Calcium-mediated binding of DNA to 1,2-distearoyl-sn-glycero-3-phosphocholine-containing mixed lipid monolayers

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20–27 Å thick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Dietary nitrate improves vascular function in patients with hypercholesterolemia: a randomized, double-blind, placebo-controlled study

Background: The beneficial cardiovascular effects of vegetables may be underpinned by their high inorganic nitrate content. Objective: We sought to examine the effects of a 6-wk once-daily intake of dietary nitrate (nitrate-rich beetroot juice) compared with placebo intake (nitrate-depleted beetroot juice) on vascular and platelet function in untreated hypercholesterolemics. Design: A total of 69 subjects were recruited in this randomized, double-blind, placebo-controlled parallel study. The primary endpoint was the change in vascular function determined with the use of ultrasound flow-mediated dilatation (FMD). Results: Baseline characteristics were similar between the groups, with primary outcome data available for 67 patients. Dietary nitrate resulted in an absolute increase in the FMD response of 1.1% (an ∼24% improvement from baseline) with a worsening of 0.3% in the placebo group (P < 0.001). A small improvement in the aortic pulse wave velocity (i.e., a decrease of 0.22 m/s; 95% CI: −0.4, −0.3 m/s) was evident in the nitrate group, showing a trend (P = 0.06) to improvement in comparison with the placebo group. Dietary nitrate also caused a small but significant reduction (7.6%) in platelet-monocyte aggregates compared with an increase of 10.1% in the placebo group (P = 0.004), with statistically significant reductions in stimulated (ex vivo) P-selectin expression compared with the placebo group (P < 0.05) but no significant changes in unstimulated expression. No adverse effects of dietary nitrate were detected. The composition of the salivary microbiome was altered after the nitrate treatment but not after the placebo treatment (P < 0.01). The proportions of 78 bacterial taxa were different after the nitrate treatment; of those taxa present, 2 taxa were responsible for >1% of this change, with the proportions of Rothia mucilaginosa trending to increase and Neisseria flavescens (P < 0.01) increased after nitrate treatment relative to after placebo treatment. Conclusions: Sustained dietary nitrate ingestion improves vascular function in hypercholesterolemic patients. These changes are associated with alterations in the oral microbiome and, in particular, nitrate-reducing genera. Our findings provide additional support for the assessment of the potential of dietary nitrate as a preventative strategy against atherogenesis in larger cohorts. This trial was registered at clinicaltrials.gov as NCT01493752.

Fish oil intakes providing dietary attainable levels of EPA and DHA reduces blood pressure in adults with systolic hypertension in a retrospective analysis

Background: Although a large number of randomized controlled trials (RCTs) have examined the impact of the n-3 (ω-3) fatty acids EPA (20:5n-3) and DHA (22:6n-3) on blood pressure and vascular function, the majority have used doses of EPA+DHA of > 3 g per d,which are unlikely to be achieved by diet manipulation. Objective: The objective was to examine, using a retrospective analysis from a multi-center RCT, the impact of recommended, dietary achievable EPA+DHA intakes on systolic and diastolic blood pressure and microvascular function in UK adults. Design: Healthy men and women (n = 312) completed a double-blind, placebo-controlled RCT consuming control oil, or fish oil providing 0.7 g or 1.8 g EPA+DHA per d in random order each for 8 wk. Fasting blood pressure and microvascular function (using Laser Doppler Iontophoresis) were assessed and plasma collected for the quantification of markers of vascular function. Participants were retrospectively genotyped for the eNOS rs1799983 variant. Results: No impact of n-3 fatty acid treatment or any treatment * eNOS genotype interactions were evident in the group as a whole for any of the clinical or biochemical outcomes. Assessment of response according to hypertension status at baseline indicated a significant (P=0.046) fish oil-induced reduction (mean 5 mmHg) in systolic blood pressure specifically in those with isolated systolic hypertension (n=31). No dose response was observed. Conclusions: These findings indicate that, in those with isolated systolic hypertension, daily doses of EPA+DHA as low as 0.7 g bring about clinically meaningful blood pressure reductions which, at a population level, would be associated with lower cardiovascular disease risk. Confirmation of findings in an RCT where participants are prospectively recruited on the basis of blood pressure status is required to draw definite conclusions. The Journal of Nutrition NUTRITION/2015/220475 Version 4

Fat mass-and obesity-associated genotype, dietary intakes and anthropometric measures in European adults: the Food4Me study

The interplay between the fat mass- and obesity-associated (FTO) gene variants and diet has been implicated in the development of obesity. The aim of the present analysis was to investigate associations between FTO genotype, dietary intakes and anthropometrics among European adults. Participants in the Food4Me randomised controlled trial were genotyped for FTO genotype (rs9939609) and their dietary intakes, and diet quality scores (Healthy Eating Index and PREDIMED-based Mediterranean diet score) were estimated from FFQ. Relationships between FTO genotype, diet and anthropometrics (weight, waist circumference (WC) and BMI) were evaluated at baseline. European adults with the FTO risk genotype had greater WC (AAv. TT: +1·4 cm; P=0·003) and BMI (+0·9 kg/m2; P=0·001) than individuals with no risk alleles. Subjects with the lowest fried food consumption and two copies of the FTO risk variant had on average 1·4 kg/m2 greater BMI (Ptrend=0·028) and 3·1 cm greater WC (Ptrend=0·045) compared with individuals with no copies of the risk allele and with the lowest fried food consumption. However, there was no evidence of interactions between FTO genotype and dietary intakes on BMI and WC, and thus further research is required to confirm or refute these findings.

