Species variation in the effects of dewatering treatment on macroalgae

Seaweeds can be a valuable resource for biorefinery and biotechnology applications, but their high water content is a recurrent problem and one of the key bottlenecks for their sustainable use. Treatments to increase dry matter content of the kelp Laminaria digitata were recently described by the authors. However macroalgae are an extremely diverse group of organisms and compositional variation between species may influence the effects of particular treatments. In this study, potential dewatering treatments including drying, osmotic media, and the application of both organic and mineral acids all followed by screw-pressing have been tested on two other species of kelp (Laminaria hyperborea and Saccharina latissima) and a red seaweed (Palmaria palmata). Conditions that dewatered these species were identified and the data have been combined with the previous results for L. digitata. There were significant differences between species across all the traits of interest. However dewatering was highly dependent on specific interactions with both treatment and season of collection. Nevertheless, the dry matter content of brown seaweeds was widely and successfully increased by air drying or acid treatment followed by screw-pressing. The results for P. palmata were quite different, particularly with regard to juice production. For this species, acid treatment did not result in dewatering, but dry matter content could be increased by screw-pressing immediately after harvest. Together the data presented here demonstrate that dewatering pre-treatments need to be specific for the type of seaweed to be processed; important knowledge for the future use of this sustainable biomass resource.     

Transcriptomic Identification of ADH1B as a Novel Candidate Gene for Obesity and Insulin Resistance in Human Adipose Tissue in Mexican Americans from the Veterans Administration Genetic Epidemiology Study (VAGES)

Type 2 diabetes (T2D) is a complex metabolic disease that is more prevalent in ethnic groups such as Mexican Americans, and is strongly associated with the risk factors obesity and insulin resistance. The goal of this study was to perform whole genome gene expression profiling in adipose tissue to detect common patterns of gene regulation associated with obesity and insulin resistance. We used phenotypic and genotypic data from 308 Mexican American participants from the Veterans Administration Genetic Epidemiology Study (VAGES). Basal fasting RNA was extracted from adipose tissue biopsies from a subset of 75 unrelated individuals, and gene expression data generated on the Illumina BeadArray platform. The number of gene probes with significant expression above baseline was approximately 31,000. We performed multiple regression analysis of all probes with 15 metabolic traits. Adipose tissue had 3,012 genes significantly associated with the traits of interest (false discovery rate, FDR ≤ 0.05). The significance of gene expression changes was used to select 52 genes with significant (FDR ≤ 10^-4) gene expression changes across multiple traits. Gene sets/Pathways analysis identified one gene, alcohol dehydrogenase 1B (ADH1B) that was significantly enriched (P < 10^-60) as a prime candidate for involvement in multiple relevant metabolic pathways. Illumina BeadChip derived ADH1B expression data was consistent with quantitative real time PCR data. We observed significant inverse correlations with waist circumference (2.8 x 10^-9), BMI (5.4 x 10^-6), and fasting plasma insulin (P < 0.001). These findings are consistent with a central role for ADH1B in obesity and insulin resistance and provide evidence for a novel genetic regulatory mechanism for human metabolic diseases related to these traits.     

Hydrophobic-domain-dependent protein-protein interactions mediate the localization of GPAT enzymes to ER subdomains

The endoplasmic reticulum (ER) is a dynamic organelle that consists of numerous regions or 'subdomains' that have discrete morphological features and functional properties. Although it is generally accepted that these subdomains differ in their protein and perhaps lipid compositions, a clear understanding of how they are assembled and maintained has not been well established. We previously demonstrated that two diacylglycerol acyltransferase enzymes (DGAT1 and DGAT2) from tung tree (Vernicia fordii) were located in different subdomains of ER, but the mechanisms responsible for protein targeting to these subdomains were not elucidated. Here we extend these studies by describing two glycerol-3-phosphate acyltransferase-like (GPAT) enzymes from tung tree, GPAT8 and GPAT9, that both colocalize with DGAT2 in the same ER subdomains. Measurement of protein-protein interactions using the split-ubiquitin assay revealed that GPAT8 interacts with itself, GPAT9 and DGAT2, but not with DGAT1. Furthermore, mutational analysis of GPAT8 revealed that the protein's first predicted hydrophobic region, which contains an amphipathic helix-like motif, is required for interaction with DGAT2 and for DGAT2-dependent colocalization in ER subdomains. Taken together, these results suggest that the regulation and organization of ER subdomains is mediated at least in part by higher-ordered, hydrophobic-domain-dependent homo- and hetero-oligomeric protein-protein interactions.     

Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.     

Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable It to Function as a Non-oligomerizing Chemokine

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.     

Developing the wool proteome

The wool proteome has been largely uncharted due to a lack of database coverage, poor protein extractability and dynamic range issues. Yet, investigating correlations between wool physical properties and protein content, or characterising UV-, heat- or processing-induced protein damage requires the availability of an identifiable and identified proteome.In this study we have achieved unprecedented wool proteome identification through a strategy of comprehensive data acquisition, iterative protein identification/validation and concurrent augmentation of the sequence database. Data acquisition comprised a range of different hyphenated MS techniques including LC–MS/MS, LC–MALDI, 2D-LC–MS/MS and SDS-PAGE LC–MS. Using iterative searching of databases and search result combination using ProteinScape, a systematic expansion of identifiable proteins in the sequence database was achieved. This was followed by extensive validation and rationalisation of the protein identifications. In total, 72 complete and 30 partial ovine-specific protein sequences were added to the database, and 113 wool proteins were identified.Enhanced access to ovine-specific protein identification and characterisation will facilitate all wool fibre protein chemistry and proteomics research.     

Chromosomal location of the genes for ferredoxin in wheat,barley and rye

Communicated by J.W. Snape