New CZE-DAD method for honeybee venom analysis and standardization of the product

The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 μm ID, and 360 μm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 μA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002–2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A2 from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A2, and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10(-8), in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms

Age- and Gender-Related Changes in Contractile Properties of Non-Atrophied EDL Muscle

Background: In humans, ageing causes skeletal muscles to become atrophied, weak, and easily fatigued. In rodent studies, ageing has been associated with significant muscle atrophy and changes in the contractile properties of the muscles. However, it is not entirely clear whether these changes in contractile properties can occur before there is significant atrophy, and whether males and females are affected differently. Methods and Results: We investigated various contractile properties of whole isolated fast-twitch EDL muscles from adult (2–6 months-old) and aged (12–22 months-old) male and female mice. Atrophy was not present in the aged mice. Compared with adult mice, EDL muscles of aged mice had significantly lower specific force, longer tetanus relaxation times, and lower fatiguability. In the properties of absolute force and muscle relaxation times, females were affected by ageing to a greater extent than males. Additionally, EDL muscles from a separate group of male mice were subjected to eccentric contractions of 15 % strain, and larger force deficits were found in aged than in adult mice. Conclusion: Our findings provide further insight into the muscle atrophy, weakness and fatiguability experienced by the elderly. We have shown that even in the absence of muscle atrophy, there are definite alterations in the physiological properties of whole fast-twitch muscle from ageing mice, and for some of these properties the alterations are mor

Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged

We investigate changes in human c-type lysozyme flexibility upon mutation via a Distance Constraint Model, which gives a statistical mechanical treatment of network rigidity. Specifically, two dynamical metrics are tracked. Changes in flexibility index quantify differences within backbone flexibility, whereas changes in the cooperativity correlation quantify differences within pairwise mechanical couplings. Regardless of metric, the same general conclusions are drawn. That is, small structural perturbations introduced by single point mutations have a frequent and pronounced affect on lysozyme flexibility that can extend over long distances. Specifically, an appreciable change occurs in backbone flexibility for 48% of the residues, and a change in cooperativity occurs in 42% of residue pairs. The average distance from mutation to a site with a change in flexibility is 17–20 Å. Interestingly, the frequency and scale of the changes within single point mutant structures are generally larger than those observed in the hen egg white lysozyme (HEWL) ortholog, which shares 61% sequence identity with human lysozyme. For example, point mutations often lead to substantial flexibility increases within the β-subdomain, which is consistent with experimental results indicating that it is the nucleation site for amyloid formation. However, β-subdomain flexibility within the human and HEWL orthologs is more similar despite the lowered sequence identity. These results suggest compensating mutations in HEWL reestablish desired properties

Acute-Phase-HDL Remodeling by Heparan Sulfate Generates a Novel Lipoprotein with Exceptional Cholesterol Efflux Activity from Macrophages

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDL∶HS interactions we discovered that HS caused significant remodeling of AP-HDL. The physical nature of this change in structure and its potential importance for cholesterol efflux from cholesterol-loaded macrophages was therefore investigated. In the presence of heparin, or HS, AP-HDL solutions at pH 5.2 became turbid within minutes. Analysis by centrifugation and gel electrophoresis indicated that AP-HDL was remodeled generating novel lipid poor particles composed only of apolipoprotein AI, which we designate β2. This remodeling is dependent on pH, glycosaminoglycan type, is promoted by Ca2+ and is independent of protease or lipase activity. Compared to HDL and AP-HDL, remodeled AP-HDL (S-HDL-SAA), containing β2 particles, demonstrated a 3-fold greater cholesterol efflux activity from cholesterol-loaded macrophage. Because the identified conditions causing this change in AP-HDL structure and function can exist physiologically at the surface of the macrophage, or in its endosomes, we postulate that AP-HDL contains latent functionalities that become apparent and active when it associates with macrophage cell surface/endosomal HS. In this way initial steps in the reverse cholesterol transport pathway are focused at sites of injury to mobilize cholesterol from macrophages that are actively participating in the phagocytosis of damaged membranes rich in cholesterol. The mechanism may also be of relevance to aspects of atherogenesis

