Novel role for glutathione S-transferase pi. Regulator of protein S-Glutathionylation following oxidative and nitrosative stress

Glutathione S-transferase Pi (GSTpi) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTpi potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTpi is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTpi, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTpi multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTpi and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs.     

Multi-scale mechanistic indicators of Midwestern USA stream macroinvertebrates

We developed ecological indicators of stream macroinvertebrates in two regions of the Midwestern USA dominated by row-crop agriculture. Indicators were identified in a hierarchical fashion. Reach-scale variables related to macroinvertebrate attributes were first identified, and then catchment-scale variables related to those reach-scale variables were identified. Reach-scale indicators common to both regions were % fine sediments, number of habitats, and width:depth ratio. SD of elevation and % commercial land use were selected as catchment-scale indicators in both regions. Our analyses revealed a multi-scale mechanistic relationship between macroinvertebrate attributes associated with degraded conditions (i.e., fewer taxa of Plecoptera and Trichoptera, and a higher proportion of chironomids, burrowers, and depositional taxa) and % fine sediments in stream reaches, which, in turn, was negatively related to catchment characteristics (i.e., SD of elevation) in one region. Understanding how natural variables such as topography influence channel shape and within-channel structure can help guide management options and expectations for different regions. We suggest that developing multi-scale indicators in a mechanistic fashion will be more effective than developing indicators at only one spatial scale for protecting and restoring stream structure and function.     

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

Weighted Interaction SNP Hub (WISH) network method for building genetic networks for complex diseases and traits using whole genome genotype data

Background

High-throughput genotype (HTG) data has been used primarily in genome-wide association (GWA) studies; however, GWA results explain only a limited part of the complete genetic variation of traits. In systems genetics, network approaches have been shown to be able to identify pathways and their underlying causal genes to unravel the biological and genetic background of complex diseases and traits, e.g., the Weighted Gene Co-expression Network Analysis (WGCNA) method based on microarray gene expression data. The main objective of this study was to develop a scale-free weighted genetic interaction network method using whole genome HTG data in order to detect biologically relevant pathways and potential genetic biomarkers for complex diseases and traits.

Results

We developed the Weighted Interaction SNP Hub (WISH) network method that uses HTG data to detect genome-wide interactions between single nucleotide polymorphism (SNPs) and its relationship with complex traits. Data dimensionality reduction was achieved by selecting SNPs based on its: 1) degree of genome-wide significance and 2) degree of genetic variation in a population. Network construction was based on pairwise Pearson's correlation between SNP genotypes or the epistatic interaction effect between SNP pairs. To identify modules the Topological Overlap Measure (TOM) was calculated, reflecting the degree of overlap in shared neighbours between SNP pairs. Modules, clusters of highly interconnected SNPs, were defined using a tree-cutting algorithm on the SNP dendrogram created from the dissimilarity TOM (1-TOM). Modules were selected for functional annotation based on their association with the trait of interest, defined by the Genome-wide Module Association Test (GMAT). We successfully tested the established WISH network method using simulated and real SNP interaction data and GWA study results for carcass weight in a pig resource population; this resulted in detecting modules and key functional and biological pathways related to carcass weight.

Conclusions

We developed the WISH network method which is a novel 'systems genetics' approach to study genetic networks underlying complex trait variation. The WISH network method reduces data dimensionality and statistical complexity in associating genotypes with phenotypes in GWA studies and enables researchers to identify biologically relevant pathways and potential genetic biomarkers for any complex trait of interest.

     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Research into the (Cost-) effectiveness of the ketogenic diet among children and adolescents with intractable epilepsy: design of a randomized controlled trial

Background  

Epilepsy is a neurological disorder, characterized by recurrent unprovoked seizures which have a high impact on the individual as well as on society as a whole. In addition to the economic burden, epilepsy imposes a substantial burden on the patients and their surroundings. Patients with uncontrolled epilepsy depend heavily on informal care and on health care professionals. About 30% of patients suffer from drug-resistant epilepsy. The ketogenic diet can be a treatment of last resort, especially for children. The beneficial effect of the ketogenic diet has been proven, but information is lacking about its cost-effectiveness. In the current study we will evaluate the (cost-) effectiveness of the ketogenic diet in children and adolescents with intractable epilepsy.     

Adults of European ant‐like stone beetles (Coleoptera: Staphylinidae: Scydmaeninae) Scydmaenus tarsatus Müller & Kunze and Scydmaenus hellwigii (Herbst) prey on soft‐bodied arthropods

Scydmaenine beetles are commonly described as predators specialized in capturing and feeding on armored mites of the order Oribatida, and documented cases of feeding on other live arthropods have not been known. Based on laboratory observations and a broad choice of Acari (armored and soft‐bodied) and other soil arthropods, food preferences and associated behavior of two scydmaenine species are clarified and described. Adults of Scydmaenus tarsatus ignored oribatid and mesostigmatan mites, but readily attacked and fed on a soft‐bodied Rhizoglyphus sp. (Acaridae), and on small springtails, especially on Ceratophysella denticulata (Hypogastruridae). A water drinking behavior was observed for this species, not reported previously in any Staphylinidae. Scydmaenus hellwigii ignored all tested Acari (including Rhizoglyphus) and scavenged on dead neanurine collembolans or freshly cut pieces of large springtails; a long term culture was maintained by feeding beetles with isotomid springtails. Previously reported strict specialization of Scydmaenus as a predator on Oribatida was not confirmed and it is concluded that the studied species feed on live soft‐bodied organisms and scavenge on dead arthropods.