Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles

Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.     

Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 μM, 100 μM, and 10 μM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.Lipopolysaccharide (LPS) is a major component of the outer membranes of Gram-negative bacteria (21). Typically, LPS molecules consist of a hydrophilic carbohydrate portion and a hydrophobic lipid A (or endotoxin). The lipid A molecule consists of a fatty acyl substituted β-d-GlcN-(1-6)-α-GlcN disaccharide unit that usually carries phosphate groups. Diphosphorylated lipid A is generally presumed to be phosphorylated at C-1 and C-4′ positions (9); however, lipid A moieties containing pyrophosphate (PP) groups have also been reported (13). The presence of phosphate groups in lipid A greatly affects the endotoxic properties of LPS (22). Deletion of either of these groups reduces an endotoxic activity of the resulting monophosphorylated LPS by approximately 100-fold (18). For example, monophosphorylated lipid A has been used as an adjuvant in a hepatitis B vaccine in Europe (1, 12).Mass spectrometry (MS) has been widely used to gain information about the heterogeneity, i.e., the number of different species of lipid A families and a distribution of fatty acids on each glucosamine residue (2, 3, 9, 16, 20, 23, 28, 29, 30, 32, 35, 36). Detailed structural information, including the phosphorylation pattern of lipid A, can be obtained by tandem mass spectrometry. Several matrices have been used for the analysis of lipid A using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and 6-aza-2-thiothymine (ATT) (8). Although DHB has been widely used for peptide analysis, it produces uneven crystals and leads to poor spot-to-spot reproducibility (3, 6, 11). Furthermore, the low solubility in the solvent compatible with lipid A and nonuniformity in a matrix layer (crystals) can lead to variations in the ionization yield across the sample resulting in formation of “hot” (or “sweet”) spots (14). On the other hand, 5-chloro-2-mercaptobenzothiazole (CMBT) was found to offer excellent spot-to-spot reproducibility because of the homogeneous crystallization of the analyte/matrix mixture over the sample spot (33). CMBT is soluble in methanol-chloroform-water (4:4:1, vol:vol:vol), a solvent compatible with lipid A molecules, especially hexaacylated species. Thus, it has been widely used for lipid A analysis (4, 9, 23, 35, 33). Interestingly, different preparation procedures for analysis of lipid A species dictate a selection of the preferred matrix system (10). For example, lipid A prepared using a TRI Reagent-based procedure with a CMBT matrix was preferable for the detection of phosphoethanolamine modifications (35). On the other hand, the analysis of lipid A prepared using an LPS extraction kit-based procedure with DHB was preferable for the detection of aminoarabinose modification (10). In addition, divalent cations, such as Ca²⁺ or Mg²⁺, can bridge the phosphorylated negatively charged groups between neighboring LPS molecules to form aggregates (24). Thus, there is a need for technologies capable of characterizing lipid A from biologically relevant samples in an accurate, rapid, and highly sensitive manner. Here we attempt to establish an optimized MALDI MS matrix system for the sensitive analysis of lipid A, especially its diphosphorylated forms, including both pyrophosphorylated and bisphosphorylated species. We also propose to incorporate a complex reagent (additive or comatrix) for reducing the interference of cations (5, 7, 15).     

In vitro production of cat blastocysts of predetermined sex using flow cytometrically sorted semen

Sex preselection in cats can have applications for both breeding purposes and as an experimental model for endangered felids. The present study examined the ability to produce cat embryos from in vitro fertilization (IVF) of in vitro matured (IVM) cat oocytes with flow cytometrically sorted spermatozoa and to verify the sex of the embryos obtained from sexed spermatozoa by PCR. In the first experiment, a total of 224 oocytes were fertilized with spermatozoa from six ejaculates sorted without sex separation. The sorting process did not influence the cleavage rate (sorted 44.0% versus unsorted 46.1%), day 6 morula-blastocyst rate (sorted 26.6% versus unsorted 29.6%) and day 7 blastocyst rate (sorted 16.5% versus unsorted 16.5%). In the second experiment, a total of 84 IVM oocytes were fertilized with sorted X- and Y-chromosome bearing spermatozoa from four ejaculates in order to obtain embryos of preselected sex. Embryonic sex determination by PCR revealed that 21 out of 24 embryos reaching morula/blastocyst stage (87.5%) were of the desired sex. In particular 12 out of 14 embryos (85.7%) derived from X-bearing spermatozoa were female and 9 embryos out of 10 (90%) derived from Y-bearing spermatozoa were male. Our results show, for the first time, that X- and Y-chromosome bearing spermatozoa sorted by high-speed flow cytometry can be successfully used in an IVM-IVF system to obtain cat embryos of a predetermined sex.     

pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb-/- myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3beta to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.     

Assessment of cardiac autonomic modulation during graded head-up tilt by symbolic analysis of heart rate variability

Two symbolic indexes, the percentage of sequences characterized by three heart periods with no significant variations (0V%) and that with two significant unlike variations (2UV%), have been found to reflect changes in sympathetic and vagal modulations, respectively. We tested the hypothesis that symbolic indexes may track the gradual shift of the cardiac autonomic modulation during an incremental head-up tilt test. Symbolic analysis was carried out over heart period variability series (250 cardiac beats) derived from ECG recordings during a graded head-up tilt test (0, 15, 30, 45, 60, 75, and 90 degrees ) in 17 healthy subjects. The percentage of subjects showing a significant linear correlation (Spearman rank-order correlation) with tilt angles was utilized to evaluate the performance of symbolic analysis. Spectral analysis was carried out for comparison over the same series. 0V% progressively increased with tilt angles, whereas 2UV% gradually decreased. The decline of 2UV% was greater than the increase of 0V% at low tilt angles. Linear correlation with tilt angles was exhibited in a greater percentage of subjects for 0V% and 2UV% than for any spectral index. Our findings suggest that symbolic analysis performed better than spectral analysis and, thus, is a suitable methodology for assessment of the subtle changes of cardiac autonomic modulation induced by a graded head-up tilt test. Moreover, symbolic analysis indicates that the changes of cardiac sympathetic and vagal modulations observed during this protocol were reciprocal but characterized by different absolute magnitudes.     

Physicochemical and biological study of selected hydrophobic polyethylenimine-based polycationic liposomes and their complexes with DNA

Non-viral gene therapy is based on the development of efficient and safe gene carrier systems able to transfer DNA into cells. Polyethylenimine (PEI), the most promising non-viral vector, with its high cationic-charge-density potential is able (1) to compact DNA in complexes (polyplexes) smaller than those formed by liposomes (lipoplexes) and (2) to destabilize the endosomal membrane by a 'proton sponge' effect. Several PEI's hydrophobic modifications were reported in the last several years but in some cases a reduced transfection efficiency was observed. The mechanism underlying this phenomenon is not well understood so far. In order to extensively investigate these mechanisms, we reported a physicochemical and biological study of selected hydrophobic PEI's derivatives grafted with chains of different length and percentages of substitution able to form vesicles (polycationic liposomes) and to bind DNA. Their properties were studied by means of dynamic light scattering, freeze-fracture microscopy, potentiometric titrations, gel retardation assays, polyanion exchange reactions, toxicity assays, in vitro transfections, and fluorescence microscopy. Our results indicate that even if polyplexes are able to pass through the cellular membrane, the stability of PEI's hydrophobic polyplexes likely explain their different transfection efficiency in vitro.     

Carbohydrate analysis and serological classification of typical and atypical isolates of Aeromonas salmonicida: a rationale for the lipopolysaccharide-based classification of A. salmonicida

The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis-mass spectrometry (CE-MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A-C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A-C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A.