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.
Most of the leftovers from agricultural productions and industrial processing of vegetables are currently discarded as waste, augmenting production costs and environmental impacts. Black soldier ...flies (BSF) are non-pest insects that can grow on various types of organic materials. The larvae initially act as fast and efficient bioconverters, before being further valorized as biomass rich in proteins, fats and chitin. The aim of the present study was to exploit the potential of BSF prepupae reared on vegetable leftovers with high seasonality, and to obtain compounds with high added value and further industrial and agronomic uses such as food/feed, soil improver or fuel. The optimization of BSF rearing substrates based on different leftovers combinations was performed through a Mixture Design approach. Initially, a database was built detailing the availability, seasonality and nutrient composition of the vegetable by-products. According to the seasonal availability of the agri-food leftovers, three main groups were identified: annual, summer and autumn mixtures, in order to promote the exploitation of the highest quantity of leftovers. This approach allowed the obtainment of statistically reliable correlations (R2 > 0.75) between the employed leftovers and the content of lipid and nitrogen compounds (protein and chitin) of the BSF prepupae. In particular, a mixture of vegetable leftovers available in autumn that included legume (25 wt%), cereal (20 wt%) and vegetable (25 wt%) wastes proved to be the best combination in terms of insect growth (−25% development time compared to the control group) and nutritional composition. The chemical composition of the insect biomass allowed the identification of potential applications with high added value, such as food ingredients (protein and fats) or nutraceuticals (chitin). The identification of the optimal parameters to ensure the greatest possible efficiency would promote the scale-up of BSF rearing to an industrial level.
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•Black soldier fly was employed for reduction of vegetable leftovers.•Vegetables leftover were selected and mixed considering seasonal availability.•Mixture design technique was employed for diets assessment.•BSF larvae development is favored by a mixture of legume, corn and fruits leftovers.•The best performing diet should vary in relation to the insect's final use.
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•XOS mixtures with different structures were produced starting from standard xylan.•A mixture of XOS with DP 6–9 was obtained through preparative SEC purification.•Further knowledge ...has been gained about in vitro antioxidant activity of XOS.
Antioxidant activity of oligosaccharides is very discussed, and the relationship between their chemical structure and functionality is not clear. In this work, XOS, with different degrees of polymerization, acetylation and purity were produced starting from commercial xylans by enzymatic hydrolysis coupled with tangential ultrafiltration. A purified mixture containing DP 6–9 XOS was also produced. A detailed molecular characterization of all the XOS mixtures was performed by LC-MS, HPSEC-RID and 1H NMR. In vitro antioxidant activity was then tested against the stable DPPH free-radical. It was found that standard pure xylose and DP 2–6 XOS had very low scavenging capacity, while DP 6–9 XOS had a great one. In general, unsubstituted XOS are more antioxidant than acetylated ones, and a higher DP favours an increase in this functionality at first, then a decrease when molecular weights become higher.
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.
The Presenilin1 (PSEN1) gene encodes the catalytic peptide of the γ-secretase complex, a key enzyme that cleaves the amyloid-β protein precursor (AβPP), to generate the amyloid-β (Aβ) peptides, ...involved in Alzheimer's Disease (AD). Other substrates of the γ-secretase, such as E-cadherin and Notch1, are involved in neurodevelopment and haematopoiesis. Gene-specific DNA methylation influences PSEN1 expression in AD animal models. Here we evaluated canonical and non-canonical cytosine methylation patterns of the PSEN1 5ʹ-flanking during brain development and AD progression, in DNA extracted from the frontal cortex of AD transgenic mice (TgCRND8) and post-mortem human brain. Mapping CpG and non-CpG methylation revealed different methylation profiles in mice and humans. PSEN1 expression only correlated with DNA methylation in adult female mice. However, in post-mortem human brain, lower methylation, both at CpG and non-CpG sites, correlated closely with higher PSEN1 expression during brain development and in disease progression. PSEN1 methylation in blood DNA was significantly lower in AD patients than in controls. The present study is the first to demonstrate a temporal correlation between dynamic changes in PSEN1 CpG and non-CpG methylation patterns and mRNA expression during neurodevelopment and AD neurodegeneration. These observations were made possible by the use of an improved bisulphite methylation assay employing primers that are not biased towards non-CpG methylation. Our findings deepen the understanding of γ-secretase regulation and support the hypothesis that epigenetic changes can promote the pathophysiology of AD. Moreover, they suggest that PSEN1 DNA methylation in peripheral blood may provide a biomarker for AD.
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.
This study aims to explore an advanced protocol for characterising dietary fibre (DF) fractions to meet the growing demand for accurate and reliable data. Although current enzymatic-gravimetric ...approaches, e.g., AOAC and Van Soest analysis, provide information about soluble and insoluble DF quantification, they present limitations related to the lack of fractions characterisation. To overcome these limitations, the proposed protocol integrates the official AOAC 991.43 method with the sequential fibre fractionation by exploiting the different resistance of the fibre fractions to acid hydrolysis treatments (TFA and H2SO4), utilising hazelnut shells as a case-study. Each hydrolysed fraction was quantified and characterised through GC–MS analysis of monosaccharides. The data obtained for hemicellulose, cellulose, and lignin fractions were then discussed and compared with the Van Soest method. This approach yields a comprehensive procedure applicable to different food and nutraceutical products, emphasising the importance of DF characterisation for a deeper understanding of their bio-functional properties.
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•A protocol for dietary fibre fractionation and quantification was developed.•Fibre was fractionated by exploiting the resistance to acid hydrolysis treatments.•Fibre fractions were quali- and quantified in terms of monosaccharides by GC–MS.•The developed method was compared with gravimetric and other chemical methods.•Advanced characterisation techniques are essential for enhancing knowledge of DF.
DNA methylation, the main epigenetic modification regulating gene expression, plays a role in the pathophysiology of neurodegeneration. Previous evidence indicates that 5'-flanking hypomethylation of
..., a gene involved in the amyloidogenic pathway in Alzheimer's disease (AD), boosts the AD-like phenotype in transgenic TgCRND8 mice. Supplementation with S-adenosylmethionine (SAM), the methyl donor in the DNA methylation reactions, reverts the pathological phenotype. Several studies indicate that epigenetic signatures, driving the shift between normal and diseased aging, can be acquired during the first stages of life, even in utero, and manifest phenotypically later on in life. Therefore, we decided to test whether SAM supplementation during the perinatal period (i.e., supplementing the mothers from mating to weaning) could exert a protective role towards AD-like symptom manifestation. We therefore compared the effect of post-weaning vs. perinatal SAM treatment in TgCRND8 mice by assessing
methylation and expression and the development of amyloid plaques. We found that short-term perinatal supplementation was as effective as the longer post-weaning supplementation in repressing
expression and amyloid deposition in adult mice. These results highlight the importance of epigenetic memory and methyl donor availability during early life to promote healthy aging and stress the functional role of non-CpG methylation.
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.
Amyloid beta peptide (Aβ) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase ...(PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aβ25-35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aβ25-35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aβ25-35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aβ25-35 induces DNA damage which in turn activates PARP-1. Challenge with Aβ25-35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aβ25-35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aβ and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.
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