Pre-implantation alcohol exposure induces lasting sex-specific DNA methylation programming errors in the developing forebrain.

BACKGROUND: Prenatal alcohol exposure is recognized for altering DNA methylation profiles of brain cells during development, and to be part of the molecular basis underpinning Fetal Alcohol Spectrum Disorder (FASD) etiology. However, we have negligible information on the effects of alcohol exposure during pre-implantation, the early embryonic window marked with dynamic DNA methylation reprogramming, and on how this may rewire the brain developmental program. RESULTS: Using a pre-clinical in vivo mouse model, we show that a binge-like alcohol exposure during pre-implantation at the 8-cell stage leads to surge in morphological brain defects and adverse developmental outcomes during fetal life. Genome-wide DNA methylation analyses of fetal forebrains uncovered sex-specific alterations, including partial loss of DNA methylation maintenance at imprinting control regions, and abnormal de novo DNA methylation profiles in various biological pathways (e.g., neural/brain development). CONCLUSION: These findings support that alcohol-induced DNA methylation programming deviations during pre-implantation could contribute to the manifestation of neurodevelopmental phenotypes associated with FASD.

Developmental genome-wide DNA methylation asymmetry between mouse placenta and embryo.

In early embryos, DNA methylation is remodelled to initiate the developmental program but for mostly unknown reasons, methylation marks are acquired unequally between embryonic and placental cells. To better understand this, we generated high-resolution DNA methylation maps of mouse mid-gestation (E10.5) embryo and placenta. We uncovered specific subtypes of differentially methylated regions (DMRs) that contribute directly to the developmental asymmetry existing between mid-gestation embryonic and placental DNA methylation patterns. We show that the asymmetry occurs rapidly during the acquisition of marks in the post-implanted conceptus (E3.5-E6.5), and that these patterns are long-lasting across subtypes of DMRs throughout prenatal development and in somatic tissues. We reveal that at the peri-implantation stages, the de novo methyltransferase activity of DNMT3B is the main driver of methylation marks on asymmetric DMRs, and that DNMT3B can largely compensate for lack of DNMT3A in the epiblast and extraembryonic ectoderm, whereas DNMT3A can only partially compensate in the absence of DNMT3B. However, as development progresses and as DNMT3A becomes the principal de novo methyltransferase, the compensatory DNA methylation mechanism of DNMT3B on DMRs becomes less effective.

Reduction of bacterial biofilm formation using marine natural antimicrobial peptides.

There is an important need for the development of new "environmentally-friendly" antifouling molecules to replace toxic chemicals actually used to fight against marine biofouling. Marine biomass is a promising source of non-toxic antifouling products such as natural antimicrobial peptides produced by marine organisms. The aim of this study was to demonstrate the efficiency of antimicrobial peptides extracted from snow crab (SCAMPs) to reduce the formation of marine biofilms on immerged mild steel surfaces. Five antimicrobial peptides were found in the snow crab hydrolysate fraction used in this study. SCAMPs were demonstrated to interact with natural organic matter (NOM) during the formation of the conditioning film and to limit the marine biofilm development in terms of viability and bacterial structure. Natural SCAMPs could be considered as a potential alternative and non-toxic product to reduce biofouling, and as a consequence microbial induced corrosion on immerged surfaces.

Impact of polymer-coated silver nanoparticles on marine microbial communities: a microcosm study.

The use of silver nanoparticles (AgNPs) in consumer products is increasing drastically and their potential environmental impacts on aquatic organisms from bacterial communities to vertebrates are not well understood. This study reports on changes in marine bacterial richness using denaturing gradient gel electrophoresis (DGGE), and overall community abundance determined by flow cytometry in marine microcosms exposed to polymer-coated AgNPs (20±5 nm) and ionic silver (Ag(+)). Our study clearly demonstrated that at low concentrations (5 and 50 µg L(-1) total silver), un-aggregated polymer-coated AgNPs and dissolved Ag(+) contamination produced similar effects: a longer lag phase suggesting an adaptation period for microorganisms. As richness decreased in the treated samples, this longer lag phase could correspond to the selection of a fraction of the initial community that is insensitive to silver contamination. Polymer-coated AgNPs preserved their bactericidal properties even under the high ionic strength of estuarine waters.