Analysis of dietary pattern impact on weight status for personalised nutrition through on-line advice: the Food4Me Spanish cohort

Obesity prevalence is increasing. The management of this condition requires a detailed analysis of the global risk factors in order to develop personalised advice. This study is aimed to identify current dietary patterns and habits in Spanish population interested in personalised nutrition and investigate associations with weight status. Self-reported dietary and anthropometrical data from the Spanish participants in the Food4Me study, were used in a multidimensional exploratory analysis to define specific dietary profiles. Two opposing factors were obtained according to food groups’ intake: Factor 1 characterised by a more frequent consumption of traditionally considered unhealthy foods; and Factor 2, where the consumption of “Mediterranean diet” foods was prevalent. Factor 1 showed a direct relationship with BMI (β = 0.226; r2 = 0.259; p < 0.001), while the association with Factor 2 was inverse (β = −0.037; r2 = 0.230; p = 0.348). A total of four categories were defined (Prudent, Healthy, Western, and Compensatory) through classification of the sample in higher or lower adherence to each factor and combining the possibilities. Western and Compensatory dietary patterns, which were characterized by high-density foods consumption, showed positive associations with overweight prevalence. Further analysis showed that prevention of overweight must focus on limiting the intake of known deleterious foods rather than exclusively enhance healthy products.

Interactions between cocoa flavanols and inorganic nitrate: additive effects on endothelial function at achievable dietary amounts

Dietary intervention studies have shown that flavanols and inorganic nitrate can improve vascular function, suggesting that these two bioactives may be responsible for beneficial health effects of diets rich in fruits and vegetables. We aimed to study interactions between cocoa flavanols (CF) and nitrate, focusing on absorption, bioavailability, excretion, and efficacy to increase endothelial function. In a double-blind randomized, dose-response crossover study, flow-mediated dilation (FMD) was measured in 15 healthy subjects before and at 1, 2, 3, and 4 h after consumption of CF (1.4-10.9 mg/kg bw) or nitrate (0.1-10 mg/kg bw). To study flavanol-nitrate interactions, an additional intervention trial was performed with nitrate and CF taken in sequence at low and high amounts. FMD was measured before (0 h) and at 1h after ingestion of nitrate (3 or 8.5 mg/kg bw) or water. Then subjects received a CF drink (2.7 or 10.9 mg/kg bw) or a micro- and macronutrient-matched CF-free drink. FMD was measured at 1, 2, and 4 h thereafter. Blood and urine samples were collected and assessed for CF and nitric oxide (NO) metabolites with HPLC and gas-phase reductive chemiluminescence. Finally, intragastric formation of NO after CF and nitrate consumption was investigated. Both CF and nitrate induced similar intake-dependent increases in FMD. Maximal values were achieved at 1 h postingestion and gradually decreased to reach baseline values at 4 h. These effects were additive at low intake levels, whereas CF did not further increase FMD after high nitrate intake. Nitrate did not affect flavanol absorption, bioavailability, or excretion, but CF enhanced nitrate-related gastric NO formation and attenuated the increase in plasma nitrite after nitrate intake. Both flavanols and inorganic nitrate can improve endothelial function in healthy subjects at intake amounts that are achievable with a normal diet. Even low dietary intake of these bioactives may exert relevant effects on endothelial function when ingested together.

Insulin resistance determines a differential response to changes in dietary fat modification on metabolic syndrome risk factors: the LIPGENE study

