Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.     

Comparing aspirations: intercalibration of ecological status concepts across European lakes for littoral diatoms

Eleven European countries participated in an exercise to harmonise diatom-based methods used for status assessment in lakes. Lakes were divided into low, medium and high alkalinity types for this exercise. However, it was not possible to perform a full intercalibration on low alkalinity lakes due to the short gradient and confounding factors. Values of the Trophie Index were computed for all samples in order that national datasets could all be expressed on a common scale. Not all participants had reference sites against which national methods could be standardised and, therefore, a Generalised Linear Modelling approach was used to control the effect of national differences in datasets. This enabled the high/good and good/moderate status boundaries to be expressed on a common scale and for deviations beyond ±0.25 class widths to be identified. Those countries which had relaxed boundaries were required to adjust these to within ±0.25 class widths whilst the intercalibration rules allowed those countries with more stringent boundaries to retain these. Despite biogeographical and typological differences between countries, there was broad agreement on the characteristics of high, good and moderate status diatom assemblages, and the exercise has ensured consistent application of Water Framework Directive assessments around Europe.     

Optimization of the DGGE band identification method

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.     

Recognition of human tumor necrosis factor α (TNF-α) by therapeutic antibody fragment: energetics and structural features

Human tumor necrosis factor α (TNF-α) exists in its functional state as a homotrimeric protein and is involved in inflammation processes and immune response of a human organism. Overproduction of TNF-α results in the development of chronic autoimmune diseases that can be successfully treated by inhibitors such as monoclonal antibodies. However, the nature of antibody-TNF-α recognition remains elusive due to insufficient understanding of its molecular driving forces. Therefore, we studied the energetics of binding of a therapeutic antibody fragment (Fab) to the native and non-native forms of TNF-α by employing calorimetric and spectroscopic methods. Global thermodynamic analysis of data obtained from the corresponding binding and urea-induced denaturation experiments has been supported by structural modeling. We demonstrate that the observed high affinity binding of Fab to TNF-α is an enthalpy-driven process due mainly to specific noncovalent interactions taking place at the TNF-α-Fab binding interface. It is coupled to entropically unfavorable conformational changes and accompanied by entropically favorable solvation contributions. Moreover, the three-state model analysis of TNF-α unfolding shows that at physiological concentrations, TNF-α may exist not only as a biologically active trimer but also as an inactive monomer. It further suggests that even small changes of TNF-α concentration could have a considerable effect on the TNF-α activity. We believe that this study sets the energetic basis for understanding of TNF-α inhibition by antibodies and its unfolding linked with the concentration-dependent activity regulation.     

CO2‐concentrating mechanisms in three southern hemisphere strains of Emiliania huxleyi

Rising global CO₂ is changing the carbonate chemistry of seawater, which is expected to influence the way phytoplankton acquire inorganic carbon. All phytoplankton rely on ribulose‐bisphosphate carboxylase oxygenase (RUBISCO) for assimilation of inorganic carbon in photosynthesis, but this enzyme is inefficient at present day CO₂ levels. Many algae have developed a range of energy demanding mechanisms, referred to as carbon concentrating mechanisms (CCMs), which increase the efficiency of carbon acquisition. We investigated CCM activity in three southern hemisphere strains of the coccolithophorid Emiliania huxleyi W. W. Hay & H. P. Mohler. Both calcifying and non‐calcifying strains showed strong CCM activity, with HCO₃^? as a preferred source of photosynthetic carbon in the non‐calcifying strain, but a higher preference for CO₂ in the calcifying strains. All three strains were characterized by the presence of pyrenoids, external carbonic anhydrase (CA) and high affinity for CO₂ in photosynthesis, indicative of active CCMs. We postulate that under higher CO₂ levels cocco‐lithophorids will be able to down‐regulate their CCMs, and re‐direct some of the metabolic energy to processes such as calcification. Due to the expected rise in CO₂ levels, photosynthesis in calcifying strains is expected to benefit most, due to their use of CO₂ for carbon uptake. The non‐calcifying strain, on the other hand, will experience only a 10% increase in HCO₃^?, thus making it less responsive to changes in carbonate chemistry of water.     

Influenza vaccine and learning in C57BL mice with an acute experimental autoimmune encephalomyelitis

Literature data suggest possible link between influenza vaccination and development of autoimmune processes. Therefore, the aim of the study was to investigate the effect of influenza vaccination on spatial learning in mice with experimental autoimmune encephalomyelitis (EAE). EAE was induced in eight-week-old C57BL/6J female mice by subcutaneous immunization (MOG_35–55 in complete Freund’s adjuvant) and Pertussis vaccine injected intraperitoneally. Mice were vaccinated with influenza vaccine three days before MOG immunization. The hippocampal-dependent spatial learning test, Morris Water Maze test (MWM), was performed before and after EAE induction. Significant difference (P < 0.05) in the time for completing the Morris Water Maze task was found between mice with mild clinical signs of EAE when compared to other mice. However no significant difference was observed between mice with EAE and mice with EAE that were vaccinated with influenza vaccine. Hippocampal tissue lesions in EAE mice are in correlation with memory impairment. Study shows no influence of influenza vaccine on progression of clinical signs of EAE, spatial learning and memory impairment.     

Characterisation of human embryonic stem cells conditioning media by 1H-nuclear magnetic resonance spectroscopy

Background

Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking.

Methodology/Principal Findings

Using a ¹H-Nuclear Magnetic Resonance (¹H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the ¹H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at −20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine.

Conclusions/Significance

¹H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.     

Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release

The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.