Saturated Fats: A Perspective from Lactation and Milk Composition

For recommendations of specific targets for the absolute amount of saturated fat intake, we need to know what dietary intake is most appropriate? Changing agricultural production and processing to lower the relative quantities of macronutrients requires years to accomplish. Changes can have unintended consequences on diets and the health of subsets of the population. Hence, what are the appropriate absolute amounts of saturated fat in our diets? Is the scientific evidence consistent with an optimal intake of zero? If not, is it also possible that a finite intake of saturated fats is beneficial to overall health, at least to a subset of the population? Conclusive evidence from prospective human trials is not available, hence other sources of information must be considered. One approach is to examine the evolution of lactation, and the composition of milks that developed through millennia of natural selective pressure and natural selection processes. Mammalian milks, including human milk, contain 50% of their total fatty acids as saturated fatty acids. The biochemical formation of a single double bond converting a saturated to a monounsaturated fatty acid is a pathway that exists in all eukaryotic organisms and is active within the mammary gland. In the face of selective pressure, mammary lipid synthesis in all mammals continues to release a significant content of saturated fatty acids into milk. Is it possible that evolution of the mammary gland reveals benefits to saturated fatty acids that current recommendations do not consider

A reappraisal of the impact of dairy foods and milk fat on cardiovascular disease risk

Background This review provides a reappraisal of the potential effects of dairy foods, including dairy fats, on cardiovascular disease (CVD)/coronary heart disease (CHD) risk. Commodities and foods containing saturated fats are of particular focus as current public dietary recommendations are directed toward reducing the intake of saturated fats as a means to improve the overall health of the population. A conference of scientists from different perspectives of dietary fat and health was convened in order to consider the scientific basis for these recommendations. Aims This review and summary of the conference focus on four key areas related to the biology of dairy foods and fats and their potential impact on human health: (a) the effect of dairy foods on CVD in prospective cohort studies; (b) the impact of dairy fat on plasma lipid risk factors for CVD; (c) the effects of dairy fat on non-lipid risk factors for CVD; and (d) the role of dairy products as essential contributors of micronutrients in reference food patterns for the elderly. Conclusions Despite the contribution of dairy products to the saturated fatty acid composition of the diet, and given the diversity of dairy foods of widely differing composition, there is no clear evidence that dairy food consumption is consistently associated with a higher risk of CVD. Thus, recommendations to reduce dairy food consumption irrespective of the nature of the dairy product should be made with cautionJ. Bruce German, Robert A. Gibson, Ronald M. Krauss, Paul Nestel, Benoît Lamarche, Wija A. van Staveren, Jan M. Steijns, Lisette C. P. G. M. de Groot, Adam L. Lock and Frédéric Destaillat

Genetic drivers of heterogeneity in type 2 diabetes pathophysiology.

Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P < 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care

Genome-wide association study identifies six new loci influencing pulse pressure and mean arterial pressure.

Numerous genetic loci have been associated with systolic blood pressure (SBP) and diastolic blood pressure (DBP) in Europeans. We now report genome-wide association studies of pulse pressure (PP) and mean arterial pressure (MAP). In discovery (N = 74,064) and follow-up studies (N = 48,607), we identified at genome-wide significance (P = 2.7 × 10(-8) to P = 2.3 × 10(-13)) four new PP loci (at 4q12 near CHIC2, 7q22.3 near PIK3CG, 8q24.12 in NOV and 11q24.3 near ADAMTS8), two new MAP loci (3p21.31 in MAP4 and 10q25.3 near ADRB1) and one locus associated with both of these traits (2q24.3 near FIGN) that has also recently been associated with SBP in east Asians. For three of the new PP loci, the estimated effect for SBP was opposite of that for DBP, in contrast to the majority of common SBP- and DBP-associated variants, which show concordant effects on both traits. These findings suggest new genetic pathways underlying blood pressure variation, some of which may differentially influence SBP and DBP