Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using ¹H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o⁻ under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD⁺. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o⁻ cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.     

Were Ancestral Proteins Less Specific?

Some have hypothesized that ancestral proteins were, on average, less specific than their descendants. If true, this would provide a universal axis along which to organize protein evolution and suggests that reconstructed ancestral proteins may be uniquely powerful tools for protein engineering. Ancestral sequence reconstruction studies are one line of evidence used to support this hypothesis. Previously, we performed such a study, investigating the evolution of peptide-binding specificity for the paralogs S100A5 and S100A6. The modern proteins appeared more specific than their last common ancestor (ancA5/A6), as each paralog bound a subset of the peptides bound by ancA5/A6. In this study, we revisit this transition, using quantitative phage display to measure the interactions of 30,533 random peptides with human S100A5, S100A6, and ancA5/A6. This unbiased screen reveals a different picture. While S100A5 and S100A6 do indeed bind to a subset of the peptides recognized by ancA5/A6, they also acquired new peptide partners outside of the set recognized by ancA5/A6. Our previous work showed that ancA5/A6 had lower specificity than its descendants when measured against biological targets; our new work shows that ancA5/A6 has similar specificity to the modern proteins when measured against a random set of peptide targets. This demonstrates that altered biological specificity does not necessarily indicate altered intrinsic specificity, and sounds a cautionary note for using ancestral reconstruction studies with biological targets as a means to infer global evolutionary trends in specificity.     

A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis

A threonine-12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding.     

Guest research facilities at the Biologische Anstalt Helgoland: Past and present

The Biologische Anstalt Helgoland (BAH) offers unique possibilities for research and education in marine sciences in the southern part of the North Sea. Besides its own research duties, the Institute provides research facilities and technical assistance for guest scientists, assists in the teaching and education of university student groups, and conducts its own courses. The Institute further supplies universities and research institutions on the mainland with marine organisms. The marine station on Helgoland has 14 laboratories, with a total of 32 working places available for guest scientists. The Wadden Sea Institute in List on the island of Sylt offers 6 laboratories with a total of 18 working places. Furthermore, laboratory classrooms are located on Helgoland and in List for 50 and 20 participants, respectively. For the convenience of the guest researchers staying at the BAH, guest-houses are run on Helgoland (Arthur-Hagmeier-Haus, Wilhelm-Mielk-Haus) und in List (Adolf-Bückmann-Haus). Guest researchers have been welcome since the founding of the Institute in 1892. Heincke gave a brief report on the activities of the first visitors from 1892 to 1897. Only sporadic reports are available for the first 60 years of this century. Guest scientists and their activities have only been recorded in detail in the annual reports of the BAH since 1962. The number of researchers and the length of their visits have increased continuously since 1962. The research facilities on Helgoland, in List and Hamburg have been modernized during the last 20 years. In 1971, four modern laboratories for guest researchers could be opened on Helgoland with financial support of the German Research Foundation (DFG). The number, of guest researchers in List and Hamburg increased after the completion of new buildings in 1979 and 1982. The recent increase in research activities by guest scientists is due to numerous students, from many different universities, using the superb research facilities to do their Masters thesis, or Ph.D. Guest researchers and students either perform their own research or cooperate with scientists of the BAH.     

Strategies to assess and optimize stability of endogenous amines during cerebrospinal fluid sampling

Introduction

Metabolic profiling of cerebrospinal fluid (CSF) is a promising technique for studying brain diseases. Measurements should reflect the in vivo situation, so ex vivo metabolism should be avoided.

Objective

To investigate the effects of temperature (room temperature vs. 4 °C), centrifugation and ethanol, as anti-enzymatic additive during CSF sampling on concentrations of glutamic acid, glutamine and other endogenous amines.

Methods

CSF samples from 21 individuals were processed using five different protocols. Isotopically-labeled alanine, isoleucine, glutamine, glutamic acid and dopamine were added prior to sampling to trace any degradation. Metabolomics analysis of endogenous amines, isotopically-labeled compounds and degradation products was performed with a validated LC–MS method.

