An optimized metaproteomics protocol for a holistic taxonomic and functional characterization of microbial communities from marine particles

This study aimed to establish a robust and reliable metaproteomics protocol for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MS-sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In the final optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver. As an application example, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9354 and 5034 identified protein groups for FL and PA bacteria, respectively. Our data suggest that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. Furthermore, our data points to functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response

Genetic versus Rearing-Environment Effects on Phenotype: Hatchery and Natural Rearing Effects on Hatchery- and Wild-Born Coho Salmon

With the current trends in climate and fisheries, well-designed mitigative strategies for conserving fish stocks may become increasingly necessary. The poor post-release survival of hatchery-reared Pacific salmon indicates that salmon enhancement programs require assessment. The objective of this study was to determine the relative roles that genotype and rearing environment play in the phenotypic expression of young salmon, including their survival, growth, physiology, swimming endurance, predator avoidance and migratory behaviour. Wild- and hatchery-born coho salmon adults (Oncorhynchus kisutch) returning to the Chehalis River in British Columbia, Canada, were crossed to create pure hatchery, pure wild, and hybrid offspring. A proportion of the progeny from each cross was reared in a traditional hatchery environment, whereas the remaining fry were reared naturally in a contained side channel. The resulting phenotypic differences between replicates, between rearing environments, and between cross types were compared. While there were few phenotypic differences noted between genetic groups reared in the same habitat, rearing environment played a significant role in smolt size, survival, swimming endurance, predator avoidance and migratory behaviour. The lack of any observed genetic differences between wild- and hatchery-born salmon may be due to the long-term mixing of these genotypes from hatchery introgression into wild populations, or conversely, due to strong selection in nature—capable of maintaining highly fit genotypes whether or not fish have experienced part of their life history under cultured conditions

CAG Repeats Determine Brain Atrophy in Spinocerebellar Ataxia 17: A VBM Study

Abnormal repeat length has been associated with an earlier age of onset and more severe disease progression in the rare neurodegenerative disorder spinocerebellar ataxia 17 (SCA17).To determine whether specific structural brain degeneration and rate of disease progression in SCA17 might be associated with the CAG repeat size, observer-independent voxel-based morphometry was applied to high-resolution magnetic resonance images of 16 patients with SCA17 and 16 age-matched healthy controls. The main finding contrasting SCA17 patients with healthy controls demonstrated atrophy in the cerebellum bilaterally. Multiple regression analyses with available genetic data and also post-hoc correlations revealed an inverse relationship again with cerebellar atrophy. Moreover, we found an inverse relationship between the CAG repeat length and rate of disease progression.Our results highlight the fundamental role of the cerebellum in this neurodegenerative disease and support the genotype-phenotype relationship in SCA17 patients. Genetic factors may determine individual susceptibility to neurodegeneration and rate of disease progression

17-allyamino-17-demethoxygeldanamycin treatment results in a magnetic resonance spectroscopy-detectable elevation in choline-containing metabolites associated with increased expression of choline transporter SLC44A1 and phospholipase A2

Abstract Introduction 17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model. Methods MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. 31P and 1H MRS were used to determine total cellular metabolite concentrations and 13C MRS was used to probe the metabolism of [1,2-13C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment. Results Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity. Conclusions This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. 1H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular choline, PC and GPC). As Hsp90 inhibitors enter routine clinical use, t-Cho could thus provide an easily detectable, noninvasive metabolic biomarker of Hsp90 inhibition in breast cancer patients