ω-3 oil intake during weight loss in obese women results in remodelling of plasma triglyceride and fatty acids

Previous studies have shown that a combination of weight loss and fish oil supplementation reduce cardiovascular disease and diabetes risks by increasing adiponectin and reducing triacylglyceride concentrations, while weight loss alone significantly improves insulin sensitivity and reduces inflammation. Here, a metabolomic approach, using a combination of ¹H-Nuclear Magnetic Resonance spectroscopy, and gas and liquid chromatography and mass spectrometry, was employed to elucidate the metabolic changes in blood plasma following weight loss and fish oil supplementation. The intervention study was conducted over 24 weeks, with 93 female subjects randomised to one of three groups. Two groups followed a 12-week weight loss program, followed by a 12-week weight maintenance period and were randomised to fish or placebo oil capsules; a control group did not follow the weight loss program and were given placebo oil capsules. Lipid profiles changed dramatically upon fish oil intake and subtly across the two weight loss groups. While the fish oil supplementation increased the proportion of various phospholipid species, previously reported reductions in total triacylglycerides (TAGs) upon fish oil intake were shown to be driven by a reduction in a specific subset of the measured TAGs. This remodelling of triglycerides may represent further beneficial effects of fish oil supplementation.     

A matter of fat: an introduction to lipidomic profiling methods

In recent years, lipidomics or lipid profiling, an extension of metabolomics where the lipid complement of a cell, tissue or organism is measured, has been the recipient of increasing attention as a research tool in a range of diverse disciplines including physiology, lipid biochemistry, clinical biomarker discovery and pathology. The advancement of the field has been driven by the development of analytical technologies, and in particular advances in liquid chromatography mass spectrometry and chemometric methods. In this review, we give an overview of the current methods with which lipid profiling is being performed. The benefits and shortcomings of mass spectrometry both in the presence and absence of chromatographic separation techniques such as liquid-, gas- and thin layer chromatography are explored. Alone these techniques have their limitations but through a combination many of the disadvantages may be overcome providing a valuable analytical tool for a variety of disease processes.     

Sequencing the maize genome

Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.     

Genetic polymorphism and trade-offs in the early life-history strategy of the Pacific oyster, Crassostrea gigas (Thunberg, 1795): a quantitative genetic study

We investigated genetic variability and genetic correlations in early life-history traits of Crassostrea gigas. Larval survival, larval development rate, size at settlement and metamorphosis success were found to be substantially heritable, whereas larval growth rate and juvenile traits were not. We identified a strong positive genetic correlation between larval development rate and size at settlement, and argue that selection could optimize both age and size at settlement. However, trade-offs, resulting in costs of metamorphosing early and large, were suggested by negative genetic correlations or covariances between larval development rate/size at settlement and both metamorphosis success and juvenile survival. Moreover, size advantage at settlement disappeared with time during the juvenile stage. Finally, we observed no genetic correlations between larval and juvenile stages, implying genetic independence of life-history traits between life-stages. We suggest two possible scenarios for the maintenance of genetic polymorphism in the early life-history strategy of C. gigas.     

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.     

Gamma-lactams and related compounds as cholesterol absorption inhibitors: homologs of the beta-lactam cholesterol absorption inhibitor SCH 48461

Our search for potent cholesterol absorption inhibitors led to the discovery of the β-lactam SCH 48461. Structure activity relationship studies prompted us to this study of γ-lactams, ring homologs of β-lactam SCH 48461, to determine their potential as cholesterol absorption inhibitors. The results indicate that the γ-lactams have moderate cholesterol absorption inhibitory properties.     

Recombinant antibodies for the depletion of abundant proteins from human serum

The identification of biomarkers from serum or plasma is often hindered by a few proteins present at high concentrations, which may obscure less abundant proteins. Ideal serum depletion strategies would be flexible as regards the proteins to be removed, and would feature the use of reagents with long shelf-lives. In this article, we describe a novel protein depletion methodology based on the incubation of serum samples with phage-derived recombinant antibody fragments, which are able to bind to staphylococcal Protein A, and which carry a C-terminal peptide tag capable of streptavidin binding. The resulting protein-antibody complexes can be removed by simultaneous capture on Protein A and/or streptavidin resin. The depletion methodology was exemplified by the isolation of recombinant human mAb fragments specific to abundant human serum Ags and by the simultaneous depletion of albumin, immunoglobulins, alpha2-macroglobulin, hemoglobin, transferrin and haptoglobin. The depleted serum samples were analyzed by 2-DE and by gel-free MS-based methodologies, confirming the efficiency and selectivity of the depletion process. The methodology presented is modular in nature, since several recombinant antibodies can be combined in a single depletion experiment. Furthermore, antibodies do not have to be covalently coupled to a solid support facilitating long-term storage.     

Genome-wide transposon tagging reveals location-dependent effects on transcription and chromatin organization in Arabidopsis

The interphase nucleus exists as a highly dynamic system, the physical properties of which have functional importance in gene regulation. Not only can gene expression be influenced by the local sequence context, but also by the architecture of the nucleus in three-dimensions (3D), and by the interactions between these levels via chromatin modifications. A challenging task is to resolve the complex interplay between sequence- and genome structure-based control mechanisms. Here, we created a collection of 277 Arabidopsis lines that allow the visual tracking of individual loci in living plants while comparing gene expression potential at these locations, via an identical reporter cassette. Our studies revealed regional gene silencing near a heterochromatin island, via DNA methylation, that is correlated with mobility constraint and nucleolar association. We also found an example of nucleolar association that does not correlate with gene suppression, suggesting that distinct mechanisms exist that can mediate interactions between chromatin and the nucleolus. These studies demonstrate the utility of this novel resource in unifying structural and functional studies towards a more comprehensive model of how global chromatin organization may coordinate gene expression over large scales.     

Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum

Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene‐rich regions. Gene‐enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene‐enrichment strategy, we have compared assemblies using methyl‐filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA‐seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single‐nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.