Results

Thirty-six endogenous amines were quantified. There were no statistically significant differences between sampling protocols for 31 out of 36 amines. For GABA there was primarily an effect of temperature (higher concentrations at room temperature than at 4 °C) and a small effect of ethanol (lower concentrations if added) due to possible degradation. O-phosphoethanolamine concentrations were also lower when ethanol was added. Degradation of isotopically-labeled compounds (e.g. glutamine to glutamic acid) was minor with no differences between protocols.

Conclusion

Most amines can be considered stable during sampling, provided that samples are cooled immediately to 4 °C, centrifuged, and stored at ??80 °C within 2 h. The effect of ethanol addition for more unstable metabolites needs further investigation. This was the first time that labeled compounds were used to monitor ex vivo metabolism during sampling. This is a useful strategy to study the stability of other metabolites of interest.

     

EvoSketch: Simple simulations for learning random and probabilistic processes in evolution,and effects of instructional support on learners’ conceptual knowledge

Background

Students’ knowledge of scientific principles of evolution is often inadequate, despite its recognized importance for understanding biology. Moreover, difficulties associated with underlying abstract concepts such as randomness and probability can hinder successful learning of evolutionary concepts. Studies show that visualizations, particularly simulations together with appropriate instructional support, facilitate the learning of abstract concepts. Therefore, we have developed interactive, web-based simulation software called EvoSketch in efforts to help learners grasp the nature and importance of random and probabilistic processes in evolutionary contexts. We applied EvoSketch in an intervention study comparing four self-directed study conditions: learning with EvoSketch (1) alone, (2) combined with interpretative support, (3) combined with reflective support, and (4) using texts about randomness and probability instead of EvoSketch. All conditions received no support from any instructors. Knowledge about evolution as well as randomness and probability in the context of evolution, time-on-task, and perceived cognitive load were measured. A sample of 269 German secondary school students (M_age?=?15.6 years, SD?=?0.6 years) participated in the study.

Results

Learners using EvoSketch without additional support obtained higher follow-up test scores regarding their knowledge of randomness and probability than those using the text-based approach. However, use of the simulations together with given instructional support (interpretative or reflective) did not increase students’ performance, relative to the text-based approach. In addition, no significant between-intervention differences were found concerning the knowledge of evolution, while significant differences between the groups were detected concerning students’ perceived cognitive load and time-on-task.

Conclusions

From our findings, we conclude that EvoSketch seems to have a very small positive effect on students’ understanding of randomness and probability. Contrary to our expectations, additional self-directed instructional support did not improve students’ understanding, probably because it was not necessary to understand EvoSketch simulations. When using EvoSketch in the classroom, we recommend increasing the intervention timeframe to several sessions and a variety of evolutionary examples for which EvoSketch serves as an underlying framework.

     

Acellular hemoglobin solution enters compressed lung capillaries more readily than red blood cells.

High lung inflation pressures compress alveolar septal capillaries, impede red cell transit, and interfere with oxygenation. However, recently introduced acellular hemoglobin solutions may enter compressed lung capillaries more easily than red blood cells. To test this hypothesis, we perfused isolated rat lungs with fluorescently labeled diaspirin cross-linked hemoglobin (DCLHb; 10%) and/ or autologous red cells (hematocrit, 20). Septal capillaries were compressed by setting lung inflation pressure above vascular pressures (zone 1). Examination by confocal microscopy showed that DCLHb was distributed throughout alveolar septa. Furthermore, this distribution was not affected by adding red blood cells to the perfusate. We estimated the maximum acellular hemoglobin mass within septa to be equivalent to that of 15 red blood cells. By comparison, we found an average of 2.7 +/- 4.6 red cells per septum in zone 1. These values increased to 30.4 +/- 25.8 and 50.4 +/- 22.1 cells per septum in zones 2 and 3, respectively. We conclude that perfusion in zone 1 with a 10% acellular hemoglobin solution may increase the hemoglobin concentration per septum up to fivefold compared with red cell perfusion.     

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.