Assessment of ProteoExtract subcellular fractionation kit reveals limited and incomplete enrichment of nuclear subproteome from frozen liver and heart tissue

The nuclear fraction of the ProteoExtract subcellular fractionation kit was assessed using frozen rat liver and heart tissue. Fractionation was evaluated by Western blot using protein markers for various subcellular compartments and followed up with LC/MS/MS analysis of the nuclear fractions. Of the proteins identified, nuclear proteins were in the minority (less than 15%) and there was poor representation of the various nuclear substructures when compared with liver nuclear isolations using a classical density‐based centrifugation protocol. The ProteoExtract kit demonstrated poor specificity for the nucleus and offers limited promise for proteomics investigations of the nuclear subproteome in frozen tissue samples.     

Evaluation of next generation sequencing platforms for population targeted sequencing studies

Background

Next generation sequencing (NGS) platforms are currently being utilized for targeted sequencing of candidate genes or genomic intervals to perform sequence-based association studies. To evaluate these platforms for this application, we analyzed human sequence generated by the Roche 454, Illumina GA, and the ABI SOLiD technologies for the same 260 kb in four individuals.

Results

Local sequence characteristics contribute to systematic variability in sequence coverage (>100-fold difference in per-base coverage), resulting in patterns for each NGS technology that are highly correlated between samples. A comparison of the base calls to 88 kb of overlapping ABI 3730xL Sanger sequence generated for the same samples showed that the NGS platforms all have high sensitivity, identifying >95% of variant sites. At high coverage, depth base calling errors are systematic, resulting from local sequence contexts; as the coverage is lowered additional 'random sampling' errors in base calling occur.

Conclusions

Our study provides important insights into systematic biases and data variability that need to be considered when utilizing NGS platforms for population targeted sequencing studies.     

Internal auditory canal metastasis mimicking a vestibular schwannoma at presentation – a case report and review of the literature

Metastasis to the internal auditory canal from breast carcinoma is extremely rare and difficult to diagnose. It radiologically mimics vestibular schwannoma and can occur as a first manifestation of systemic relapse after a long disease-free interval in patients previously treated for early breast cancer. The diagnosis is usually made retrospectively and the optimal management of such metastasis following complete resection remains undefined.     

Urban rat exposure to anticoagulant rodenticides and zoonotic infection risk

Anticoagulant rodenticides (ARs) deployed to control rodent pest populations can increase the risk of pathogen infection for some wildlife. However, it is unknown whether ARs also increase infection risk for target rodents, which are common hosts for zoonotic (animal-to-human transmitted) pathogens. In this study, we tested whether rats exposed to ARs were more likely to be infected with zoonotic pathogens, specifically Leptospira spp. or Escherichia coli, after controlling for known predictors of infection (i.e. sex, age, body condition). We collected biological samples from 99 rats trapped in Chicago alleys and tested these for Leptospira infection, E. coli shedding and AR exposure. We found that rats that had been exposed to ARs and survived until the time of trapping, as well as older rats, were significantly more likely to be infected with Leptospira spp. than other rats. We found no significant association between E. coli shedding and any predictors. Our results show that human actions to manage rats can affect rat disease ecology and public health risks in unintended ways, and more broadly, contribute to a growing awareness of bidirectional relationships between humans and natural systems in cities.     

Cerebrospinal Fluid Concentration of Brain-Derived Neurotrophic Factor and Cognitive Function in Non-Demented Subjects

Background

Brain-derived neurotrophic factor (BDNF) is an activity-dependent secreted protein that is critical to organization of neuronal networks and synaptic plasticity, especially in the hippocampus. We tested hypothesis that reduced CSF BDNF is associated with age-related cognitive decline.

Methodology/Principal Findings, and Conclusions/Significance

CSF concentration of BDNF, Aβ₄₂ and total tau were measured in 128 cognitively normal adults (Normals), 21 patients with Alzheimer''s disease (AD), and nine patients with Mild Cognitive Impairment. Apolipoprotein E and BDNF SNP rs6265 genotype were determined. Neuropsychological tests were performed at baseline for all subjects and at follow-up visits in 50 Normals. CSF BDNF level was lower in AD patients compared to age-matched Normals (p = 0.02). CSF BDNF concentration decreased with age among Normals and was higher in women than men (both p<0.001). After adjusting for age, gender, education, CSF Aβ₄₂ and total tau, and APOE and BDNF genotypes, lower CSF BDNF concentration was associated poorer immediate and delayed recall at baseline (both p<0.05) and in follow up of approximately 3 years duration (both p<0.01).

Conclusions/Significance

Reduced CSF BDNF was associated with age-related cognitive decline, suggesting a potential mechanism that may contribute in part to cognitive decline in older individuals.     

The MetabolomeExpress Project: enabling web-based processing,analysis and transparent dissemination of GC/MS metabolomics datasets

Background  

Standardization of analytical approaches and reporting methods via community-wide collaboration can work synergistically with web-tool development to result in rapid community-driven expansion of online data repositories suitable for data mining and meta-analysis. In metabolomics, the inter-laboratory reproducibility of gas-chromatography/mass-spectrometry (GC/MS) makes it an obvious target for such development. While a number of web-tools offer access to datasets and/or tools for raw data processing and statistical analysis, none of these systems are currently set up to act as a public repository by easily accepting, processing and presenting publicly submitted GC/MS metabolomics datasets for public re-analysis.     

N',2-diphenylquinoline-4-carbohydrazide based NK3 receptor antagonists

A new class of potent NK3R antagonists based on the N',2-diphenylquinoline-4-carbohydrazide core is described. In an ex vivo assay in gerbil, the lead compound 2g occupies receptors within the CNS following oral dosing (Occ(90) 30 mg/kg po; plasma Occ(90) 0.95 microM) and has good selectivity and promising PK properties.     

A genomic scan for divergent selection in a secondary contact zone between Atlantic and Gulf of Mexico oysters, Crassostrea virginica

The degree of population structure within species often varies considerably among loci. This makes it difficult to determine whether observed variance reflects neutral-drift stochasticity or locus-specific selection at one or more loci. This uncertainty is exacerbated when evolutionary equilibrium cannot be assumed and/or admixture potentially inflates genomic variance. Thus, the value of a 'genome scan', where locus-specific summary statistics are compared with a simulated neutral distribution among loci, may be limited in secondary contact zones if the null distribution is sensitive to the timing of secondary contact. Of particular interest here is the wide variance previously observed in locus-specific patterns of population structure between Atlantic and Gulf of Mexico populations of eastern oyster, Crassostrea virginica. To test the robustness of an equilibrium null model, we compared among-locus distributions of F(ST) simulated under migration-drift equilibrium and several nonequilibrium secondary contact histories. We then tested for evidence of divergent selection between two oyster populations on either side of a secondary contact zone using 215 amplified fragment length polymorphism (AFLP) loci. Constant-migration equilibrium and nonequilibrium secondary-contact simulations produced equivalent distributions of F(ST) when anchored by the global mean F(ST) observed in oysters, 0.0917. The 99th quantile of simulated neutral F(ST) encompassed most of the variation among oyster loci. Three AFLP loci exhibited F(ST) values higher than this threshold. Although no locus was significant after correcting for multiple tests, our results show in geographically clinal organisms: AFLPs can efficiently characterize the genomic distribution of F(ST); equilibrium models can be used to evaluate outliers; these procedures help focus research on genomic regions of interest.     

Having it all: historical energy intakes do not generate the anticipated trade-offs in fecundity

An axiom of life-history theory, and fundamental to our understanding of ageing, is that animals must trade-off their allocation of resources since energy and nutrients are limited. Therefore, animals cannot "have it all"--combine high rates of fecundity with extended lifespans. The idea of life-history trade-offs was recently challenged by the discovery that ageing may be governed by a small subset of molecular processes independent of fitness. We tested the "trade-off" and "having it all" theories by examining the fecundities of C57BL/6J mice placed onto four different dietary treatments that generated caloric intakes from -21 to +8.6% of controls. We predicted body fat would be deposited in relation to caloric intake. Excessive body fat is known to cause co-morbidities that shorten lifespan, while caloric restriction enhances somatic protection and increases longevity. The trade-off model predicts that increased fat would be tolerated because reproductive gain offsets shortened longevity, while animals on a restricted intake would sacrifice reproduction for lifespan extension. The responses of body fat to treatments followed our expectations, however, there was a negative relationship between reproductive performance (fecundity, litter mass) and historical intake/body fat. Our dietary restricted animals had lower protein oxidative damage and appeared able to combine life-history traits in a manner contrary to traditional expectations by having increased fecundity with the potential to have extended lifespans.     

Common evolutionary origin of swapped-hairpin and double-psi beta barrels

The core of swapped-hairpin and double-psi beta barrels is formed by duplication of a conserved betaalphabeta element, suggesting a common evolutionary origin. The path connecting the two folds is unclear as the two barrels are not interconvertible by a simple topological modification, such as circular permutation. We have identified a protein family whose sequence properties are intermediate to the two folds. The structure of one of these proteins, Pyrococcus horikoshii PhS018, is also built by duplication of the conserved betaalphabeta element but shows yet a third topology, which we name the RIFT barrel. This topology is widespread in the structure database and spans three folds of the SCOP classification, including the middle domain of EF-Tu and the N domain of F1-ATPase. We propose that swapped-hairpin beta barrels arose from an ancestral RIFT barrel by strand invasion and double-psi beta barrels by a strand swap. We group the three barrel types into a metafold, the cradle-loop barrels.