Online monitoring of metabolism and morphology of peptide-treated neuroblastoma cancer cells and keratinocytes

Antimicrobial peptides are promising anti-cancer agents with a unique mode of action. We established the usage of a chip-based sensor to monitor the dynamic interplay between cells on the chip and peptides and compared it with endpoint tests. Human neuroblastoma cancer cells and spontaneously immortalized non-cancer keratinocytes were perfused with representative peptides (NK-2, NK11, and melittin). The sensor system enabled continuous recording of cell layer impedance (adhesion/confluence), oxygen consumption (respiration) and extracellular acidification (glycolysis) and provided insights in cell damage, stress response and recovery. Cells responded differentially to peptide treatment. During perfusion, peptides accumulated on the cell surface until they reached a critical concentration. Preceding to cell death, melittin triggered glycolysis, suggesting stress response. NK-2 induced no change in energy metabolism, but led to an increase in impedance, i.e. a temporarily altered morphology, which appeared to be an excellent parameter to detect subtle structural changes of cell layers.     

Single Nucleotide Variants in the Protein C Pathway and Mortality in Dialysis Patients

Background

The protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients.

Methods

Single nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort.

Results

Factor V Leiden was associated with a 1.5-fold (95% CI 1.1–1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0–1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality.

Conclusion

Our study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients.     

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.     

Separation of recombinant E. coli from culture broths by adsorption to modified carriers and controlled release of an expressed protein

Investigations were carried out concerning the considerate and selective separation of intact, product including recombinant E. coli cells from culture broths by adsorption. Adsorbents were synthesized on basis of porous glass “SIRAN” (Schott, Mainz) by chemical surface modification in order to adapt surface charge density and hydrophobicity to the surface behavior of the hosts. It is possible to accumulate up to 58?mg dry biomass per gram carrier by using the fixed bed circulation technique and by simultaneous dosage of small amounts of polycationic reagent Polyethyleneimine (PEI). The method is especially useful for the separation of shear stress sensible microorganisms. Preferred release of the recombinant product staphylokinase (SAK) from the cytoplasmatic space of the adsorbed cells was done by permeabilization of the cells and elution of the column with suitable agents. Thus, product enrichment was achieved simultaneously with the clearence of host cell proteins and other cell components or fragments.     

Model-based Conservation Decision Aiding in the Presence of Goal Conflicts and Uncertainty

Goal conflicts and uncertainty are two major problems in decision-making for conservation and species protection. Conflicts can often be found between ecological goals on the one hand and socio-economic goals on the other, but also among different ecological goals. They can be formally analysed by methods of multi-criteria analysis. As the solution of a multi-criteria decision problem usually depends on the weights put on the individual criteria (objectives), sensitivity and robustness analyses are necessary to understand the decision problem, concentrate on the essential aspects, and support actual decision processes fully. Uncertainty in the decision problem is often caused by scarcity of information needed to predict the consequences of management actions. The so-called outranking concept proved very useful in the consideration of such uncertainty. Based on a simple fictitious case study the paper demonstrates how multi-criteria decision analysis (in particular the PROMETHEE outranking method) in combination with population model analysis can assist in conservation biological decision-making.     

Genetic fingerprinting proves cross‐correlated automatic photo‐identification of individuals as highly efficient in large capture–mark–recapture studies

Capture–mark–recapture (CMR) approaches are the backbone of many studies in population ecology to gain insight on the life cycle, migration, habitat use, and demography of target species. The reliable and repeatable recognition of an individual throughout its lifetime is the basic requirement of a CMR study. Although invasive techniques are available to mark individuals permanently, noninvasive methods for individual recognition mainly rest on photographic identification of external body markings, which are unique at the individual level. The re‐identification of an individual based on comparing shape patterns of photographs by eye is commonly used. Automated processes for photographic re‐identification have been recently established, but their performance in large datasets (i.e., > 1000 individuals) has rarely been tested thoroughly. Here, we evaluated the performance of the program AMPHIDENT, an automatic algorithm to identify individuals on the basis of ventral spot patterns in the great crested newt (Triturus cristatus) versus the genotypic fingerprint of individuals based on highly polymorphic microsatellite loci using GENECAP. Between 2008 and 2010, we captured, sampled and photographed adult newts and calculated for 1648 samples/photographs recapture rates for both approaches. Recapture rates differed slightly with 8.34% for GENECAP and 9.83% for AMPHIDENT. With an estimated rate of 2% false rejections (FRR) and 0.00% false acceptances (FAR), AMPHIDENT proved to be a highly reliable algorithm for CMR studies of large datasets. We conclude that the application of automatic recognition software of individual photographs can be a rather powerful and reliable tool in noninvasive CMR studies for a large number of individuals. Because the cross‐correlation of standardized shape patterns is generally applicable to any pattern that provides enough information, this algorithm is capable of becoming a single application with broad use in CMR studies for many species.