Low Degree Of Methylation Research Articles

Epigenetics of the non-coding RNA nc886 across blood, adipose tissue and skeletal muscle in offspring exposed to diabetes in pregnancy

BackgroundDiabetes in pregnancy is associated with increased risk of long-term metabolic disease in the offspring, potentially mediated by in utero epigenetic variation. Previously, we identified multiple differentially methylated single CpG sites in offspring of women with gestational diabetes mellitus (GDM), but whether stretches of differentially methylated regions (DMRs) can also be identified in adolescent GDM offspring is unknown. Here, we investigate which DNA regions in adolescent offspring are differentially methylated in blood by exposure to diabetes in pregnancy. The secondary aim was to characterize the RNA expression of the identified DMR, which contained the nc886 non-coding RNA.MethodsTo identify DMRs, we employed the bump hunter method in samples from young (9–16 yr, n = 92) offspring of women with GDM (O-GDM) and control offspring (n = 94). Validation by pyrosequencing was performed in an adult offspring cohort (age 28–33 years) consisting of O-GDM (n = 82), offspring exposed to maternal type 1 diabetes (O-T1D, n = 67) and control offspring (O-BP, n = 57). RNA-expression was measured using RT-qPCR in subcutaneous adipose tissue and skeletal muscle.ResultsOne significant DMR represented by 10 CpGs with a bimodal methylation pattern was identified, located in the nc886/VTRNA2-1 non-coding RNA gene. Low methylation status across all CpGs of the nc886 in the young offspring was associated with maternal GDM. While low methylation degree in adult offspring in blood, adipose tissue, and skeletal muscle was not associated with maternal GDM, adipose tissue nc886 expression was increased in O-GDM compared to O-BP, but not in O-T1D. In addition, adipose tissue nc886 expression levels were positively associated with maternal pre-pregnancy BMI (p = 0.006), but not with the offspring’s own adiposity.ConclusionsOur results highlight that nc886 is a metastable epiallele, whose methylation in young offspring is negatively correlated with maternal obesity and GDM status. The physiological effect of nc886 may be more important in adipose tissue than in skeletal muscle. Further research should aim to investigate how nc886 regulation in adipose tissue by exposure to GDM may contribute to development of metabolic disease.

Sequential extraction of hesperidin, pectin, lignin, and cellulose from orange peels: towards valorization of agro‐waste

AbstractTo maximize the reuse of citrus fruit processing waste, which is generated in large amounts due to juice production, this work shows the results of a new renewable approach for the extraction of high‐added‐value compounds present in orange biomass, specifically in orange peels (OP). The method is based on the use of diluted Ca2+, acid, and base solutions for the extraction of hesperidin (HSD), pectin, lignin, and cellulose sequentially, without generating any organic solid waste at the end, and without the need for air drying the starting biomass. Good yields of hesperidin were recovered (~1.56%), with excellent purity (up to 95%), as well as lignin (yield ~2.2%) and cellulose (yield ~12%). Pectin obtained by this process showed a high amount of galacturonic acid (~88%) and a low degree of methylation (~16%). All products were thoroughly characterized by applying Fourier transform infrared (FTIR) spectroscopy and solid‐state cross‐polarization/magic angle spinning carbon‐13 nuclear magnetic resonance spectroscopy (CP/MAS 13C‐NMR). The products recovered from OP can be applied in a wide array of fields, such as food, medicine, and pharmaceuticals, among others.

Effects of extraction methods on the physicochemical properties and functionalities of pectic polysaccharides from burdock (Arctium lappa L.)

In this work, the effects of four different extraction methods, acid (HCl), alkali (NaOH), enzymes (cellulase/pectinase), and buffer (pH 7.0) on the physicochemical properties and functionalities of burdock pectin were systematically investigated and compared. Buffer extraction gave a low yield (2.8 %) and is therefore limited in its application. The acid treatment hydrolyzed the neutral sidechains and gave a homogalacturonan content of 72.6 %. By contrast, alkali and enzymes preserved the sidechains while degrading the polygalacturonan backbone, creating a rhamnogalacturonan-I dominant structure. The branched structure, low molecular weight, and high degree of methylation (42.3 %) contributed to the interfacial adsorption, emulsifying capacity, and cellular antioxidant activity of the enzyme-extracted product. For the acid-extracted product, the strong intramolecular electrostatic repulsion restricted the formation of a contact interface to prevent coalescence of the emulsion. In addition, they did not have sufficient reducing ends to scavenge free radicals. Although a high branching size (5.0) was adopted, the low degree of methylation (19.5 %) affected the emulsifying capacity of the alkali-extracted products. These results provide useful information for pectic polysaccharides production with tailored properties.

Ultrasound-assisted extraction (UAE) and characterization of citrus peel pectin: Comparison between pummelo (Citrus grandis L. Osbeck) and sweet lime (Citrus limetta Risso)

Citrus peels are a commercial source for pectin extraction which is widely used in the food industry as a hydrocolloid, fat mimetic, and gelling agent. Acid extraction is an established method for extraction; however, environmental and consumer concerns are pushing processors to explore sustainable and green methods to provide clean solutions. The present study was designed to optimize pectin extraction from pummelo and sweet lime peels using ultrasound-assisted extraction (UAE). The optimized conditions of UAE obtained through response surface methodology (RSM) were: sonication time = 9.38 min, power = 44.01%, and liquid-solid ratio (L:S) of 46.8:1. These levels resulted in maximum pectin yield (26.35%) and high galacturonic acid content (69.11%; GalA) from pummelo peels as compared to sweet lime peels. The pectin extracted via UAE was food-grade with a low degree of methylation (<50%) and high functional quality. Characterization of pectin through Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and rheology confirmed its similarity to commercial pectin. Pummelo peel pectin demonstrated high biological activity in inhibiting lipid deposition in HepG2 cells and low cytotoxicity in 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Overall, based on rheological and functional properties, pummelo peels were found to be superior to sweet lime for commercial pectin extraction. The findings suggest that citrus peel pectin with low degree of esterification (DE<50%) is a valuable ingredient for formulating low glycemic functional foods. Overall UAE seems to be an efficient, economic, and sustainable method for extraction of high-quality pectin.

Dual crosslinking of low-methoxyl pectin by calcium and europium for the simultaneous removal of pharmaceuticals and divalent heavy metals

The contamination of water resources by heavy metals and pharmaceuticals is a significant threat to human health and the environment. Pectin is a promising material for removing heavy metals from water. However, its removal efficacy for other types of pollutants is limited. In this study, we developed a novel approach to enhance the remediation capacity of pectin (with a low degree of methylation) by crosslinking it with different agents: calcium, europium, and their combination. We performed scanning electron microscopy, infrared spectroscopy, and X-ray diffraction experiments to understand the molecular structure of pectin after gelation with the three agents. Our results showed that calcium, europium, and their combination all induce the gelation of pectin. However, the reticulated pectin structures exhibited significant structural differences depending on the type of crosslinking agent used, which affected the adsorption capacity. Specifically, calcium cations partially formed a crystalline “egg-box” structure, whereas europium cations produced a more homogeneous network without crystalline regions. The dual-crosslinking system comprising calcium and europium cations resulted in an intermediate network with both crystalline and amorphous regions. Our findings suggest that dual-cross-linked pectin is a highly effective adsorbent for the simultaneous removal of both heavy metals and pharmaceutical products. This novel approach of crosslinking pectin with multiple agents has the potential to significantly enhance its remediation capacity, offering a promising solution for the simultaneous removal of multiple pollutants from water.

Film Properties of Pectin Obtained from Various Fruits’ (Lemon, Pomelo, Pitaya) Peels

Food by-products, as sources of high-value compounds, have shown great promise for formulating novel food packaging and edible films for food preservation. This work focused on extracting pectin from lemon, pomelo, and pitaya peels using an ethanol precipitation method. The degree of esterification and the monosaccharide composition of the extracted pectin were determined, and the antioxidant activity of the pectin was assessed. Subsequently, pectin-based films were prepared using the casting method, employing pectin from various sources. These films’ physical and mechanical properties (tensile strength and elongation at break) were assessed, and their microstructure and intermolecular interactions were examined using scanning electron microscopy and Fourier transform infrared spectroscopy analysis. The results of this study demonstrate variations in the degree of esterification among the three pectin samples, with pomelo peel and lemon peel pectins exhibiting higher degrees of methylation compared to pectin from pitaya peel, which had a lower degree of methylation. However, no significant differences were observed regarding the films’ moisture content and water vapor permeability. Notably, the films made from pitaya peel pectin exhibited superior tensile strength and elongation at break to those derived from pomelo peel and lemon peel pectins. The pectin recovered from these fruit peels had great film properties and thus could be utilized for food packaging and edible films.

Tuning and Coupling Irreversible Electroosmotic Water Flow in Ionic Diodes: Methylation of an Intrinsically Microporous Polyamine (PIM-EA-TB).

Molecularly rigid polymers with internal charges (positive charges induced by amine methylation) allow electroosmotic water flow to be tuned by adjusting the charge density (the degree of methylation). Here, a microporous polyamine (PIM-EA-TB) is methylated to give a molecularly rigid anion conductor. The electroosmotic drag coefficient (the number of water molecules transported per anion) is shown to increase with a lower degree of methylation. Net water transport (without charge flow) in a coupled anionic diode circuit is demonstrated based on combining low and high electroosmotic drag coefficient materials. The AC-electricity-driven net process offers water transport (or transport of other neutral species, e.g., drugs) with net zero ion transport and without driver electrode side reactions.

Human milk oligosaccharides and non-digestible carbohydrates reduce pathogen adhesion to intestinal epithelial cells by decoy effects or by attenuating bacterial virulence

This work investigated the effects of different chemical structures of human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) on pathogen adhesion by serving as decoy receptors. Pre-exposure of pathogens to inulins and low degree of methylation (DM) pectin prevented binding to gut epithelial Caco2-cells, but effects were dependent on the molecules’ chemistry, pathogen strain and growth phase. Pre-exposure to 3-fucosyllactose increased E. coli WA321 adhesion (28%, p < 0.05), and DM69 pectin increased E. coli ET8 (15 fold, p < 0.05) and E. coli WA321 (50%, p < 0.05) adhesion. Transcriptomics analysis revealed that DM69 pectin upregulated flagella and cell membrane associated genes. However, the top 10 downregulated genes were associated with lowering of bacteria virulence. DM69 pectin increased pathogen adhesion but bacterial virulence was attenuated illustrating different mechanisms may lower pathogen adhesion. Our study illustrates that both hMOs and NDCs can reduce adhesion or attenuate virulence of pathogens but that these effects are chemistry dependent.

The Impact of DNA Methylation on IL6 mRNA Levels in Hematinic Deficiency and Atopy-Associated Recurrent Aphthous Stomatitis Patients.

Objective To investigate the DNA methylation using pyrosequencing and its effects on the upregulation of IL6 mRNA in patients with recurrent aphthous stomatitis (RAS) in connection with hematinic deficiency and atopy. Material and Methods. This cross-sectional study was conducted at Dr. Hasan Sadikin Hospital, Bandung, from January–March 2019 and was approved by the Health Research Ethics Committee of Universitas Padjadjaran (Ethics No. 990/UN6.KEP/EC/2018). Furthermore, the subjects had RAS ulcers with a history of at least twice a year along with atopy and dietary imbalance with no history of recurrent intraoral herpes or any systemic diseases. This study was performed on 23 RAS patients and 21 healthy subjects, and the sampling was carried out consecutively. The blood samples were collected from all the subjects, and then, the DNA and RNA were extracted from the peripheral blood mononuclear cells (PBMCs). Consequently, the bisulfite-modified DNA was used to confirm the methylation status of the IL6 gene promoter through the pyrosequencing method. The methylation levels of the IL6 promoter were assessed by a reverse transcriptase-polymerase chain reaction technique. The gene expression of RAS and the control group was analyzed by the 2−ΔΔCT method. The statistical analysis using the Mann–Whitney U test was conducted to evaluate IL6 mRNA levels and DNA methylation with p value <0.05 considered to be statistically significant. Result The IL6 mRNA levels were approximately 1.88-fold in RAS patients, and there was a significant relationship between the expression of the IL6 gene and the increased risk of RAS (p < 0.001). It was reported that four out of six sites in the cytosine phosphate guanine (CpG) island IL6 promoter had a lower degree of methylation, and two other sites in patients with RAS had greater methylation compared with control, but not statistically significant. Conclusion This study showed the upregulation of IL6 mRNA levels in RAS patients compared to control. DNA methylation in the present study is at sites 566–658, whereas the location of the IL6 promoter is at sites 1–1684. Thus, it would be necessary conducting some research at other CpG sites of IL6 promoter islands to determine the status of DNA methylation.

An immune regulatory 3D-printed alginate-pectin construct for immunoisolation of insulin producing β-cells

Different bioinks have been used to produce cell-laden alginate-based hydrogel constructs for cell replacement therapy but some of these approaches suffer from issues with print quality, long-term mechanical instability, and bioincompatibility. In this study, new alginate-based bioinks were developed to produce cell-laden grid-shaped hydrogel constructs with stable integrity and immunomodulating capacity. Integrity and printability were improved by including the co-block-polymer Pluronic F127 in alginate solutions. To reduce inflammatory responses, pectin with a low degree of methylation was included and tested for inhibition of Toll-Like Receptor 2/1 (TLR2/1) dimerization and activation and tissue responses under the skin of mice. The viscoelastic properties of alginate-Pluronic constructs were unaffected by pectin incorporation. The tested pectin protected printed insulin-producing MIN6 cells from inflammatory stress as evidenced by higher numbers of surviving cells within the pectin-containing construct following exposure to a cocktail of the pro-inflammatory cytokines namely, IL-1β, IFN-γ, and TNF-α. The results suggested that the cell-laden construct bioprinted with pectin-alginate-Pluronic bioink reduced tissue responses via inhibiting TLR2/1 and support insulin-producing β-cell survival under inflammatory stress. Our study provides a potential novel strategy to improve long-term survival of pancreatic islet grafts for Type 1 Diabetes (T1D) treatment.

Effects of pH and temperature on the structure, rheological and gel-forming properties of sugar beet pectins

Sugar beet pectin (SBP) was modified by alkaline at both 25 °C and 3 °C to produce gel-type pectins. The structure, rheological and gelation properties of series modified SBPs were then assessed. The results showed that low temperature effectively inhibited the β-elimination of SBP during alkali treatment, and the average molecular weight (Mw) and galacturonic acid (GalA) content of the resulting SBP were much higher than that of the alkali-treated SBP at 25 °C. Due to the low degree of methylation (DM) (≦ 21.2) and degree of acetylation (DA) (≦ 14.6), SBP treated under pH 11 and 12 at 3 °C could successfully form stable elastic gels, and these gels were further confirmed to be frequency-independent through rheological analysis. The SEM and TG/DTG results showed that the corresponding gel of SBP treated under pH 12 at 3 °C for 120 min had the most uniform micro-network structure and the best thermal stability. This work for the first time demonstrated the effect of a wide pH range on the chemical structure, rheological and physical gelation properties of SBP, especially under alkaline conditions at a low temperature (3 °C). Findings from this work suggest that alkali treatment at low-temperature allows SBP to have the potential to become a gel-type commercial pectin.

Perennial halophyte Salicornia neei Lag.: Cell wall composition and functional properties of its biopolymers

Salicornia neei halophyte extends in Argentina seashores. To envisage potential applications, cell wall sequential extraction performed on dry plant yielded 1.1, 2.4, 0.3 and 0.9% of pectin fractions respectively extracted by room temperature water, 90 °C-water, CDTA and Na2CO3. They contained 21–33% uronic acids (UA) with low degree of methylation and 0.5–1.2 M ratios of neutral sugars to UA. High arabinose level suggests that long arabinan side-chains maintain cell wall flexibility in water deficit. Fractions also contained 10–36% of proteins. The KOH-soluble fractions (4.3%) were mainly arabinoxylans. At 2.0% w/v, pectin fractions developed “weak gel”-type networks with Ca2+, while arabinoxylans generated “dilute solutions”. Cellulose (28%) and lignin (45.1%) were the main biopolymers in the final residue, which showed low water swelling capacity (3.6 mL/g) due to lignin, increasing when arabinoxylans were also present. Phenolics (9.8%) were mainly water-extractable. Salicornia is a source of biopolymers and antioxidants potentially useful for food applications.

Manipulation of β-carotene levels in tomato fruits results in increased ABA content and extended shelf life.

SummaryTomato fruit ripening is controlled by the hormone ethylene and by a group of transcription factors, acting upstream of ethylene. During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit‐specific overexpression of LYCOPENE β‐CYCLASE (LCYb) resulted in increased β‐carotene (provitamin A) content. Unexpectedly, LCYb‐overexpressing fruits also exhibited a diverse array of ripening phenotypes, including delayed softening and extended shelf life. These phenotypes were accompanied, at the biochemical level, by an increase in abscisic acid (ABA) content, decreased ethylene production, increased density of cell wall material containing linear pectins with a low degree of methylation, and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoid compounds were also altered in the transgenic fruits, which could be attributed to delayed fruit ripening and/or to ABA. Network correlation analysis and pharmacological experiments with the ABA biosynthesis inhibitor, abamine, indicated that altered ABA levels were a direct effect of the increased β‐carotene content and were in turn responsible for the extended shelf life phenotype. Thus, manipulation of β‐carotene levels results in an improvement not only of the nutritional value of tomato fruits, but also of their shelf life.

Trimethylated H3K27, and Di- and Trimethylated H3K4 Proteomic Profiling Distinguishes Acute Lymphoid Leukemia (ALL) from Acute Myeloid Leukemia (AML) and Associates with Overall Survival and Tyrosine Kinase Inhibitor Sensitivity in Adult ALL

Background: Lineage-specific gene transcription signatures between AML and ALL are recognized, but post-translational phenotype-specific protein expression profiles remain undefined. We hypothesized that functional proteomic patterns vary between AML and ALL and that the activity state of cells correlates with response to therapy within subgroups, complementing cytogenetic and molecular data. Methods: Reverse phase protein arrays (RPPA) were generated using bone marrow (BM) and peripheral blood (PB) samples from newly diagnosed B-ALL (n=114), T-ALL (n=14), and AML (n=241) adult patients admitted at the MD Anderson Cancer Center. RPPA allowed simultaneous expression measurement of 229 highly validated protein antibodies including 3 Histone 3 (H3) post-translational methylation regulatory modifications; H3K4Me2, H3K4Me3 and H3K27Me3. Results: Unsupervised clustering of histone modification protein expressions distinguished AML from ALL in freshly prepared lysates from BM (n=241) and PB (n=127) as well as when BM and PB samples were combined (fig. 1A). The ALL-enriched cluster was dominated by high H3K27Me3. Elevated H3K27Me3 levels were found in the BM derived leukemic blasts compared to PB blasts in ALL (P &amp;lt; 0.001), but not AML (P = 0.35). Trimethylation of the repressive mark H3K27 is catalyzed by the polycomb group protein Ezh2. Oncogenic gain-of-functions of Ezh2 are seen in patients with lymphoid malignancies and others have shown that mutated Ezh2 increased H3K27Me3 in B-cells which associated with tumorigenesis. H3K27Me3 and Ezh2 antibody expressions were highly correlated in another RPPA of ALL and AML we created (R2=0.49, P &amp;lt; 0.001). Profiling of methylation marks using unsupervised clustering in ALL divided patients in 2 clusters that correlated with survival (fig. 1B-C, P = 0.02). Cluster 1 (C1) with higher H3K27Me3, H3K4Me2 and H3K4Me3 was associated with better outcome. In ALL, Ph+ historically associated with poor prognosis but outcomes have improved substantially with the use of tyrosine kinase inhibitors (TKI). In our cohort, 11/26 Ph+ ALL patients were treated with TKIs and it is notable that sensitivity to TKIs correlated with cluster membership; all C1 patients (high degree of methylation) were alive after 7 years of follow-up in contrast to none of the TKI-treated Ph+ ALL patients in cluster 2 (C2, low degree of methylation) (fig. 1D, P = 0.01). Recently, TKI resistance in Ph+ ALL has been proposed to associate with smoking due to altered DNA methylation patterns caused by chemical components of cigarette smoke. Retrospectively, we identified that 2 of 11 TKI treated patients were smokers. Both had membership in C2, were resistant against TKIs and died after 1 year. Thus, 2 out of 3 resistant TKI treated Ph+ ALL were smokers compared to none of the 8 responders. We then aimed to identify proteins that are potentially downregulated by increased expression of the repressive mark H3K27Me3. Pathway enrichment analysis of 59 significant negatively correlated proteins with H3K27Me3 revealed that these are involved in tyrosine kinase activity and resistance, including Jak/STAT and PI3K/Akt signaling pathways. If these pathways are less activated in patients with high H3K27Me3, then this can partially explain the increased sensitivity to TKIs in this subgroup. Clinically, no differences were found in age, BM and PB blast counts between TKI-treated C1 and C2 patients to provide an explanation for the higher death rate in C2. Conclusion: ALL and AML share some pathophysiology and the identification of differences in the functional activity of cells may contribute to a better understanding of the etiology of both diseases. Here we report that high H3K27Me3 protein levels in BM and PB distinguish ALL from AML and are related to TKI sensitivity in Ph+ ALL. Histone methylation status defines a group of Ph+ ALL patients that does not benefit from the addition of TKI therapy. The idea that smoking alters the epigenetic machinery in TKI resistant Ph+ ALL has been proposed and warrants further investigation. Fig. 1 A) Heatmap showing histone methylation levels in BM and PB from AML and ALL patients. B) Heatmap showing histone methylation levels in ALL BM and PB. Unsupervised clustering divided samples into 2 clusters. C) ALL patients in C1 survived longer than patients in C2 (P = 0.02). D) Increased long-term sensitivity for TKI therapy in C1 Ph+ ALL patients compared to C2 (100 vs. 0%, P = 0.01). Figure.1 Disclosures Jabbour: AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Amgen: Consultancy, Research Funding.

Valorization of Cocoa Husks: Pectin Recovery.

Food processing by-products are usually cheap and abundant and can be source of valuable molecules of great interest to various industries like the pharmaceutical or the food ones. In this frame, the husks of roasted cocoa beans, that are a by-product of the cocoa processing industry, can constitute a source of pectin. The recovery process has been already defined at laboratory scale with boiling acid extraction (pH 2.5). This process is suitable to recover a quantity of pectin, expressed as anhydro-galacturonic acid (AGA), around 8 g AGA/100 g dry husks; this pectin is characterized by low degree of methylation (%DM around 31) and acetylation degree lower than 2%. In this paper the effects of some operative conditions on pectin quantity and quality were studied, in order to optimize the parameters that can make the process economically competitive: the in-excess quantities of solvents and operation time were reduced, without altering yield and pectin characteristics. In particular, the extract was concentrated by 13.3%, the ethanol for pectin precipitation was reduced (ratio extract to ethanol equal to 1:4), and it was also demonstrated that a single washing with 40% ethanol is sufficient to obtain a purified product.

Rheological behavior of soluble dietary fiber fractions isolated from artichoke residues

Cynara cardunculus var. scolymus agroindustrial by-products (stems, outer bracts, leaves) constitute 80% of the plant. These by-products may be recovered for the extraction of food additives and nutraceuticals. In this research, the rheological behavior of soluble dietary fiber fractions isolated from bracts (B) and stems (S) of artichoke was studied. Fractions were isolated by means of a heat treatment followed by processing with sodium citrate buffer (S1, B1) or buffer/protease (S2, B2) or hemicellulase (S3, B3) or both enzymes (S4, B4). They were composed, mainly, by pectin (degree of methylation, DM ≤ 53%) and inulin. When dissolved in water (2.00% w/v) with Ca2+ (40 mg/g pectin), fractions were sensitive to the ion showing shear thinning flow, with initial yield stress in the case of B2, B3 or S3. Citrate buffer extracted fractions (B1, S1) showed the highest viscosities, whereas extraction with buffer and both enzymes (B4, S4) produced the least viscous fiber fractions. At rest assays revealed the formation of gel-like networks that were stronger, in general, in the presence of higher pectin and inulin content, homogalacturonan proportion, and low methylation degree. Leftovers of artichoke can be used to extract soluble dietary fiber fractions that might be utilized as natural thickeners and gelling agents in foods, pharmaceutical and cosmetics, helping to add value to raw materials and to close the bioeconomy loop in relation to this plant.

Characterization of the structural and emulsifying properties of sugar beet pectins obtained by sequential extraction

In this study, we examine how the extractant type used and extraction steps effect the structural and emulsifying properties of the resulting sugar beet pectins (SBPs). Using ammonium oxalate and sulfuric acid as the extractants, two oxalate-extracted pectins (denoted OEP1 and OEP2) and four acid-solubilized pectins (denoted AEP1, AEP2, AEP3 and AEP4) were sequentially extracted and characterized regarding their structures and emulsifying properties. All six pectins shared common features such as having a high galacturonic acid content and a low methylation degree, however, OEP1 and OEP2 not only had lower amounts of ferulic acid (FA) and protein but their molecular sizes were also smaller in comparison to the remainder. Moreover, the oxalate-extracted sample series was significantly different from the acid-extracted sample series in regards to amino acid compositions. Due probably to the hydrophobicity of the proteinaceous components and the lower contents of FA and protein, OEP1 failed to stabilize oil-in-water emulsions containing 15% middle chain triglyceride (MCT). In contrast, AEP1-4 were shown to have better emulsifying activities, producing fine emulsions under favorable conditions (e.g. pectin concentration 1.5%). The d43 values of the emulsions prepared at a 1.5% concentration of AEP2, AEP3, or AEP4 did not markedly change throughout the period of storage (28 days) at 30 °C, whereas the AEP1 emulsion was relatively less stable. The correlation between emulsifying properties and the structures among the acid-extracted pectins highlights that the extraction sequence plays an important role in the resulting compositional and emulsifying properties of SBP.

Yield and structural composition of pomelo peel pectins extracted under acidic and alkaline conditions

Extraction of pectin from pomelo peel was performed using water, acid and alkaline solutions with a microwave power of 1100 W for 2 min, and yield and structural/chemical characteristics of the extracted pectins were investigated. Extraction of pectin with water gave the lowest yield (6.5%, dry weight basis). The water-extracted pectin contained the highest amount of neutral sugar (29.5%), while equivalent molecular weight (MWeq) and degree of methylation (DM) were 43 kDa and 29.7%, respectively. Extraction of pectin with HCl solutions at concentrations of 25, 50, 100 and 200 mM resulted in a significant increase of pectin yields (12.1–20.5%) and pectins with high amount of galacturonic acid (83.3–85.6%). The MWeq and DM of the acid-extracted pectins ranged from 171 to 368 kDa and 53.9–82.5%, respectively. Compared to the acid, extraction with NaOH solutions at the same concentrations provided higher yields (13.9–24.2%), smaller size (142–187 kDa) and lower DM (<1.7%) pectins, while amount of galacturonic acid was comparable. The highest yield of extraction was found at a concentration of 50 mM for both HCl and NaOH solutions. Extraction of pomelo peel with 50 mM NaOH solution by reduction of the microwave power to 550 W and extending the extraction time to 15 min resulted in an increase of pectin yield (29.2%) and pectin with smaller size (76 kDa).

Genetic and epigenetic characterization of the cry1Ab coding region and its 3′ flanking genomic region in MON810 maize using next-generation sequencing

MON810 maize was first commercialized in 1997 and it is one of the most marketed genetically modified crops worldwide. Although MON810 maize has been studied extensively, its genetic stability and epigenetics have not been studied very well. We used next-generation sequencing to investigate the genetics and epigenetics of the cry1Ab coding region and its 3′ flanking genomic region in three different MON810 maize varieties. Genetic characterization of the cry1Ab coding region allowed us to identify and quantify several sequence variants. Samples from seeds containing a stacked MON810 event had more variants than MON810 single event varieties. Specifically, position 71 of the analyzed region varied in 15 of 600 samples tested and thus appears to be a mutational hotspot. In addition, position 71 varied at very different frequencies in the samples. Epigenetic analysis revealed a low degree of methylation, making it difficult to associate the coding region variants with methylation status. In conclusion, the variation in the coding region is either due to the increased age of the seeds from the tested maize varieties, which is known to increase the mutation rate, or due to the presence of a second (non-functional) cry1Ab fragment in the genome of the MON810 maize variety.

DNA methyltransferases and tRNA methyltransferase DNMT2 in developing pig brain - expression and promoter methylation

DNA methylation constitutes an epigenetic mechanism used by cells to control gene expression and chromatin organization. DNA methylation is a biochemical process, catalyzed by DNA methyltransferases, where a methyl group is added to a nucleotide base. In mammals, DNA methylation is essential for normal development. Here we report on the developmental expression of DNA methyltransferases and DNMT2, a tRNA methyltransferase, in various brain tissues in porcine embryos. Expressions of DNMT1, DNMT2, DMNT3A and DNMT3B transcripts were determined by qRT-PCR in five different brain tissues at four or five time points in embryo development. Low transcript levels from all these genes were detected as early as 60 days of gestation in all tissues examined. From 60 to 115 days of gestation, a reduction in DNMT1 and DMNT3A and DNMT3B mRNA was seen in brain stem, hippocampus, basal ganglia, cortex and cerebellum. No significant changes were seen for DNMT2 mRNA. In heart, a bimodal expression was observed for all four DNMTs analyzed. Analysis of DNA methylation status of two selected regions in the DNMT3A promoter revealed a very low degree of methylation (<3%). It was concluded that DNA methylation of the examined region of the DNMT3A gene is not determining expression of its transcript.