Background: Previous data support the benefits of reducing dietary saturated fatty acids (SFAs) on insulin resistance (IR) and other metabolic risk factors. However, whether the IR status of those suffering from metabolic syndrome (MetS) affects this response is not established. OBJECTIVE: Our objective was to determine whether the degree of IR influences the effect of substituting high-saturated fatty acid (HSFA) diets by isoenergetic alterations in the quality and quantity of dietary fat on MetS risk factors. DESIGN: In this single-blind, parallel, controlled, dietary intervention study, MetS subjects (n = 472) from 8 European countries classified by different IR levels according to homeostasis model assessment of insulin resistance (HOMA-IR) were randomly assigned to 4 diets: an HSFA diet; a high-monounsaturated fatty acid (HMUFA) diet; a low-fat, high-complex carbohydrate (LFHCC) diet supplemented with long-chain n-3 polyunsaturated fatty acids (1.2 g/d); or an LFHCC diet supplemented with placebo for 12 wk (control). Anthropometric, lipid, inflammatory, and IR markers were determined. RESULTS: Insulin-resistant MetS subjects with the highest HOMA-IR improved IR, with reduced insulin and HOMA-IR concentrations after consumption of the HMUFA and LFHCC n-3 diets (P < 0.05). In contrast, subjects with lower HOMA-IR showed reduced body mass index and waist circumference after consumption of the LFHCC control and LFHCC n-3 diets and increased HDL cholesterol concentrations after consumption of the HMUFA and HSFA diets (P < 0.05). MetS subjects with a low to medium HOMA-IR exhibited reduced blood pressure, triglyceride, and LDL cholesterol levels after the LFHCC n-3 diet and increased apolipoprotein A-I concentrations after consumption of the HMUFA and HSFA diets (all P < 0.05). CONCLUSIONS: Insulin-resistant MetS subjects with more metabolic complications responded differently to dietary fat modification, being more susceptible to a health effect from the substitution of SFAs in the HMUFA and LFHCC n-3 diets. Conversely, MetS subjects without IR may be more sensitive to the detrimental effects of HSFA intake. The metabolic phenotype of subjects clearly determines response to the quantity and quality of dietary fat on MetS risk factors, which suggests that targeted and personalized dietary therapies may be of value for its different metabolic features.

Production of acid whey hydrolysates applying an integrative process: effect of calcium on process performance

High ionic calcium concentration and the absence of caseinmacropeptides (CMP) in acid whey could influence the production of angiotensin-I-converting enzyme (ACE)-inhibitory hydrolysate and its bioactivity through the application of the integrative process. Therefore, the aim of the present study was to produce a hydrolysate from acid whey applying the integrative process. Process performance was evaluated based on protein adsorption capacity and conversion in relation to ACE-inhibitory activity (ACEi%) and ionic calcium concentration. Hydrolysates with high potency of their biological activity were produced (IC50 = 206-353 μg mL-1). High ionic calcium concentration in acid whey contributed to ACE-inhibitory activity. However, low β-lactoglobulin adsorption and conversion was observed. Optimisation of the resin volume increased the adsorption of β-lactoglobulin significantly but with lower selectivity. The changes in conversion value were not significant even at higher concentration of enzyme. Several ACE inhibitors derived from β-lactoglobulin that were identified before in sweet whey hydrolysates such as, IIAEKT, IIAE, IVTQ, LIVTQ, LIVTQT, LDAQ and LIVT were found. New peptides such as, SNICNI and ECCHGD derived from α-lactalbumin and BSA respectively were identified.

Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17beta-estradiol (E(2)), can potentiate the transcription mediated by E(2) from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E(2) demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E(2) in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens. Copyright (c) 2005 S. Karger AG, Basel.

Reproducibility of the online Food4Me food-frequency questionnaire for estimating dietary intakes across Europe

Background: Accurate dietary assessment is key to understanding nutrition-related outcomes and is essential for estimating dietary change in nutrition-based interventions. Objective: The objective of this study was to assess the pan-European reproducibility of the Food4Me food-frequency questionnaire (FFQ) in assessing the habitual diet of adults. Methods: Participantsfromthe Food4Me study, a 6-mo,Internet-based, randomizedcontrolled trial of personalized nutrition conducted in the United Kingdom, Ireland, Spain, Netherlands, Germany, Greece, and Poland were included. Screening and baseline data (both collected before commencement of the intervention) were used in the present analyses, and participants were includedonly iftheycompleted FFQs at screeningand at baselinewithin a 1-mo timeframebeforethe commencement oftheintervention. Sociodemographic (e.g., sex andcountry) andlifestyle[e.g.,bodymass index(BMI,inkg/m2)and physical activity] characteristics were collected. Linear regression, correlation coefficients, concordance (percentage) in quartile classification, and Bland-Altman plots for daily intakes were used to assess reproducibility. Results: In total, 567 participants (59% female), with a mean 6 SD age of 38.7 6 13.4 y and BMI of 25.4 6 4.8, completed bothFFQswithin 1 mo(mean 6 SD: 19.26 6.2d).Exact plus adjacent classification oftotal energy intakeinparticipants was highest in Ireland (94%) and lowest in Poland (81%). Spearman correlation coefficients (r) in total energy intake between FFQs ranged from 0.50 for obese participants to 0.68 and 0.60 in normal-weight and overweight participants, respectively. Bland-Altman plots showed a mean difference between FFQs of 210 kcal/d, with the agreement deteriorating as energy intakes increased. There was little variation in reproducibility of total energy intakes between sex and age groups. Conclusions: The online Food4Me FFQ was shown to be reproducible across 7 European countries when administered within a 1-mo period to a large number of participants. The results support the utility of the online Food4Me FFQ as a reproducible tool across multiple European populations. This trial was registered at clinicaltrials.gov as NCT01530139.

Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste

Aim. To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Design. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Results. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P < 0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Conclusion. Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.