Black Soldier Fly (BSF) is one of the most utilized, thanks to its ability to live on many leftovers. Vegetable processing industries produce huge amounts of by-products, and it is important to efficiently rear BSF on different substrates to assure an economical advantage in bioconversion and to overcome the seasonality of some leftovers. This work evaluated how different substrates affect the protein and amino acid content of BSF. BSF prepupae reared on different substrates showed total protein content varying between 35% and 49% on dry matter. Significant lower protein contents were detected in BSF grown on fruit by-products, while higher contents were observed when autumnal leftovers were employed. BSF protein content was mainly correlated to fibre and protein content in the diet. Among amino acids, lysine, valine and leucine were most affected by the diet. Essential amino acids satisfied the Food and Agricultural Organization (FAO) requirements for human nutrition, except for lysine in few cases. BSF could be a flexible tool to bio-convert a wide range of vegetable by-products of different seasonality in a high-quality protein-rich biomass, even if significant differences in the protein fraction were observed according to the rearing substrate.

Alzheimer’s disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of Aβ across the blood–brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.

Exopolysaccharides (EPS) are complex molecules produced by some microorganisms and used in foods as texturizers and stabilizers, their properties depending on their chemical structure. In this work, three different lactic acid bacteria (LAB), were tested for their ability to produce EPS, by using five different mono- and disaccharides as their sole carbon source. The growth and acidifying ability were analysed, the EPSs were quantified by the official method AOAC 991.43, and their chemical structure was investigated. The amount of EPS varied from 0.71 g/L to 2.38 g/L, and maltose was the best sugar for EPS production by Lacticaseibacillus paracasei 2333. Lacticaseibacillus rhamnosus 1019 produced the highest amount when fed with lactose, whereas the EPS amount of Lactobacillus bulgaricus 1932 was not significantly different depending on the sugar type. The EPS chains consisted of fructose, galactose, glucose, mannose, ribose, glucosamine, galactosamine, and in some cases rhamnose in different proportions, depending on the strain and carbon source. The molecular weight of EPS ranged from <10 KDa to >500 KDa and was again highly dependent on the strain and the sugar used, suggesting the possibility of growing different strains under different conditions to obtain EPS with different potential applications in the food system.

This study explores methods to isolate high-pure monocytes and optimize the best growth factor concentration to generate monocytes-derived dendritic cells (mo-DCs), subset DC1, which is crucial in immune responses. Three protocols for monocyte isolation from peripheral blood mononuclear cells (PBMCs) were evaluated: three-hour incubation on FBS-coated flasks; an overnight incubation on FBS-coated flasks; and Magnetic Activated Cell Sorting (MACS). Additionally, five different concentrations of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and human recombinant interleukin-4 (hrIL-4) were compared. We used Flow cytometry to assess the isolation, purification, and generation of pure monocytes characterized as CD14 + , and expression of mo-DC classical markers (HLA-DR, CD80, CD83, and CD86). The obtained results show that monocytes isolated with the second method (overnight incubation) had the highest purity ( P  < 0.0001) but the lowest yield ( P  > 0.05), balancing purity and cost-effectiveness. A combination of hrGM-CSF and hrIL-4 at 400 U/mL produced the most favorable outcomes, leading to the highest rate of mo-DC generation ( P  < 0.05). Notably, this concentration resulted in increasing expression of HLA-DR, CD80, and CD86 surface markers in the generated DCs ( P  < 0.0001), with no changes in CD83 expression levels. In conclusion, this study offers valuable insights into selecting the optimal approach for monocyte isolation and mo-DC generation in various research contexts, providing a foundation for more effective immunological studies.

Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of \"Methylation-Insensitive Primers\" (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

Recent research has increasingly focused on the health benefits of dietary fibre (DF), including improved digestion, blood glucose and cholesterol regulation, satiety, and prebiotic effects, which depend on the specific DF type. Traditional gravimetric methods (e.g. Van Soest and AOAC) quantify DF fractions but lack molecular or monosaccharide detail. Advanced chromatographic methods offer more insights but require extensive sample preparation. To address this limitation, the study developed a method using proton Nuclear Magnetic Resonance ( 1 H NMR) spectroscopy to directly analyse DF hydrolysed fractions (mainly pectin, hemicellulose, and cellulose) without the need for derivatisation or neutralisation. It provides detailed structural insights, including the monosaccharide composition, carbohydrate modifications (methylation and acetylation), and the degradation products. The method was validated and then applied to hydrolysed DF fractions obtained from the AOAC process. 1 H NMR shows a comparable monosaccharide distribution to GC-MS, but yields higher recoveries, particularly for uronic acids. In addition, it offers faster sample preparation and acquisition, making it a powerful tool for comprehensive DF analysis. Dietary fibre (DF) analysis is crucial for understanding its health benefits, yet traditional methods lack molecular detail. Here, the authors develop a 1H NMR spectroscopy method to directly analyze hydrolysed DF fractions with high recovery and fast preparation while revealing detailed structural information including monosaccharide composition, carbohydrate modifications and the degradation products.