Comments and suggestions on fundamental principles of radiation protection

Radiation and Environmental Biophysics -     

The puzzle of TRPV4 channelopathies

Hereditary channelopathies, that is, mutations in channel genes that alter channel function and are causal for the pathogenesis of the disease, have been described for several members of the transient receptor potential channel family. Mutations in the TRPV4 gene, encoding a polymodal Ca²⁺ permeable channel, are causative for several human diseases, which affect the skeletal system and the peripheral nervous system, with highly variable phenotypes. In this review, we describe the phenotypes of TRPV4 channelopathies and overlapping symptoms. Putative mechanisms to explain the puzzle, and how mutations in the same region of the channel cause different diseases, are discussed and experimental approaches to tackle this surprising problem are suggested.     

Lysosomal Membrane Proteins and Their Central Role in Physiology

The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non‐mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways.      

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal,Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.     

A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.     

Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

Background

Human mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens.

Methodology/Principal Findings

We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments.

Conclusions/significance

HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo studies.     

Homooligomeric dA·dU and dA·dT Sequences in Parallel and Antiparallel Strand Orientation: Consequence of the 5-methyl Groups on Stability,Structure and Interaction with the Minor Groove Binding Drug HOECHST 33258

Abstract

Oligodeoxyribonucleotides containing dA·dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA·oligomer. Thermal denaturation studies show that these parallel dAdU sequences are significantly less stable than their dA·analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5- methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA·and APS dA·dU sequences. However, binding to the PS dA·hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmoF reduced free energy of binding for the PS dA·sequence. Replacement of the bulky methyl group with a hydrogen (ie. T -> U) results in significantly stronger Hoechst 33258 binding to the parallel dA·dU sequences with a penalty of only 4.1 kJmol^?1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA·dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

Minor contribution of leaf litter to N nutrition of beech (Fagus sylvatica) seedlings in a mountainous beech forest of Southern Germany

Aims

Our aims were to characterize the fate of leaf-litter-derived nitrogen in the plant-soil-microbe system of a temperate beech forest of Southern Germany and to identify its importance for N nutrition of beech seedlings.

Methods

¹⁵N-labelled leaf litter was traced in situ into abiotic and biotic N pools in mineral soil as well as into beech seedlings and mycorrhizal root tips over three growing seasons.

Results

There was a rapid transfer of ¹⁵N into the mineral soil already 21 days after tracer application with soil microbial biomass initially representing the dominant litter-N sink. However, ¹⁵N recovery in non-extractable soil N pools strongly increased over time and subsequently became the dominant ¹⁵N sink. Recovery in plant biomass accounted for only 0.025 % of ¹⁵N excess after 876 days. After three growing seasons, ¹⁵N excess recovery was characterized by the following sequence: non-extractable soil N?>>?extractable soil N including microbial biomass?>>?plant biomass?>?ectomycorrhizal root tips.

Conclusions

After quick vertical dislocation and cycling through microbial N pools, there was a rapid stabilization of leaf-litter-derived N in non-extractable N pools of the mineral soil. Very low ¹⁵N recovery in beech seedlings suggests a high importance of other N sources such as root litter for N nutrition of beech understorey.