Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in cultured mammalian cells are not independently reproducible

Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Using the comet assay, the micronucleus test and the chromosome aberration test with human fibroblasts (ES1 cells), the EU-funded "REFLEX" project (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) reported clearly positive effects for various exposure conditions. Because of the ongoing discussion on the biological significance of the effects observed, it was the aim of the present study to independently repeat the results using the same cells, the same equipment and the same exposure conditions. We therefore exposed ES1 cells to RF-EMF (1800 MHz; SAR 2 W/kg, continuous wave with intermittent exposure) for different time periods and then performed the alkaline (pH>13) comet assay and the micronucleus test (MNT). For both tests, clearly negative results were obtained in independently repeated experiments. We also performed these experiments with V79 cells, a sensitive Chinese hamster cell line that is frequently used in genotoxicity testing, and also did not measure any genotoxic effect in the comet assay and the MNT. Appropriate measures of quality control were considered to exclude variations in the test performance, failure of the RF-EMF exposure or an evaluation bias. The reasons for the difference between the results reported by the REFLEX project and our experiments remain unclear.     

Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null) mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.     

TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host.     

CyaC,a redox‐regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme‐B membrane anchor domain

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane‐integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra‐histidine signature that is universally present in the membrane anchors of bacterial diheme‐B succinate‐quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme‐B protein and carried a binuclear iron‐sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme‐B in the membrane and loss of AC activity. Heme‐B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q₀ or Q₀H₂). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q₀ and O₂, or NADH and Q₀H₂ respectively. We conclude that CyaC‐like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.     

Imaging nutrient distributions in plant tissue using time-of-flight secondary ion mass spectrometry and scanning electron microscopy

A new approach to trace the transport routes of macronutrients in plants at the level of cells and tissues and to measure their elemental distributions was developed for investigating the dynamics and structure-function relationships of transport processes. Stem samples from Phaseolus vulgaris were used as a test system. Shock freezing and cryo-preparation were combined in a cryogenic chain with cryo-time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS) for element and isotope-specific imaging. Cryo-scanning electron microscopy (cryo-SEM) was integrated into the cryogenic workflow to assess the quality of structural preservation. We evaluated the capability of these techniques to monitor transport pathways and processes in xylem and associated tissues using supplementary sodium (Na) and tracers for potassium (K), rubidium (Rb), and (41)K added to the transpiration stream. Cryo-ToF-SIMS imaging produced detailed mappings of water, K, calcium, magnesium, the K tracers, and Na without quantification. Lateral resolutions ranged from 10 microm in survey mappings and at high mass resolution to approximately 1 microm in high lateral resolution imaging in reduced areas and at lower mass resolution. The tracers Rb and (41)K, as well as Na, were imaged with high sensitivity in xylem vessels and surrounding tissues. The isotope signature of the stable isotope tracer was utilized for relative quantification of the (41)K tracer as a fraction of total K at the single pixel level. Cryo-SEM confirmed that tissue structures had been preserved with subcellular detail throughout all procedures. Overlays of cryo-ToF-SIMS images onto the corresponding SEM images allowed detailed correlation of nutrient images with subcellular structures.     

Hydroxyethyl Starch (HES 130/0.4) Impairs Intestinal Barrier Integrity and Metabolic Function: Findings from a Mouse Model of the Isolated Perfused Small Intestine

BackgroundThe application of hydroxyethyl starch (HES) for volume resuscitation is controversially discussed and clinical studies have suggested adverse effects of HES substitution, leading to increased patient mortality. Although, the intestine is of high clinical relevance and plays a crucial role in sepsis and inflammation, information about the effects of HES on intestinal function and barrier integrity is very scarce. We therefore evaluated the effects of clinically relevant concentrations of HES on intestinal function and barrier integrity employing an isolated perfused model of the mouse small intestine.MethodsAn isolated perfused model of the mouse small intestine was established and intestines were vascularly perfused with a modified Krebs-Henseleit buffer containing 3% Albumin (N=7) or 3% HES (130/0.4; N=7). Intestinal metabolic function (galactose uptake, lactate-to-pyruvate ratio), edema formation (wet-to-dry weight ratio), morphology (histological and electron microscopical analysis), fluid shifts within the vascular, lymphatic and luminal compartments, as well as endothelial and epithelial barrier permeability (FITC-dextran translocation) were evaluated in both groups.ResultsCompared to the Albumin group, HES perfusion did not significantly change the wet-to-dry weight ratio and lactate-to-pyruvate ratio. However, perfusing the small intestine with 3% HES resulted in a significant loss of vascular fluid (p<0.01), an increased fluid accumulation in the intestinal lumen (p<0.001), an enhanced translocation of FITC-dextran from the vascular to the luminal compartment (p<0.001) and a significantly impaired intestinal galactose uptake (p<0.001). Morphologically, these findings were associated with an aggregation of intracellular vacuoles within the intestinal epithelial cells and enlarged intercellular spaces.ConclusionA vascular perfusion with 3% HES impairs the endothelial and epithelial barrier integrity as well as metabolic function of the small intestine.     

A complex rearrangement on chromosome 22 affecting both homologues; haplo-insufficiency of the Cat eye syndrome region may have no clinical relevance

The presence of highly homologous sequences, known as low copy repeats, predisposes for unequal recombination within the 22q11 region. This can lead to genomic imbalances associated with several known genetic disorders. We report here a developmentally delayed patient carrying different rearrangements on both chromosome 22 homologues, including a previously unreported rearrangement within the 22q11 region. One homologue carries a deletion of the proximal part of chromosome band 22q11. To our knowledge, a ‘pure’ deletion of this region has not been described previously. Four copies of this 22q11 region, however, are associated with Cat eye syndrome (CES). While the phenotypic impact of this deletion is unclear, familial investigation revealed five normal relatives carrying this deletion, suggesting that haplo-insufficiency of the CES region has little clinical relevance. The other chromosome 22 homologue carries a duplication of the Velocardiofacial/DiGeorge syndrome (VCFS/DGS) region. In addition, a previously undescribed deletion of 22q12.1, located in a relatively gene-poor region, was identified. As the clinical features of patients suffering from a duplication of the VCFS/DGS region have proven to be extremely variable, it is impossible to postulate as to the contribution of the 22q12.1 deletion to the phenotype of the patient. Additional patients with a deletion within this region are needed to establish the consequences of this copy number alteration. This study highlights the value of using different genomic approaches to unravel chromosomal alterations in order to study their phenotypic impact.