Heat-induced protein structure and subfractions in relation to protein degradation kinetics and intestinal availability in dairy cattle.

The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included 1) protein structure alpha-helix to beta-sheet ratio; 2) protein subfractions profiles; 3) protein degradation kinetics and effective degradability; 4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120 degrees C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120 degrees C for 40 and 60 min increased the protein structure alpha-helix to beta-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences between the treatments. There was a linear effect of heating time on the DVE and a cubic effect on the OEB value. Our results showed that heating changed chemical profiles, protein structure alpha-helix to beta-sheet ratio, and protein subfractions; decreased rumen-degradable protein and rumen-degradable dry matter; and increased potential nutrient supply to dairy cattle. The protein structure alpha-helix to beta-sheet ratio had a significant positive correlation with total intestinally absorbed protein supply and negative correlation with degraded protein balance.

Understanding the differences in molecular conformation of carbohydrate and protein in endosperm tissues of grains with different biodegradation kinetics using advanced synchrotron technology.

Conventional "wet" chemical analyses rely heavily on the use of harsh chemicals and derivatization, thereby altering native seed structures leaving them unable to detect any original inherent structures within an intact tissue sample. A synchrotron is a giant particle accelerator that turns electrons into light (million times brighter than sunlight) which can be used to study the structure of materials at the molecular level. Synchrotron radiation-based Fourier transform IR microspectroscopy (SR-FTIRM) has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of the brightness of synchrotron light and a small effective source size, is capable of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional 'wet' chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of plant seed tissue in relation to nutrient utilization. The objective of this study was to use novel synchrotron radiation-based technology (SR-FTIRM) to identify the differences in the molecular chemistry and conformation of carbohydrate and protein in various plant seed endosperms within intact tissues at cellular and subcellular level from grains with different biodegradation kinetics. Barley grain (cv. Harrington) with a high rate (31.3%/h) and extent (78%), corn grain (cv. Pioneer) with a low rate (9.6%/h) and extent of (57%), and wheat grain (cv. AC Barrie) with an intermediate rate (23%/h) and extent (72%) of ruminal DM degradation were selected for evaluation. SR-FTIRM evaluations were performed at the National Synchrotron Light Source at the Brookhaven National Laboratory (Brookhaven, NY). The molecular structure spectral analysis involved the fingerprint regions of ca. 1720-1485 cm(-1) (attributed to protein amide I C=O and C-N stretching; amide II N-H bending and C-N stretching), ca. 1650-950 cm(-1) (non-structural CHO starch in endosperms), and ca. 1185-800 cm(-1) (attributed to total CHO C-O stretching vibrations) together with agglomerative hierarchical cluster and principal component analyses. Analyses involving the protein amide I features consistently identified differences between all three grains. Other analyses involving carbohydrate features were able to differentiate between wheat and barley but failed however to differentiate between wheat and corn. These results suggest that SR-FTIRM plus the multivariate analyses can be used to identify spectral features associated with the molecular structure of endosperm from grains with different biodegradation kinetics, especially in relation to protein structure. The Novel synchrotron radiation-based bioanalytical technique provides a new approach for plant seed structural molecular studies at ultraspatial resolution and within intact tissue in relation to nutrient availability.

Rapid characterization of molecular chemistry, nutrient make-up and microlocation of internal seed tissue.

