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Polysaccharides were extracted from corn bran (CBPs) and then modified by a sulfating reaction to obtain sulfated corn bran polysaccharides (SCBPs). Synthesized sulfated polysaccharides were located in the equatorial bonds of monosaccharide residues. A more orderly and smooth surface of SCBPs was observed. Single factor experiment results showed 2.0 g of aminosulfonic acid, 60 mL of N-dimethylformamide and 2.5 h of reaction time were the optimum conditions for SCBPs preparation. Phenolic content of SCBPs significantly decreased with increasing concentration of aminosulfonic acid, while it increased with increasing DMF concentration and reaction time. SCBPs had higher DPPH, ABTS and iron chelating capacity under the optimum conditions. SCBPs significantly inhibited the proliferation of A549 and HepG2 cell lines, which was associated with the up-regulation at the mRNA expression level of pro-apoptotic genes CASP3, CASP8, CASP9 and reduced expression of p53, gene Bcl-2 and gene iNOS.

Summary Pulses are an important food source in many countries and also widely used as feed for livestock. Discovery of novel health benefits in pulses may contribute to improved diet and potential added value to crops. In this study, we examined the ability of extracts from faba bean, field pea and chickpea to inhibit the formation of fat droplets in an in vitro cell culture system that mimics the process of fat cell formation or adipogenesis. Both seed and hull extracts of all three pulses showed an inhibition of adipogenesis and the hull fraction and also contained higher levels of polyphenols compared with the seed extracts. At the molecular level, adipogenic inhibition was associated with a marked reduction in peroxisome proliferator-activated receptor gamma (PPARG) gene expression. Furthermore, the field pea pulse extract was shown to inhibit pancreatic lipase activity at a similar level to the drug ‘orlistat’, which is commonly used to treat obesity.

Recent studies have revealed that diabetes mellitus caused gut bacterial dysbiosis, and intervention studies aiming to selectively alter the composition and metabolism of the gut microbiota are crucial next steps for investigating the links between the microbiome and diabetes. The sequences encompassing V1–3 16S rDNA hypervariable regions were PCR amplified from rat fecal samples fed with resistant starch (RS). In total, 13 different phyla and 107 different genus were obtained with a 3% distance cut off. The microbiota profile of normal rats was significantly different from that of rats in diabetic control and RS intervention group. The most prominent phyla were Firmicutes and Bacteroidetes in the three groups. The proportion of Proteobacteria was significantly higher in the rats of diabetic control compared to normal group, and RS intervention could inhibit the proliferation of Proteobacteria phyla, including a wide variety of pathogenic species, such as Escherichia-Shige, Klebsiella, and Pseudomona. At genus level, Lactobacillus in the diabetic control was significantly lower than that in the normal group, whereas the feeding of RS increased the amount of Lactobacillus. More importantly, the consumption of RS reduced the composition of Ruminococcus and increased S24-7 norank comparing with the diabetic control. This study might indicate that RS is an effective food ingredient for manipulating the gut microbiota.

The application of bioactive lutein in the food industry is limited because of its poor water-solubility, instability, and low bioavailability. Nano-sized ferritin and chitosan provide a platform for fabricating shell–core system to encapsulate lutein. Herein, soybean seed ferritin (Glycine max) and chitosan stabilized lutein composites (FCLs) were fabricated by a unique reversible disassembly-reassembly character of apoferritin and ferritin–chitosan interaction. Results showed that lutein molecules were successfully encapsulated within the apoferritin with a molar ratio of 25.2 to 1 (lutein/ferritin), and the encapsulation efficiency and loading capacity were 16.8% and 2.50% (w/w), respectively. It was calculated that approximately 10 chitosan molecules were bound to a ferritin with a binding constant of 6.3 × 105 M−1, suggesting electrostatic interaction played an important role in ferritin–chitosan interaction. Results also indicated as much as 74.1% (w/w) of lutein was retained in FCLs after storage at 20 °C for 7 days. In addition, the photo- and heat-stability of lutein in FCLs were greatly improved as compared to free lutein. Furthermore, FCLs exhibited prolonged release of lutein in simulated gastrointestinal tract (GI) digestion as a result of ferritin coating and chitosan binding. Interestingly, food components exerted different effects in lutein release, EGCG, grape seed proanthocyanidin, and milk could inhibit while pectin could facilitate the release of lutein from FCLs. This work demonstrates an innovative strategy to solubilize, stabilize and control release of functional food nutrients.

