Lysozyme purification with dye-affinity beads under magnetic field

Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg.     

Diversity of Toxoplasma gondii strains shaped by commensal communities of small mammals

Commensal rodent species are key reservoirs for Toxoplasma gondii in the domestic environment. In rodents, different T. gondii strains show variable patterns of virulence according to host species. Toxoplasma gondii strains causing non-lethal chronic infections in local hosts will be more likely to persist in a given environment, but few studies have addressed the possible role of these interactions in shaping the T. gondii population structure. In addition, the absence of validated techniques for upstream detection of T. gondii chronic infection in wild rodents hinders exploration of this issue under natural conditions. In this study, we took advantage of an extensive survey of commensal small mammals in three coastal localities of Senegal, with a species assemblage constituted of both native African species and invasive species. We tested 828 individuals for T. gondii chronic infection using the modified agglutination test for antibody detection in serum samples and a quantitative PCR assay for detection of T. gondii DNA in brain samples. The infecting T. gondii strains were genotyped whenever possible by the analysis of 15 microsatellite markers. We found (i) a very poor concordance between molecular detection and serology in the invasive house mouse, (ii) significantly different levels of prevalence by species and (iii) the autochthonous T. gondii Africa 1 lineage strains, which are lethal for laboratory mice, only in the native African species of commensal small mammals. Overall, this study highlights the need to reconsider the use of MAT serology in natural populations of house mice and provides the first known data about T. gondii genetic diversity in invasive and native species of small mammals from Africa. In light of these results, we discuss the role of invasive and native species, with their variable adaptations to different T. gondii strains, in shaping the spatial structure of T. gondii genetic diversity in Africa.     

Density of insect‐pollinated grassland plants decreases with increasing surrounding land‐use intensity

Pollinator declines have raised concerns about the persistence of plant species that depend on insect pollination, in particular by bees, for their reproduction. The impact of pollinator declines remains unknown for species‐rich plant communities found in temperate seminatural grasslands. We investigated effects of land‐use intensity in the surrounding landscape on the distribution of plant traits related to insect pollination in 239 European seminatural grasslands. Increasing arable land use in the surrounding landscape consistently reduced the density of plants depending on bee and insect pollination. Similarly, the relative abundance of bee‐pollination‐dependent plants increased with higher proportions of non‐arable agricultural land (e.g. permanent grassland). This was paralleled by an overall increase in bee abundance and diversity. By isolating the impact of the surrounding landscape from effects of local habitat quality, we show for the first time that grassland plants dependent on insect pollination are particularly susceptible to increasing land‐use intensity in the landscape.     

Proteomic Analysis of Chinook Salmon (Oncorhynchus tshawytscha) Ovarian Fluid

The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon (Oncorhynchus tshawytscha) ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.     

Meta-analysis identified the TNFA -308G > A promoter polymorphism as a risk factor for disease severity in patients with rheumatoid arthritis

Introduction

The goal of this study is to investigate whether the -308G > A promoter polymorphism in the tumor necrosis factor alpha (TNFA) gene is associated with disease severity and radiologic joint damage in a large cohort of patients with rheumatoid arthritis (RA).

Methods

A long-term observational early RA inception cohort (n = 208) with detailed information about disease activity and radiologic damage after 3, 6 and 9 years of disease was genotyped for the TNFA -308G > A promoter polymorphism (rs1800629). A longitudinal regression analysis was performed to assess the effect of genotype on RA disease severity and joint damage. Subsequently, a meta-analysis, including all publically available data, was performed to further test the association between joint erosions and the TNFA polymorphism. To learn more about the mechanism behind the effect of the polymorphism, RNA isolated from peripheral blood from RA patients (n = 66) was used for TNFA gene expression analysis by quantitative PCR.

Results

Longitudinal regression analysis with correction for gender and disease activity showed a significant difference in total joint damage between GG and GA+AA genotype groups (P = 0.002), which was stable over time. The meta-analysis, which included 2,053 patients, confirmed an association of the genetic variant with the development of erosions (odds ratio 0.78, 95% CI 0.62, 0.98). No significant differences in TNFA gene expression were observed for the different genotypes, confirming earlier findings in healthy individuals.

Conclusions

Our data confirm that the TNFA -308G > A promoter polymorphism is associated with joint damage in patients with RA. This is not mediated by differences in TNFA gene expression between genotypes.     

Aging effects on DNA methylation modules in human brain and blood tissue

Background

Several recent studies reported aging effects on DNA methylation levels of individual CpG dinucleotides. But it is not yet known whether aging-related consensus modules, in the form of clusters of correlated CpG markers, can be found that are present in multiple human tissues. Such a module could facilitate the understanding of aging effects on multiple tissues.

Results

We therefore employed weighted correlation network analysis of 2,442 Illumina DNA methylation arrays from brain and blood tissues, which enabled the identification of an age-related co-methylation module. Module preservation analysis confirmed that this module can also be found in diverse independent data sets. Biological evaluation showed that module membership is associated with Polycomb group target occupancy counts, CpG island status and autosomal chromosome location. Functional enrichment analysis revealed that the aging-related consensus module comprises genes that are involved in nervous system development, neuron differentiation and neurogenesis, and that it contains promoter CpGs of genes known to be down-regulated in early Alzheimer's disease. A comparison with a standard, non-module based meta-analysis revealed that selecting CpGs based on module membership leads to significantly increased gene ontology enrichment, thus demonstrating that studying aging effects via consensus network analysis enhances the biological insights gained.

Conclusions

Overall, our analysis revealed a robustly defined age-related co-methylation module that is present in multiple human tissues, including blood and brain. We conclude that blood is a promising surrogate for brain tissue when studying the effects of age on DNA methylation profiles.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.     

Vinyl imidazole carrying metal-chelated beads for reversible use in yeast invertase adsorption

Poly(ethylene glycol dimethacrylate-n-vinyl imidazole) [poly(EGDMA–VIM)] hydrogel (average diameter 150–200 μm) was prepared copolymerizing ethylene glycol dimethacrylate (EGDMA) with n-vinyl imidazole (VIM). Poly(EGDMA–VIM) beads had a specific surface area of 59.8 m²/g. Poly(EGDMA–VIM) beads were characterized by swelling studies and scanning electron microscope (SEM). Cu²⁺ ions were chelated on the poly(EGDMA–VIM) beads (452 μmol Cu²⁺/g), then the metal-chelated beads were used in the adsorption of yeast invertase in a batch system. The maximum invertase adsorption capacity of the poly(EGDMA–VIM)–Cu²⁺ beads was observed as 35.2 mg/g at pH 4.5. The adsorption isotherm of the poly(EGDMA–VIM)–Cu²⁺ beads can be well fitted to the Langmuir model. Adsorption kinetics data were tested using pseudo-first- and -second-order models. Kinetic studies showed that the adsorption followed a pseudo-second-order reaction. The value of the Michaelis constant K_m of invertase was significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the adsorbed invertase. The optimum temperature for the adsorbed preparation of poly(EGDMA–VIM)–Cu²⁺-invertase at 50 °C, 10 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with adsorption. Adsorbed invertase retains an activity of 82% after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications.     

One-step separation of IgG subclasses of DNA hydrolyzing autoantibodies: a study on sera of patients with systemic lupus erythematosus and primary antiphospholipid syndrome by histidine ligand affinity chromatography

A method based on histidine ligand affinity chromatography has been utilized for the separation of DNA hydrolyzing autoantibodies from sera of patients suffering from systemic lupus erythematosus and primary antiphospholipid syndrome using the gel, histidyl-aminohexyl-sepharose. The separation of autoantibodies was carried out under mild chromatographic conditions. Human IgG subclass distribution in the different fractions separated on the column was studied by enzyme-linked immunosorbent assay. The purified DNA hydrolyzing autoantibodies were shown to hydrolyze plasmid DNA.