Wheat differs from corn in biodegradation kinetics and fermentation characteristics. Wheat exhibits a relatively high rate (23% h(-1)) and extent (78% DM) of biodegradation, which can lead to metabolic problems such as acidosis and bloat in ruminants. The objective of this study was to rapidly characterize the molecular chemistry of the internal structure of wheat (cv. AC Barrie) and reveal both its structural chemical make-up and nutrient component matrix by analyzing the intensity and spatial distribution of molecular functional groups within the intact seed using advanced synchrotron-powered Fourier transform infrared (FTIR) microspectroscopy. The experiment was performed at the U2B station of the National Synchrotron Light Source at Brookhaven National Laboratory, New York, USA. The wheat tissue was imaged systematically from the pericarp, seed coat, aleurone layer and endosperm under the peaks at approximately 1732 (carbonyl C=O ester), 1515 (aromatic compound of lignin), 1650 (amide I), 1025 (non-structural CHO), 1550 (amide II), 1246 (cellulosic material), 1160, 1150, 1080, 930, 860 (all CHO), 3350 (OH and NH stretching), 2928 (CH(2) stretching band) and 2885 cm(-1) (CH(3) stretching band). Hierarchical cluster analysis and principal component analysis were applied to analyze the molecular FTIR spectra obtained from the different inherent structures within the intact wheat tissues. The results showed that, with synchrotron-powered FTIR microspectroscopy, images of the molecular chemistry of wheat could be generated at an ultra-spatial resolution. The features of aromatic lignin, structural and non-structural carbohydrates, as well as nutrient make-up and interactions in the seeds, could be revealed. Both principal component analysis and hierarchical cluster analysis methods are conclusive in showing that they can discriminate and classify the different inherent structures within the seed tissue. The wheat exhibited distinguishable differences in the structural and nutrient make-up among the pericarp, seed coat, aleurone layer and endosperm. Such information on the molecular chemistry can be used for grain-breeding programs for selecting a superior variety of wheat targeted for food and feed purposes and for predicting wheat quality and nutritive value in humans and animals. Thus advanced synchrotron-powered FTIR technology can provide a greater understanding of the plant-animal interface.

Characterization of functional interactions among the Escherichia coli mismatch repair proteins using a bacterial two-hybrid assay.

Vsr mediates very short patch repair in Escherichia coli, correcting T/G mismatches caused by deamination of 5-methylcytosine to thymine. MutS and MutL, part of the post-replication mismatch repair system, stimulate VSP repair. In this study, we use a bacterial two-hybrid assay to show that MutL interacts with Vsr. We also show that interaction between Vsr and MutL inhibits the ability of MutL to dimerize, to interact with MutS and MutH and to mediate a previously unknown interaction between MutS and MutH. This inhibition may explain why high levels of Vsr are mutagenic in vivo. In addition, we show that the Mut fusion proteins are repair proficient in the bacterial two-hybrid assay, making it possible to study their interactions in various genetic backgrounds, or in the presence of DNA damaging agents.

Effect of interaction between 5-azacytidine and DNA (cytosine-5) methyltransferase on C-to-G and C-to-T mutations in Escherichia coli.

The purpose of this study was to determine the effect of the Dcm cytosine methyltransferase on 5-azacytidine (5-azaC) mutagenesis in Escherichia coli. We used a Lac reversion assay to measure C-to-G and C-to-T mutations at a single, methylatable cytosine in the lacZ gene, in the presence and absence of Dcm. C-to-G mutations are stimulated by 5-azaC but are largely independent of Dcm. In contrast, C-to-T mutations are not stimulated by 5-azaC in either wild type or dcm cells. However, in cells which contain Dcm but are defective in very short patch repair, the normally high frequency of spontaneous C-to-T mutations is decreased by the analog in a dose-dependent manner.

The Vsr endonuclease of Escherichia coli: an efficient DNA repair enzyme and a potent mutagen.

The Vsr endonuclease of Escherichia coli initiates the repair of T/G mismatches caused by deamination of 5-methylcytosine to thymine. In this paper, we examine the capacity of Vsr to prevent CG-to-TA mutations in cells with increased transcription of the cytosine methylase gene (dcm). We find that sufficient Vsr is produced by a single chromosomal copy of vsr to prevent mutagenesis. We also investigate the cause of the transition and frameshift mutations in cells overproducing Vsr. Neither the absence of the dcm methylase nor its overproduction affects Vsr-stimulated mutagenesis. However, addition of mutS, mutL, or mutH on multicopy plasmids has a significant effect: mutL or mutH decreases the number of mutations, while mutS stimulates mutagenesis. The mut-containing plasmids have the same effect in cells treated with 2-aminopurine and in cells made defective in DNA proofreading, two experimental situations known to cause transition and frameshift mutations by saturating mismatch repair.

Overexpression of vsr in Escherichia coli is mutagenic.

Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.