This study investigated effects of triterpenoids derived from Ganoderma lucidum (GLT) on the ability to attenuate cognitive impairment and reduce oxidative stress and inflammation induced by D-galactose using an aged rats model. The data revealed that dietary GLT intervention improved the memory of aged rats in a water maze experiment. A reduction in inflammation was also achieved as GLT decreased the levels of MDA, AGEs, NO, TNF-α and AChE activity, and increased the activity of T-AOC, GSH-Px, T-SOD, CAT and IL-2 level in serum. The GLT intervention revised hepatocyte cellular arrangement and binucleation of the liver status, and even hippocampal neurons morphology. GLT upregulated the expression of FOXO4, SIRT1, and downregulated the expression of GFAP, iNOS, PI3K, AKT, mTOR and IL-6 with activation of PI3K/AKT/mTOR pathway. The current study highlights a promising dietary approach for prevention or alleviation of oxidative stress, inflammation and cognitive impairment induced by age-related conditions.

Integrated physiological and proteomic analyses using japonica rice and glutinous rice were applied to investigate the proteome regulation related to rice yellowing during natural storage. The changes of soluble sugar and soluble protein combined with the up-regulation expression of related proteins indicated the enhancement of carbohydrate, protein and lipid metabolism in yellowed rice. The expression of heat shock protein was up-regulated, indicating that rice yellowing occurred under heat, dry or other abiotic stress. Nevertheless, this study noted that the rice yellowing might be associated with the emergence of secondary metabolites, because the gene ontology analysis showed that the biosyntheses of secondary metabolites and phenylpropanoid were significantly enriched in yellowed rice. Based on above results, a possible proteome regulation of rice yellowing is proposed in the current study.

Differences in volatile compounds and the changes in physicochemical properties during the yellowing of glutinous rice were analyzed for investigating its association with water content during storage. The physicochemical analysis indicated that the yellowing led to an enhanced rice viscosity and reduced pasting temperature. More importantly, rice yellowing at higher moisture content resulted in a greater difference in the pasting properties between fresh and the yellowed rice. The changes in the rheological properties of rice appeared to be more sensitive to the initial moisture content, in particular at high moisture condition. In addition, the parameter tanδ might be a key indicator in highlighting the association of rice yellowing with its corresponding dynamic rheological properties. Specially, the volatile compounds detected by gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography-mass spectrometry (GC-MS) showed that most of volatile compounds were decreased following the rice yellowing process, which was independent to the initial moisture content of the storage, and the increased level of the storage moisture enhanced the loss of the volatile compounds. Nevertheless, the newly formed volatile compounds during the rice yellowing still noted, and the increase of 1,2,4-trimethyl-bnzene and the decrease of phenol may be the potent to indicating of rice yellowing.

Considering that the content of resistant starch (RS) in original starch is very low, chemical modification is one of choices to increase RS level for enhancing its application. The current study applied debranched waxy maize starch (DBS) to produce a citrate-esterified debranched starch (CADS) and investigated impact of the modification on RS formation. Native starch was also citrate esterified without debranching treatment as the control (CANS). Physicochemical and digestion properties of each sample were characterized, and the results indicated an absorption at 1724 cm −1 in FTIR spectrum was determined in either CADS or CANS, indicating the occurrence of the esterification. Debranching of starch molecules led to a higher degree of substitution (DS) of 0.793 for CADS than CANS. Furthermore, re-association of debranched starch achieved a B type crystalline pattern rather than an A type from its native starch granules. Esterification greatly destroyed crystalline regions until completely disappeared in CADS. The loss of crystalline region was highly consistent with the absence of endothermic peak both in CADS and CANS as revealed by DSC. Hydrolysis rate and digestibility of each sample followed the order: native starch > DBS > CANS > CADS, which may indicate that the introduction of the citric anhydride onto the starch molecules led to an increased space steric hindrance that delayed the enzyme contacting within glucosidic bonds. It could be the first report to prepare a citrate-esterified debranched starch with a higher resistance to amylolysis, and the current investigation may highlight a potent to produce a starch derivative with a higher RS content.

Pasting properties indicated that the yellowing process led to a reduced peak viscosity and breakdown value of the postharvest yellowing (PHY) rice compared to corresponding normal one. More importantly, the current study revealed that both moduli of G′ and G″ of the gel formed from the PHY rice was lower than that of its corresponding rice and the yellowing significantly reduced the area of the hysteresis curve. Thus, it is proposed that PHY may inhibit the formation of a continuous network structure. Studies of moisture absorption dynamics and low-field NMR suggested that the yellowing process led to a faster moisture absorption and de-absorption, indicating a faster moisture fluidity in rice kernels induced by yellowing process. Potent of the “capillary channels” might be formed during rice yellowing, and these structural characteristics may be associated with its higher digestibility due to the accelerated diffusion of the digestive enzymes in rice kernels.

In this study, the level of gamma-aminobutyric acid (GABA) in wheat bran was increased to be six times higher through the action of endogenous glutamate decarboxylase compared with untreated bran. The process of GABA formation in wheat bran also led to an increased level of phenolic compounds with enhanced antioxidant capacity 2 times higher than the untreated status. The interventional effect of a diet containing GABA-enriched bran on hyperinsulinemia induced by a high-fat diet (HFD) was investigated in a rat model. The results showed that, when compared with animals fed with HFD-containing untreated bran (NB group), the consumption of HFD-containing GABA-enriched bran (GB group) demonstrated a greater improvement of insulin resistance/sensitivity as revealed by the changes in the homeostatic model assessment for insulin resistance index (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). The expression of hepatic genes, cytochrome P450 family 7 subfamily A member 1 (Cyp7a1) and ubiquitin C (Ubc), which are involved in the adipogenesis-associated PPAR signalling pathway, was found to be significantly down-regulated in the GB group compared with the HFD group (P = 0.0055). Meanwhile, changes in the expression of a number of genes associated with lipid metabolism and gluconeogenesis were also noted in the GB group versus the HFD group, but not in the NB group, indicating different regulatory patterns between the two brans in a high-fat diet. More importantly, the analysis of key genes related to glucose metabolism further revealed that the expression of insulin-induced gene 1/2 (Insig-1/2) was increased following GB intervention with a corresponding reduction in phosphoenolpyruvate carboxykinase 1 (Pepck) and glucose-6-phosphatase, catalytic subunit (G6pc) expression, suggesting that glucose homeostasis is greatly improved through the intervention of GABA-enriched bran in the context of a high-fat diet.

Rice bran, a valuable byproduct of the rice milling process, has limitations in food industrial applications due to its instability during storage. This review summaries the methodology for stabilization and its impact on the nutritional properties of rice bran. A variety of treatments have been used and these include heat treatment, low-temperature storage, biological and chemical approaches and these will be discussed in terms of their ability to destroy/inhibit enzyme activity and improve storage performance of rice bran. More importantly, changes in the nutritional value of rice bran in terms of vitamins, polyphenols, tocopherols, flavonoids, free fatty acids caused by stabilization of rice bran will also be discussed. This review highlights the importance of appropriate design of processes for stabilization and controlling storage conditions to ensure quality of the rice bran and enhancing levels of phytochemicals in the bran for novel applications in functional foods.

Difference in thermal stability of two commercially available canola oils prepared by either expeller-extraction (EE) or solvent-extraction (SE) method was investigated. After 5 days consecutive deep-fry, content of oxidized-triacylglycerols (oxTAGs) in SE oil increased by 250.0% compared to its original status. However, 62.5% increase of oxTAGs in EE oil occurred, indicating that EE oil exhibits superior thermal stability to SE oil. Antioxidant capacity of EE oil was highly retained and loss rate of tocopherols in EE oil was much slower than in SE oil during deep-fry. Lipidomics showed that although there was no significant difference in molecular profile of either triacylglycerols or diacylglycerols between two oils, EE oil was characterized with 19 times higher phosphatidylcholine contents than SE oil. Considering no difference in antioxidant capacity between the two oils in their original status, it is proposed that synergetic mechanism is simultaneously initiated by antioxidant compounds and phosphatidylcholines, which plays key roles for maintaining better thermo-stability of vegetable oil during deep-fry.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

Effect of high pressure processing (HPP) on buckwheat nutritional properties is investigated in this study. The results indicated that the digestion of buckwheat starches of untreated and HPP treated at room temperature (RT) showed an “internal corrosion” pattern. However, an “exocorrosion” digestion behavior was observed for the starches of HPP treated sample at 45 °C, which might be due to the formation of amylose–lipid complexes during the process as revealed by X-ray diffraction (XRD). HPP treatment at 45 °C increased the antioxidant activity of the buckwheat as measured using 1,1′-diphenyl-2-picrylhydrazylvalue (DPPH) and iron chelating capacity (ICC) compared to untreated sample ( P

This study assessed the ability of canola protein isolate (CPI) and enzymatic hydrolysates (CPHs) to inhibit adipogenic differentiation of C3H10T1/2 murine mesenchymal stem cells in vitro. Cell viability was maintained at concentrations of 60 μg/ml of sample. Cells treated with Alcalase hydrolysate demonstrated a higher reduction in anti-adipogenic differentiation through quantitation by oil-red O staining. qPCR analysis showed that CPI and CPH-treated cells significantly inhibited PPARγ expression, a key transcription factor involved in adipocyte differentiation, as evident in an ∼ 60-80% fold reduction of PPARγ mRNA. Immunofluorescence staining for PPARγ protein also showed a reduced expression in some treated cells when compared to differentiated untreated cells. The 50% inhibition concentration (IC50) of CPI, CPHs and their membrane ultrafiltration fractions on pancreatic lipase (PL) activity ranged between 0.75 and 2.5 mg/ml, (p0.05) for the hydrolysed and unhydrolysed samples. These findings demonstrate that CPI and CPHs contain bioactive components which can modulate in vitro adipocyte differentiation.

Rutin is a common dietary flavonoid with important pharmacological activities. However, its application in the food industry is limited mainly because of its poor water-solubility. The nano-scale ferritin cage provides an ideal space for subtle encapsulation of hydrophobic rutin molecules. This study describes the preparation of novel homogeneous soybean seed ferritin stabilized rutin nanodispersions (FRNs) by a unique reversible dissociation and reassembly of the apoferritin. The characteristics including the water-solubility, morphology, leakage kinetics, and stability of the FRNs were investigated. Results indicated that the rutin molecules could be successfully encapsulated within the protein cages with a rutin/protein molar ratio of 30.1 to 1, and the encapsulation and loading efficiency were 25.1% (w/w) and 3.29% (w/w), respectively. In vitro experiments of rutin release demonstrated that the entrapment of rutin was effective, with more than 75% (w/w) still encapsulated in the ferritin cage after storage for 15 days. Furthermore, the thermal and UV radiation stability of ferritin trapped rutin was greatly improved due to the encapsulation as compared to free rutin. Additionally, the antioxidant activity of FRNs was partly retained as compared to free rutin molecules. This study provides a novel strategy for the design and fabrication of nanocarriers providing water-insoluble molecules with protection and stabilization.

The current study analyzed the different effects of intervention in high-fat diet fed rats using chito-oligosaccharides (CO group), resistant starch (RS group) and their complexes (CO-RS group), respectively. Properties such as fecal fat composition, gut derived fecal microbiota and fecal metabolites were investigated. The results show that supplementation with CO-RS in the high-fat diet led to the highest level of fecal fat excretion, followed by the CO and RS groups, and its fatty acid composition was characterized by a lower ∑UFA and higher ∑SFA levels. Furthermore, CO-RS consumption significantly enhanced the excretion of bile acids in the feces, which might be associated with a higher conversion of cholesterol into bile acids and the enhanced binding capacity with the bile acids. The fecal microbiota profile using amplified V4 rDNA suggested that rats in the CO-RS group developed an increased richness and diversity in the gut bacterial community compared to the high-fat diet group. More importantly, the CO-RS intake significantly increased the abundance of both Lactobacillus and Bifidobacterium, indicating their potential use as prebiotics. Furthermore, the highest abundance of Allobaculum and Blautia genus in the feces of the CO-RS group was also found in this study, which is highly related to the highest production of short-chain fatty acids (SCFAs) in the colon among all the animals groups. A histological analysis of colon tissue revealed that the CO-RS supplemented diet was associated with a greater degree of thickness of the mucosa layer compared to the rats in the untreated high-fat diet group. The resulting shift in the gut microbiome, increased metabolite (SCFAs) production and increased thickness of the mucosal layer may provide profound influences of CO-RS consumption on gut protection.

Lupin seeds are rich in proteins, which are utilized in the food industry. There is an increased interest in lupin research due to its association with health-related benefits, such as reduction of hypertension and hyperglycemia. However, studies on the peptides derived from lupin proteins are rare.Lupin protein hydrolysates (LPHs) were prepared by proteolysis using alcalase, trypsin and pepsin, respectively. All the hydrolysates demonstrated higher antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities compared to lupin proteins. The hydrolysates were fractionated into three fractions based on molecular weight (MW), and the peptides with MW 3 kDa (LPH3) had the highest antioxidant and ACE inhibitory activities compared to other fractions. Cell model study revealed that LPH3 fraction had the highest protection against the generation of reactive oxygen species in HepG2 cells, which was associated with increased activities of superoxide dismutase and glutathione peroxidase through upregulation of SOD1, GPX1, GCLM, SLC7A11 and SRXN1 expression.The analysis of amino acid composition indicated that the peptides were characterized with high content of hydrophobic amino acids, which may be responsible for the greatest antioxidant activity. This study highlights the promising potential of lupin peptides as a functional ingredient in healthy foods. © 2018 Society of Chemical Industry.

cope A recent study revealed that the accumulation of gut microbiota-produced acetate (GMPA) led to insulin over-secretion and obesity symptom. To further develop this scientific point, the effect of resistant starch (RS) or exogenous acetate carried by RS (RSA) in the gut on metabolic syndrome is investigated using diet-induced obese rats. Methods and results The metabonomics analysis shows that the gut of rats in the RSA group generate more butyrate in both serum and feces rather than acetate compared to the rats in RS group, indicating the conversion among metabolites, in particular from acetate to butyrate via gut microbiota. Consistently, the gut microbiome uses acetate as a substrate to produce butyrate, such as Coprococcus, Faecalibacterium, Roseburia, and Eubacterium and was highly promoted in RSA group, which further supports the metabolic conversion. This is the first report to reveal the accumulation of gut microbiota-produced butyrate (GMPB) but not GMPA significantly enriched AMPK signaling pathway with reduced expression of lipogenesis-associated genes for suppressing sphingosines and ceramides biosynthesis to trigger insulin sensitivity. Conclusion Gut microbiome profile and lipogenesis pathway are regulated by GMPB, which substantially influences energy harvesting in the gut from patterns opposed to GMPA.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role.

The extraction of phenolic compounds from canola meal produces functional health products and renders the canola meal a more digestible animal feed. The extracted phenolics may have novel bioactivity worth investigation. In this study, several solvents were evaluated for their ability to extract phenolic compounds from canola meal: water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The in vitro antioxidant and anti-obesity properties of various extracts were investigated. Anti-obesity properties were studied using adipogenic differentiation inhibition of a murine mesenchymal stem cell line (C3H10T1/2) and a pancreatic lipase inhibition assay. AE, ME, and BE showed significant (p &lt, 0.05) adipogenesis and pancreatic lipase inhibitory activities and may have more pharmacological properties. AE down-regulated the gene expression of the major adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPAR&gamma, ), correlating to phenolic content in a dose-dependent manner. The chemical characterization of AE revealed the presence of sinapic acid, ferulic acid, and kaempferol derivatives as main bioactive phenols.

Scope The characteristics of gut microbiota and host metabolism are hypothesized to be associated with constipation status, but the regulation mechanism is not fully understood. Thus, the current study investigates the effect of constipation symptoms on gut functionality following the modulation of gut microbiota and metabolites via dietary fiber intervention. Methods and results Constipation causes a significantly reduced short-chain fatty acids (SCFAs) production and a higher level of iso-butyrate. The feces of constipated people are characterized with inhibited Faecalibacterium, Ruminococcaceae and Roseburia abundance. Desulfovibrionaceae is identified to be an important endotoxin producer in constipated patients, and a butyrate-enriched SCFAs profile achieved by dietary fiber supplement accelerates gastrointestinal transit and increases the thickness of the mucosal layer, possibly through triggering the secretion of colonic hormones and enhancing the expression of tight junction proteins for maintaining intestinal barrier integrity. More importantly, an interacting regulatory mechanism among SCFAs, in particular butyrate and propionate, may be involved in signaling between the microbiome and host cells in the colon. Conclusion Gut microbiota, characterized with enriched butyrate-producing and depressed Desulfovibrionaceae bacteria, attenuates constipation symptoms through promoting intestinal hormones secretion and maintaining gut barrier integrity.

The anti-obesity effects of two types of resistant starch (RS) in high-fat-diet-induced obese rats were investigated. The serum triglycerides, total cholesterol and malondialdehyde concentrations were significantly reduced, and the total antioxidant capacity, superoxide dismutase levels and glutathione peroxidase activity were increased by RS2 and RS4 consumption compared to the obesity group. A significant reduction in the serum glucose level and elevations in hepatic lipid metabolic enzyme activities were observed only for RS4 administration. Moreover, the expression levels of the fatty acid synthesis associated genes ACC and Fads1, the triglyceride synthesis and metabolism-related gene SREBP-1, the adipocyte differentiation gene PPARγ, the cholesterol synthesis associated gene HMGCR, and the gluconeogenesis associated gene GAPDH were all significantly down-regulated, whilst the lipid oxidation gene Acox1 and the liver function genes Gsta2, Nqo1, and Gclm were up-regulated in both administered groups. Additionally, RS4 performed well in up-regulating the expressions of Gsta2, Gsta3, Nqo1, and Egfr, and down-regulating LXRα, Igfbp1, and Pml. RS4 exhibited great advantages in reducing oxidative stress compared with RS2.

Rice bran albumin protein (RAP) is sensitive to thermal changes and tends to degrade when exposed to high-temperature processing. In this work, RAP-epigallocatechin-3-gallate (EGCG) complex (RAPE) was prepared and the thermal stability was evaluated. Fluorescence results showed that EGCG could interact with RAP with a binding number n of 0.0885:1 (EGCG:RAP, w/w) and a binding constant K of 1.02 (± 0.002) ×10

Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P 0.0001) and up regulation of the hypertrophic marker collagen X (P 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ-3 (P 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P 0.05), while up-regulating collagen II and Aggrecan mRNA (P 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201-1215, 2017. © 2016 Wiley Periodicals, Inc.

Rarefaction Curve showing the rationality of bacterial community in different groups. Control: basal diet without extra oil consumption; NEO: basal diet with unheated canola oil; DFEO: basal diet with deep-fried canola oil. Figure S2. Clustering and annotation of the OTUs of samples. The X-axis is different sample names. The first Y-axis is Tags number, and the second Y-axis is OTUs number. Table S1. Components of basal diets. Table S2. MRPP for difference among control, NEO and DFEO groups. (DOC 198Â kb)

Primary antibody controls for Fig. 1. As a control for specific primary antibody binding, immunostaining was performed in the absence of primary antibodies, with only fluorophore tagged secondary antibody present. We confirm no florescence signal was detected these controls for eNOS and CAV-1. (a) Detection of nuclear staining by Dapi and (b) immunostaining performed without mouse monoclonal anti-eNOS antibody but with an anti-mouse IgG secondary antibody conjugated with Alexa 488. (c) Detection of nuclear staining by Dapi and (d) immunostaining performed without rabbit polyclonal anti-CAV-1 antibody but with an anti-rabbit IgG secondary antibody conjugated with Alexa 555. (e) Detection of nuclear staining by Dapi and (d) fluorescence detection with no mouse monoclonal anti-eNOS antibody with an anti-mouse IgG secondary antibody conjugated with Alexa 555. (f) Detection of nuclear staining by Dapi and (g) fluorescence detection with no rabbit polyclonal anti-CAV-1 antibody with an anti-rabbit IgG secondary antibody conjugated with Alexa 488. (PPT 182 kb)

Primary antibody controls for Fig. 11. As a control for specific β-catenin primary antibody binding, immunostaining was carried out in the absence of the primary antibody with no florescence signal detected. (a) Detection of nuclear staining by Dapi and (b) fluorescence detection without rabbit monoclonal anti-β-catenin antibody with an anti-mouse IgG secondary antibody conjugated with Alexa 488. (PPT 102 kb)

The purpose of this review is to discuss the achievements and progress that has been made in the use of atomic force microscopy in DNA related research in the last 25 years. For this review DNA related research is split up in chromosomal-, chromatin- and DNA focused research to achieve a logical flow from large- to smaller structures. The focus of this review is not only on the AFM as imaging tool but also on the AFM as measuring tool using force spectroscopy, as therein lays its greatest advantage and future. The amazing technological and experimental progress that has been made during the last 25 years is too extensive to fully cover in this review but some key developments and experiments have been described to give an overview of the evolution of AFM use from 'imaging tool' to 'measurement tool' on chromosomes, chromatin and DNA.

Background The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/β-glycerol phosphate (β-GP) as a carrier. Methods Three chitosan/β-GP scaffolds were prepared with varying concentrations of chitosan and β-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/β-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre-seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth. Results Collagen displayed its most significant elution of virus on day 1 and chitosan/β-GP (with a final concentration of 2.17% chitosan) on day 3. Conclusions The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system. Copyright © 2011 John Wiley & Sons, Ltd.

Background Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses. Methods Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction. Results Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRα and FGFR2, respectively. Conclusions A novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with βIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

Background Nitric oxide (NO) is generated in endothelial cells by endothelial nitric oxide synthase (eNOS). Caveolin-1 (Cav1) inhibits eNOS function and NO production. Modifying Cav1 scaffold domain, in particular Phenylalanine at position 92 (F92) is critical for the inhibitory actions of Cav1 toward eNOS. The aims of this study were to investigate the effect of enhanced NO production in term of in vitro angiogenesis on rat bone marrow derived mesenchymal stem cells (BMSCs) transduced with a novel bicistronic lentiviral vector co-expressing eNOS and mutant Cav1 (F92A). Methods A bicistronic eNOS/F92-Cav1 lentiviral vector was constructed, and used to transduce rat BMSCs. The expression of eNOS and VEGF protein were confirmed by western-blot. NO production was detected by the greiss assay and in vitro angiogenesis was assessed by matrigel assisted capillary tube formation. The cell viability was evaluated using a Cell Counting Kit (CCK)-8. Results The bicistronic eNOS/F92A-Cav1 lentiviral vector increased eNOS and VEGF protein expression, NO production compared to controls. In vitro capillary formation was increased in eNOS-F92A transduced cells and cell viability was not affected by transduction. Conclusion Transduction of rat BMSCs with an eNOS-F92A-Cav1 lentiviral vector can increase NO production by enhancing eNOS protein expression. The increased NO production did not reduce cell viability. This study demonstrates that genetic modification of BMSCs to enhance NO producton could be applied in stem cell based therapeutic approaches to treat diseases such as pulmonary arterial hypertension (PAH) which is characterized by decreased endothelial NO release.

We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p

One of the major limitations of adenovirus as an efficient gene delivery vehicle is its transient nature of transgene expression. At present, no biodegradable release system exists that permits the sustained release of gene delivery. This study aimed to optimize a biodegradable fibrin scaffold for sustained adenoviral mediated gene delivery. Scaffold formulations were fabricated incorporating an adenoviral vector encoding β-galactosidase, and each scaffold was incubated in elution fluid. The level of transfection of cells was measured to assess virus diffusion from each scaffold at different time periods. Results showed that viral particles were present in the elution fluid and had the ability to transfect cells at different time points. Depending on the scaffold formulation, variable rates of diffusion were seen. Analysis of scaffold microarchitecture using stereo pairs showed that variable pore depth is seen in different fibrin scaffold concentration, which may be a determining factor in the diffusion rate. It was concluded that sustained release of the adenovirus could be attained up to 192 h at the optimal concentration of 60 mg/mL fibrinogen and 4 IU thrombin. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006

Adenovirus (Ad) and Adeno-associated virus (AAV) are efficient gene delivery systems; manipulation of the wild-type genome allows their use as vectors for the overexpression of desirable transgenes. Generation and purification of such viral vectors can be labour intensive, costly and require specialized equipment, but a new generation of membrane-mediated ion exchange kits for purification of recombinant virus may facilitate this process. Here, we examine the yields, transgene expression and purity of preparations of Ad and AAV purified using commercially available kits in comparison to other established techniques for purification of recombinant viral vectors. We demonstrate comparable results for Ad and AAV respectively in all parameters investigated, with a substantial reduction in purification time for the kit-based technology. Such approaches are attractive methods for small-scale purification of recombinant Ad and AAV viral vectors.

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals –Ψ) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Ψ+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Ψ– RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Ψ does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Ψ+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Ψ thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.

In this study, rats with a fresh oil diet, a deep-fried oil diet, and a deep-fried oil and resistant starch (RS) diet were investigated for revealing the effects of deep-fried oil to the metabolic system and if RS could effectively attenuate metabolic dysfunction caused by deep-fried oil. The results showed that DO feeding led to significant increases of liver biomarkers of alanine aminotransferase (ALT) and aspartate transaminase (AST), accompanied by consistent reduction of total antioxidation (T-AOC) and glutathione peroxidase (GSH-Px) activity compared to the rats feeding with unheated canola oil (FO group) (p

Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential.

Pulmonary arterial hypertension (PAH) is a progressive disease characterised by lung endothelial cell dysfunction and vascular remodelling. A number of studies now suggest that endothelial progenitor cells (EPCs) may induce neovascularisation and could be a promising approach for cell based therapy for PAH. On the contrary EPCs may contribute to pulmonary vascular remodelling, particularly in end-stage pulmonary disease. This review article will provide a brief summary of the relationship between PAH and EPCs, the application of the EPCs to PAH and highlight the potential clinical application of the EPCs cell therapy to PAH.

In vitro, β-herpesviruses can stimulate or inhibit HIV replication under particular circumstances. In order to investigate the effects of β-herpesvirus infection on HIV replication and vice versa at an organ level, we determined the quantitative relationships between cytomegalovirus (CMV), human herpesviruses (HHV) 6 and 7, and HIV-1 proviral DNA using quantitative competitive PCR methods in 141 organs collected at autopsy from 11 AIDS patients. The presence of HHV-6 DNA in an organ was significantly associated with elevated HIV-1 proviral DNA (difference in HIV median loads, 1.3 log10 genomes; P = 0.004). Consistent with this, there was a trend for the presence of HIV-1 proviral DNA to be associated with an elevated HHV-6 load (0.44 log10 difference; P = 0.07). In contrast, there were no significant differences between viral loads in the combinations of either CMV or HHV-7 with HIV-1 proviral DNA load. Pairwise combinations of the β-herpesviruses revealed that the quantity of HHV-7 was increased in the presence of HHV-6 (difference in median loads, 1.3 log10; P = 0.001) and the quantity of HHV-6 was increased in the presence of HHV-7 (difference in median loads, 0.7 log10; P = 0.002). These results demonstrate that the presence of HHV-6 in an organ is significantly associated with an elevated HIV-1 proviral load and have implications for understanding HIV pathogenesis in the human host and the role that β-herpesviruses, especially HHV-6, might play as cofactors in the HIV disease process. J. Med. Virol. 57:278–282, 1999. © 1999 Wiley-Liss, Inc.

Replication of HIV introduces errors into the genome which are responsible for conferring a growth advantage over wildtype virus when drugs such as zidovudine (ZDV) exert a selective pressure. The molecular basis for HIV-1 resistance to ZDV has been mapped to codons 41, 67, 70, 215 and 219 of the reverse transcriptase gene both in vitro and in clinical samples of blood. This study has investigated the relationship between the quantitative prevalence of ZDV resistance in multiple organs of the same individual. Proviral HIV-1 load was measured by quantitative-competitive PCR in 90 samples from organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine. The distribution of wildtype and mutant sequences at the positions 41, 67, 70, 215 and 219 of the reverse transcriptase was assessed using a point mutation assay. The results showed that the highest proviral loads were predominately found in lymph node, spleen and lung and there was a significant association between viral load and resistance to ZDV (P = 0.008). Inter-organ distribution of wildtype and mutant sequences at codons 41, 67, 70, 215 and 219 was frequently not uniform and in some patients differed markedly between the lymphoreticular system and other organs. These results demonstrate that treatment of HIV-1 infection with zidovudine does not exert uniform selective pressures in multiple organs. These findings have implications for the interpretation of resistance data and design of treatment strategies for HIV, arguing in particular that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in multiple sites within the same individual. (C) 1998 Wiley-Liss, Inc.

A direct in situ polymerase chain reaction (IS-PCR) assay is described for the detection of HIV-1 proviral DNA in formalin fixed paraffin embedded brain tissue. Biotin-16-dUTP is incorporated during the PCR process and microwave pretreatment of tissue sections ensures that no non-specific incorporation into damaged or nicked genomic DNA occurs. Two methods are compared to detect the biotinylated amplified product, the use of an avidin-biotin-alkaline phosphatase complex (ABC) and the application of tyramide signal amplification (TSA) which allows both chromogenic and fluorescence detection. TSA detection enhances the sensitivity of IS-PCR, permitting fewer PCR cycles and preserving tissue morphology.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been demonstrated in formalin fixed, paraffin embedded post-mortem brain tissue (PM) by chromogenic immunohistochemistry for the HIV p24 antigen. The sensitivity of antigen detection is increased significantly by tyramide signal amplification (TSA) compared to the conventional peroxidase labelled Avidin-Biotin complex (ABC) technique. The TSA method also permitted the use of a lower concentration of primary antibody than is conventionally used. Sensitivity was enhanced further by microwave irradiation of the paraffin embedded tissues in citrate buffer. HIV-1 p24 antigen was also detected in PM brain tissue by TSA enhanced immunofluorescence and demonstrated increased sensitivity compared to the conventional immunofluorescence technique with a greatly reduced autofluorescence background.