Oxidant Sensing by Reversible Disulfide Bond Formation

Maintenance of the cellular redox balance is crucial for cell survival. An increase in reactive oxygen, nitrogen, or chlorine species can lead to oxidative stress conditions, potentially damaging DNA, lipids, and proteins. Proteins are very sensitive to oxidative modifications, particularly methionine and cysteine residues. The reversibility of some of these oxidative protein modifications makes them ideally suited to take on regulatory roles in protein function. This is especially true for disulfide bond formation, which has the potential to mediate extensive yet fully reversible structural and functional changes, rapidly adjusting the protein''s activity to the prevailing oxidant levels.     

Anti-Inflammatory Effects of Adult Stem Cells in Sustained Lung Injury: A Comparative Study

Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-β following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury.     

Body Position Influences Which Neural Structures Are Recruited by Lumbar Transcutaneous Spinal Cord Stimulation

Transcutaneous stimulation of the human lumbosacral spinal cord is used to evoke spinal reflexes and to neuromodulate altered sensorimotor function following spinal cord injury. Both applications require the reliable stimulation of afferent posterior root fibers. Yet under certain circumstances, efferent anterior root fibers can be co-activated. We hypothesized that body position influences the preferential stimulation of sensory or motor fibers. Stimulus-triggered responses to transcutaneous spinal cord stimulation were recorded using surface-electromyography from quadriceps, hamstrings, tibialis anterior, and triceps surae muscles in 10 individuals with intact nervous systems in the supine, standing and prone positions. Single and paired (30-ms inter-stimulus intervals) biphasic stimulation pulses were applied through surface electrodes placed on the skin between the T11 and T12 inter-spinous processes referenced to electrodes on the abdomen. The paired stimulation was applied to evaluate the origin of the evoked electromyographic response; trans-synaptic responses would be suppressed whereas direct efferent responses would almost retain their amplitude. We found that responses to the second stimulus were decreased to 14%±5% of the amplitude of the response to the initial pulse in the supine position across muscles, to 30%±5% in the standing, and to only 80%±5% in the prone position. Response thresholds were lowest during standing and highest in the prone position and response amplitudes were largest in the supine and smallest in the prone position. The responses obtained in the supine and standing positions likely resulted from selective stimulation of sensory fibers while concomitant motor-fiber stimulation occurred in the prone position. We assume that changes of root-fiber paths within the generated electric field when in the prone position increase the stimulation thresholds of posterior above those of anterior root fibers. Thus, we recommend conducting spinal reflex or neuromodulation studies with subjects lying supine or in an upright position, as in standing or stepping.     

Emerging from the ice-fungal communities are diverse and dynamic in earliest soil developmental stages of a receding glacier

We used amplicon sequencing and isolation of fungi from in-growth mesh bags to identify active fungi in three earliest stages of soil development (SSD) at a glacier forefield (0–3, 9–14, 18–25 years after retreat of glacial ice). Soil organic matter and nutrient concentrations were extremely low, but the fungal diversity was high [220 operational taxonomic units (OTUs)/138 cultivated OTUs]. A clear successional trend was observed along SSDs, and species richness increased with time. Distinct changes in fungal community composition occurred with the advent of vascular plants. Fungal communities of recently deglaciated soil are most distinctive and rather similar to communities typical for cryoconite or ice. This indicates melting water as an important inoculum for native soil. Moreover, distinct seasonal differences were detected in fungal communities. Some fungal taxa, especially of the class Microbotryomycetes, showed a clear preference for winter and early SSD. Our results provide insight into new facets regarding the ecology of fungal taxa, for example, by showing that many fungal taxa might have an alternative, saprobial lifestyle in snow-covered, as supposed for a few biotrophic plant pathogens of class Pucciniomycetes. The isolated fungi include a high proportion of unknown species, which can be formally described and used for experimental approaches.     

Identification and characterization of cells with high angiogenic potential and transitional phenotype in calcific aortic valve

Recent data suggest that angiogenesis plays an important role in the pathogenesis of valvular disease. However, the cellular mechanisms underlying this process remain unknown. This study aimed at identifying and characterizing the cellular components responsible for pathological neovascularization in calcific aortic valves (CAV). Immunohistochemical analysis of uncultured CAV tissues revealed that smooth muscle alpha-actin (alpha-SMA)-positive cells, which coexpressed Tie-2 and vascular endothelial growth factor receptor-2 (VEGFR-2), can be identified prior to the initiation of capillary-like tube formation. In a second step, leaflets of CAV and non-calcific aortic valves (NCAV) were cultured and the cells involved in capillary-like tube formation were isolated. The majority of these cells displayed the same phenotype as non-cultured cells identified in CAV tissues, i.e., expression of alpha-SMA, Tie-2, and VEGFR-2. In comparison to cells isolated from cultures of NCAV leaflets, these cells showed enhanced angiogenic activity as demonstrated by migration and tube assays. The coexpression of VEGFR-2 and Tie-2 together with alpha-SMA suggests both endothelial and mesenchymal properties of the angiogenically activated cells involved in valvular neovascularization. Hence, our findings might provide new insights into the process of pathological angiogenesis in cardiac valves.     

O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: studies addressing safety issues

As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.     

BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea

The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H(2)O(2) and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Deltabcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs.