Milk fat miRNome changes in response to LPS challenge in Holstein cows.

Mastitis is an inflammatory disease in dairy cows, causing economic losses and reducing animal welfare. In order to contribute for the discovery of early and noninvasive indicators, our objective was to determine the effects of a lipopolysaccharide (LPS) challenge on the microRNA profile (miRNome) of milk fat, using microarray analyses in cows. Cows were fed a lactation diet at ad libitum intake (n = 6). At 27 ± 3 days in milk, cows were injected with 50 µg of LPS Escherichia coli in one healthy rear mammary quarter. Milk samples were collected just before LPS challenge (LPS-) and 6.5 h after LPS challenge (LPS +) from the same cows. Microarray analysis was performed using customized 8 × 60 K ruminant miRNA microarrays to compare LPS- to LPS + miRNome. In silico functional analyses were performed using OmicsNet and Mienturnet software. MiRNome comparison between LPS- and LPS + identified 37 differentially abundant miRNAs (q-value ≤ 0.05). The predicted target genes of the 37 differentially abundant miRNAs are mostly involved in cell life including apoptosis, cell cycle, proliferation and differentiation and in gene expression processes. MiRNome analyses suggest that miRNAs profile is related to the inflammation response of the mammary gland. In conclusion, we demonstrated that milk fat might be an easy and rapid source of miRNAs that are potential indicators of early mastitis in cows.

Comparison of goat and cow milk-derived extracellular vesicle miRNomes.

miRNAs present in milk are mainly found in extracellular vesicles (EVs), which are nanosized membrane vesicles released by most of the cell types to ensure intercellular communication. The majority of the studies performed so far on these vesicles have been conducted on human and cow's milk and focused on their miRNA content. The objectives of this study were to profile the miRNA content of purified EVs from five healthy goats and to compare their miRNome to those obtained from five healthy cows, at an early stage of lactation. EV populations were morphologically characterized using Transmission Electron Microscopy and Nanoparticle Tracking Analysis. The presence of EV protein markers checked by Western blotting and the absence of contamination of preparations by milk proteins. The size distribution and concentration of bovine and goat milk-derived EVs were similar. RNA-sequencing were performed, and all sequences were mapped to the cow genome identifying a total of 295 miRNAs. This study reports for the first-time a goat miRNome from milk EVs and its validation using cow miRNomes.

Comparative transcriptome analysis of goat (Capra hircus) adipose tissue reveals physiological regulation of body reserve recovery after the peak of lactation.

Adipose tissue is the energy storage organ providing energy to other tissues, including mammary gland, that supports the achievement of successive lactation cycles. Our objective was to investigate the ability of goats to restore body fat reserves by comparing lipogenic enzyme activities and by transcriptomic RNA-Seq data at two different physiological stages, mid- and post-lactation. Key lipogenic enzyme activities were higher in goat omental adipose tissue during mid-lactation (74 days in milk) than during the post-lactation period (300 days postpartum). RNA-Sequencing analysis revealed 19,271 expressed genes in the omental adipose tissue. The comparison between adipose transcriptome analysis from mid- and post-lactation goats highlighted 252 differentially expressed genes (padj < 0.05) between these two physiological stages. The differential expression of 11 genes was confirmed by RT-qPCR. Functional genomic analysis revealed that 31% were involved in metabolic processes among which 38% in lipid metabolism. Most of the genes involved in lipid synthesis and those in lipid transport and storage were upregulated in adipose tissue of mid- compared to post-lactation goats. In addition, adipose tissue plasticity was emphasized by genes involved in cellular signaling and tissue integrity. Network analyses also highlighted three key regulators of lipid metabolism (LEP, APOE and HNF4A) and a key target gene (VCAM1). The greatest lipogenic enzyme activities with the upregulation of genes involved in lipid metabolism highlighted a higher recovery of lipid reserves after the lactation peak than 4 months post-lactation. This study contributes to a better understanding of the molecular mechanisms controlling the body lipid reserves management during the successive lactations.

Perspective: Milk microRNAs as Important Players in Infant Physiology and Development.

Evolutionary selective pressure on lactation has resulted in milk that provides far more than simply essential nutrients, delivering a complex repertoire of agents from hormones to intact cells. Human infants are born with low barrier integrity of their gut, which means that many of the complex biopolymer components of milk enter and circulate in lymph and blood, reaching organs throughout the body. Due to this state of gut maturation, all components of milk are potentially part of the crosstalk between mother and infants. This article highlights the functions of milk's complex biopolymers, more specifically the potential role of microRNAs (miRNAs) contained in extracellular vesicles in human milk. miRNAs are key effectors in the regulation of many biological processes during early-age development, and consequently milk-sourced miRNAs must be considered to provide unique biological assets to the infant during breastfeeding. This article interprets the evidence of the potential action of human milk miRNAs on infant development, taking into account their abundance in milk based on the literature and current knowledge. Human milk miRNAs appear to influence lipid and glucose metabolism, gut maturation, neurogenesis, and immunity. We also show growing evidence that human milk miRNAs are epigenetic modulators that play a pivotal role in the regulation of tissue-specific gene expression throughout life. Furthermore, this article addresses the ongoing debate regarding the potential influence of human milk miRNAs on viral infection as a new research area. This article highlights that these bioactive molecules are now being incorporated into our overall understanding of nutrient needs for healthy infant development, preparing each individual infant to succeed as a healthy and protected adult throughout its life. In essence, miRNAs are a new language in the Rosetta stone of health that is mammalian lactation.

Nutrigenomic analyses reveal miRNAs and mRNAs affected by feed restriction in the mammary gland of midlactation dairy cows.

The objective of this study was to investigate the effects of feed restriction on mammary miRNAs and coding gene expression in midlactation cows. Five Holstein cows and 6 Montbéliarde cows underwent 6 days of feed restriction, during which feed allowance was reduced to meet 50% of their net energy for lactation requirements. Mammary biopsies were performed before and at the end of the restriction period. Mammary miRNA and mRNA analyses were performed using high-throughput sequencing and microarray analyses, respectively. Feed restriction induced a negative energy balance and decreased milk production and fat and protein yields in both breeds. Feed restriction modified the expression of 27 miRNAs and 374 mRNAs in mammary glands from Holstein cows, whereas no significant miRNA change was observed in Montbéliarde cows. Among the 27 differentially expressed miRNAs, 8 miRNAs were associated with dairy QTL. Analysis of target genes indicate that the 8 most abundantly expressed miRNAs control transcripts related to lipid metabolism, mammary remodeling and stress response. A comparison between the mRNAs targeted by the 8 most strongly expressed miRNAs and 374 differentially expressed mRNAs identified 59 mRNAs in common. The bioinformatic analyses of these 59 mRNAs revealed their implication in lipid metabolism and endothelial cell proliferation. These effects of feed restriction on mammary miRNAs and mRNAs observed in Holstein cows suggest a potential role of miRNAs in mammary structure and lipid biosynthesis that could explain changes in milk production and composition.

Nutritional Regulation of Mammary Gland Development and Milk Synthesis in Animal Models and Dairy Species.

In mammals, milk is essential for the growth, development, and health. Milk quantity and quality are dependent on mammary development, strongly influenced by nutrition. This review provides an overview of the data on nutritional regulations of mammary development and gene expression involved in milk component synthesis. Mammary development is described related to rodents, rabbits, and pigs, common models in mammary biology. Molecular mechanisms of the nutritional regulation of milk synthesis are reported in ruminants regarding the importance of ruminant milk in human health. The effects of dietary quantitative and qualitative alterations are described considering the dietary composition and in regard to the periods of nutritional susceptibly. During lactation, the effects of lipid supplementation and feed restriction or deprivation are discussed regarding gene expression involved in milk biosynthesis, in ruminants. Moreover, nutrigenomic studies underline the role of the mammary structure and the potential influence of microRNAs. Knowledge from three lactating and three dairy livestock species contribute to understanding the variety of phenotypes reported in this review and highlight (1) the importance of critical physiological stages, such as puberty gestation and early lactation and (2) the relative importance of the various nutrients besides the total energetic value and their interaction.

Different miRNA contents between mammary epithelial cells and milk fat globules: a random or a targeted process?

MicroRNAs (miRNAs) are small noncoding RNAs present in milk-derived extracellular vesicles and milk fat globules (MFG). Nucleic acid content between the lactating mammary tissue (MT) and MFG are quite similar but discrepancies exist in their miRNA content. Our objective was to identify the origin of these discrepancies, and to evaluate the existence of a possible mechanism sorting miRNAs that will or will not be exported from the mammary epithelial cells (MECs) in bovine MFG. miR-125b-5p, miR-126-3p, miR-141-3p, and miR-204-5p, chosen on the basis of their abundance in the MT, were quantified using RT-qPCR in lactating cow MT, MFG, and laser capture-microdissected MECs. Two miRNAs (miR-125b-5p and miR-141-3p) were detected in the MT as well as in MFG and MECs. miR-204-5p was detected only in the MT, suggesting that it is very likely expressed in a cell type other than MECs. miR-126-3p was detected both in the MT and in MECs but not in MFG, suggesting a targeting mechanism for miRNAs in MECs. These results highlights differences in miRNA content between MECs and MFG may be due to a possibly not random mechanism for loading MFG with miRNA cargos that could involve a variable distribution in MECs or a sorting mechanism.

Si Doping of Vapor-Liquid-Solid GaAs Nanowires: n-Type or p-Type?

The incorporation of Si into vapor-liquid-solid GaAs nanowires often leads to p-type doping, whereas it is routinely used as an n-dopant of planar layers. This property limits the applications of GaAs nanowires in electronic and optoelectronic devices. The strong amphoteric behavior of Si in nanowires is not yet fully understood. Here, we present the first attempt to quantify this behavior as a function of the droplet composition and temperature. It is shown that the doping type critically depends on the As/Ga ratio in the droplet. In sharp contrast to vapor-solid growth, the droplet contains very few As atoms, which enhance their reverse transfer from solid to liquid. As a result, Si atoms preferentially replace As in GaAs, leading to p-type doping in nanowires. Hydride vapor phase epitaxy provides the highest As concentrations in the catalyst droplets during their vapor-liquid-solid growth, resulting in n-type dopant behavior of Si. We present experimental data on n-doped Si-doped GaAs nanowires grown by this method and explain the doping within our model. These results give a clear route for obtaining n-type or p-type Si doping in GaAs nanowires and may be extended to other III-V nanowires.

Mammary Gland Transcriptome and Proteome Modifications by Nutrient Restriction in Early Lactation Holstein Cows Challenged with Intra-Mammary Lipopolysaccharide.

: The objective is to study the effects of nutrient restrictions, which induce a metabolic imbalance on the inflammatory response of the mammary gland in early lactation cows. The aim is to decipher the molecular mechanisms involved, by comparing a control, with a restriction group, a transcriptome and proteome, after an intra-mammary lipopolysaccharide challenge. Multi-parous cows were either allowed ad libitum intake of a lactation diet (n = 8), or a ration containing low nutrient density (n = 8; 48% barley straw and dry matter basis) for four days starting at 24 ± 3 days in milk. Three days after the initiation of their treatments, one healthy rear mammary quarter of 12 lactating cows was challenged with 50 µg of lipopolysaccharide (LPS). Transcriptomic and proteomic analyses were performed on mammary biopsies obtained 24 h after the LPS challenge, using bovine 44K microarrays, and nano-LC-MS/MS, respectively. Restriction-induced deficits in energy, led to a marked negative energy balance (41 versus 97 ± 15% of Net Energy for Lactation (NEL) requirements) and metabolic imbalance. A microarray analyses identified 25 differentially expressed genes in response to restriction, suggesting that restriction had modified mammary metabolism, specifically ß-oxidation process. Proteomic analyses identified 53 differentially expressed proteins, which suggests that the modification of protein synthesis from mRNA splicing to folding. Under-nutrition influenced mammary gland expression of the genes involved in metabolism, thereby increasing ß-oxidation and altering protein synthesis, which may affect the response to inflammation.

Defects of the endoplasmic reticulum and changes to lipid droplet size in mammary epithelial cells due to miR-30b-5p overexpression are correlated to a reduction in Atlastin 2 expression.

During lactation, mammary epithelial cells secrete fat in the form of milk fat globules that originate from intracellular lipid droplets. These droplets may form de novo from the endoplasmic reticulum or be derived from existing lipid droplets; they then either grow because enzymes of triacylglycerol synthesis relocate from the reticulum to their surface, or due to fusion and fission with other droplets. The overexpression of miR-30b-5p in the developing mouse mammary gland impairs lactation, which includes an increase in lipid droplet size. This study was performed to understand the origin of this defect affecting lipid droplets observed in transgenic mice. Electron microscopy analyses revealed a fragmented and discontinued tubular network of endoplasmic reticulum in the mammary epithelial cells of transgenic mice. The milk fatty acid composition was modified, with lower levels of medium-chain saturated fatty acids and a proportional increase in long-chain monounsaturated fatty acids in transgenic versus wild-type mice. Further, investigations of microRNA targets revealed a significant downregulation of ATLASTIN 2 (a GTPase described as playing a key role in lipid droplet formation) due to miR-30b-5p overexpression. Our results suggest that the increase in lipid droplet size observed in the mammary epithelial cells of transgenic mice might result from changes to lipid droplet formation and secretion because of direct modifications to Atl2 expression and indirect changes to endoplasmic reticulum morphology resulting from the overexpression of miR-30b-5p.

Effects of Azgp1-/-on mammary gland, adipose tissue and liver gene expression and milk lipid composition in lactating mice.

The expression of Azgp1 gene, an adipokine involved in the mobilization of body reserves, was observed in mammary gland of ruminants. Its regulation by different dietary conditions suggests a potential role in the mechanisms controlling the composition of milk fat. The aim of this study was to evaluate the role of Azgp1 during lactation. Azgp1-/- mice were compared to wild-type to determine its effects on milk fatty acid composition and offspring growth. To determine its effects on mammary gland, adipose tissue and liver gene expression, gene expression was analyzed using RT-qPCR via TLDA analyses. The body weight of Azgp1-/- mothers was slightly higher after parturition and at 10 days of lactation compared to the wild type. The milk polyunsaturated fatty acid content was increased in Azgp1-/- mice. Among the 40 genes studied, Azgp1-/- modified the expression of 9, 10 and 3 genes in mammary gland, adipose tissue and liver, respectively. These genes, involved in fatty acid synthesis, transport and triglyceride synthesis, were downregulated in Azgp1-/- mice showing a particularity during lactation. Changes in mammary gland gene expression may explain the modifications observed in milk fatty acid composition. This study supports a role of Azgp1 on lipid metabolism, in particular in mammary gland, during lactation function.

Sunflower oil supplementation affects the expression of miR-20a-5p and miR-142-5p in the lactating bovine mammary gland.

Oil supplementation in dairy cattle diets is used to modulate milk fat composition, as well as the expression of mammary lipogenic genes, whose regulation remains unclear. MiRNAs are small non-coding RNA considered as crucial regulators of gene expression, offering clues to explain the mechanism underlying gene nutriregulation. The present study was designed to identify miRNAs whose expression in the cow mammary gland is modulated by sunflower oil supplementation. MiRNomes were obtained using RNAseq technology from the mammary gland of lactating cows receiving a low forage diet, supplemented or not with 4% sunflower oil. Among the 272 miRNAs characterized, eight were selected for RT-qPCR validations, showing the significant down-regulation of miR-142-5p and miR-20a-5p by sunflower supplementation. These two miRNAs are predicted to target genes whose expression was reported as differentially expressed by sunflower supplementation. Among their putative targets, ELOVL6 gene involved in lipid metabolism has been studied. However, a first analysis did not show its significant down-regulation, in response to the over-expression of miR-142-5p, of miR-20a-5p, or both, in a bovine mammary epithelial cell line. However, a clearer understanding of the miRNA expression by lipid supplementation would help to decipher the regulation of lactating cow mammary gland in response to nutrition.

Sr1/2Ce5/14□1/7WO4: a new modulated ternary scheelite compound.

For the first time, a ternary tetragonal scheelite structure tungstate with strontium and cerium cations, (Sr,Ce)WO4, was synthesized. As much as 35% Ce could be inserted into the structure, leaving 1\over 7 of the (Sr,Ce) cation sites vacant. Partial ordering of Sr and Ce, with atomic displacements, were shown by high-resolution electron microscopy. Two-dimensional incommensurate modulations occur in this material, in small domains 20 nm in size. The band gap of this compound is significantly lower than the band gap of SrWO4 and this was related to the distortions of WO4 and (Sr,Ce)O8 polyhedra. The band gap value of 3.2 eV makes Sr1/2Ce5/14□1/7WO4 a promising candidate for violet luminescence.

Self-catalyzed GaAs nanowires on silicon by hydride vapor phase epitaxy.

Gold-free GaAs nanowires on silicon substrates can pave the way for monolithic integration of photonic nanodevices with silicon electronic platforms. It is extensively documented that the self-catalyzed approach works well in molecular beam epitaxy but is much more difficult to implement in vapor phase epitaxies. Here, we report the first gallium-catalyzed hydride vapor phase epitaxy growth of long (more than 10 µm) GaAs nanowires on Si(111) substrates with a high integrated growth rate up to 60 µm h-1 and pure zincblende crystal structure. The growth is achieved by combining a low temperature of 600 °C with high gaseous GaCl/As flow ratios to enable dechlorination and formation of gallium droplets. GaAs nanowires exhibit an interesting bottle-like shape with strongly tapered bases, followed by straight tops with radii as small as 5 nm. We present a model that explains the peculiar growth mechanism in which the gallium droplets nucleate and rapidly swell on the silicon surface but then are gradually consumed to reach a stationary size. Our results unravel the necessary conditions for obtaining gallium-catalyzed GaAs nanowires by vapor phase epitaxy techniques.

Bovine Mammary Nutrigenomics and Changes in the Milk Composition due to Rapeseed or Sunflower Oil Supplementation of High-Forage or High-Concentrate Diets.

BACKGROUND: Fatty acid (FA) composition plays a crucial role in milk nutritional quality. Despite the known nutritional regulation of ruminant milk composition, the overall mammary mechanisms underlying this regulation are far from being understood. The aim of our study was to determine nutritional regulation of mammary transcriptomes in relation to the cow milk composition. METHODS: Twelve cows received diets differing in the forage-to-concentrate ratio [high forage (HF) and low forage (LF)] supplemented or not with lipids [HF with whole intact rapeseeds (RS) and LF sunflower oil (SO)] in a 4 × 4 Latin square design. Milk production and FA composition were determined. The gene expression profile was studied using RT-qPCR and a bovine microarray. RESULTS: Our results showed a higher amplitude of milk composition and mammary transcriptome responses to lipid supplementation with the LF-SO compared with the LF diet than with the HF-RS compared with the HF diet. Forty-nine differentially expressed genes, including genes involved in lipid metabolism, were identified with LF-SO versus LF, whereas RS supplementation to the HF diet did not affect the mammary transcriptome. CONCLUSIONS: This study highlights different responses to lipid supplementation of milk production and composition and mammary transcriptomes depending on the nature of lipid supplementation and the percentage of dietary concentrate.

Food Deprivation Affects the miRNome in the Lactating Goat Mammary Gland.

BACKGROUND: Nutrition affects milk composition thus influencing its nutritional properties. Nutrition also modifies the expression of mammary genes, whose regulation is not fully understood. MicroRNAs (miRNA) are small non coding RNA which are important post-transcriptional regulators of gene expression by targeting messenger RNAs. Our goal was to characterize miRNA whose expression is regulated by nutrition in the lactating goat mammary gland, which may provide clues to deciphering regulations of the biosynthesis and secretion of milk components. METHODOLOGY/PRINCIPAL FINDINGS: Using high-throughput sequencing technology, miRNomes of the lactating mammary gland were established from lactating goats fed ad libitum or deprived of food for 48 h affecting milk production and composition. High throughput miRNA sequencing revealed 30 miRNA with an expression potentially modulated by food deprivation; 16 were down-regulated and 14 were up-regulated. Diana-microT predictive tools suggested a potential role for several nutriregulated miRNA in lipid metabolism. Among the putative targets, 19 were previously identified as differently expressed genes (DEG). The functions of these 19 DEG revealed, notably, their involvement in tissue remodelling. CONCLUSION/SIGNIFICANCE: In conclusion, this study offers the first evidence of nutriregulated miRNA in the ruminant mammary gland. Characterization of these 30 miRNA could contribute to a clearer understanding of gene regulation in the mammary gland in response to nutrition.

Annotation of the goat genome using next generation sequencing of microRNA expressed by the lactating mammary gland: comparison of three approaches.

BACKGROUND: MicroRNAs (miRNA) are small endogenous non-coding RNA involved in the post-transcriptional regulation of specific mRNA targets. The first whole goat genome sequence became available in 2013, with few annotations. Our goal was to establish a list of the miRNA expressed in the mammary gland of lactating goats, thus enabling implementation of the goat miRNA repertoire and considerably enriching annotation of the goat genome. RESULTS: Here, we performed high throughput RNA sequencing on 10 lactating goat mammary glands. The bioinformatic detection of miRNA was carried out using miRDeep2 software. Three different methods were used to predict, quantify and annotate the sequenced reads. The first was a de novo approach based on the prediction of miRNA from the goat genome only. The second approach used bovine miRNA as an external reference whereas the last one used recently available goat miRNA. The three methods enabled the prediction and annotation of hundreds of miRNA, more than 95% were commonly identified. Using bovine miRNA, 1,178 distinct miRNA were detected, together with the annotation of 88 miRNA for which corresponding precursors could not be retrieved in the goat genome, and which were not detected using the de novo approach or with the use of goat miRNA. Each chromosomal coordinate of the precursors determined here were generated and depicted on a reference localisation map. Forty six goat miRNA clusters were also reported. The study revealed 263 precursors located in goat protein-coding genes, amongst which the location of 43 precursors was conserved between human, mouse and bovine, revealing potential new gene regulations in the goat mammary gland. Using the publicly available cattle QTL database, and cow precursors conserved in the goat and expressed in lactating mammary gland, 114 precursors were located within known QTL regions for milk production and composition. CONCLUSIONS: The results reported here represent the first major identification study on miRNA expressed in the goat mammary gland at peak lactation. The elements generated by this study will now be used as references to decipher the regulation of miRNA expression in the goat mammary gland and to clarify their involvement in the lactation process.

Record pure zincblende phase in GaAs nanowires down to 5 nm in radius.

We report the Au catalyst-assisted synthesis of 20 µm long GaAs nanowires by the vapor-liquid-solid hydride vapor phase epitaxy (HVPE) exhibiting a polytypism-free zincblende phase for record radii lower than 15 nm down to 5 nm. HVPE makes use of GaCl gaseous growth precursors at high mass input of which fast dechlorination at the usual process temperature of 715 °C results in high planar growth rate (standard 30-40 µm/h). When it comes to the vapor-liquid-solid growth of nanowires, fast solidification at a rate higher than 100 µm/h is observed. Nanowire growth by HVPE only proceeds by introduction of precursors in the catalyst droplets from the vapor phase. This promotes almost pure axial growth leading to nanowires with a constant cylinder shape over unusual length. The question of the cubic zincblende structure observed in HVPE-grown GaAs nanowires regardless of their radius is at the heart of the paper. We demonstrate that the vapor-liquid-solid growth in our conditions takes place at high liquid chemical potential that originates from very high influxes of both As and Ga. This yields a Ga concentration systematically higher than 0.62 in the Au-Ga-As droplets. The high Ga concentration decreases the surface energy of the droplets, which disables nucleation at the triple phase line thus preventing the formation of wurtzite structure whatever the nanowire radius is.

Vapor liquid solid-hydride vapor phase epitaxy (VLS-HVPE) growth of ultra-long defect-free GaAs nanowires: ab initio simulations supporting center nucleation.

High aspect ratio, rod-like and single crystal phase GaAs nanowires (NWs) were grown by gold catalyst-assisted hydride vapor phase epitaxy (HVPE). High resolution transmission electron microscopy and micro-Raman spectroscopy revealed polytypism-free zinc blende (ZB) NWs over lengths of several tens of micrometers for a mean diameter of 50 nm. Micro-photoluminescence studies of individual NWs showed linewidths smaller than those reported elsewhere which is consistent with the crystalline quality of the NWs. HVPE makes use of chloride growth precursors GaCl of which high decomposition frequency after adsorption onto the liquid droplet catalysts, favors a direct and rapid introduction of the Ga atoms from the vapor phase into the droplets. High influxes of Ga and As species then yield high axial growth rate of more than 100 µm/h. The diffusion of the Ga atoms in the liquid droplet towards the interface between the liquid and the solid nanowire was investigated by using density functional theory calculations. The diffusion coefficient of Ga atoms was estimated to be 3 × 10(-9) m(2)/s. The fast diffusion of Ga in the droplet favors nucleation at the liquid-solid line interface at the center of the NW. This is further evidence, provided by an alternative epitaxial method with respect to metal-organic vapor phase epitaxy and molecular beam epitaxy, of the current assumption which states that this type of nucleation should always lead to the formation of the ZB cubic phase.

Dietary flavanols modulate the transcription of genes associated with cardiovascular pathology without changes in their DNA methylation state.

BACKGROUND: In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. CONCLUSION: Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans.