GAPDH is not regulated in human glioblastoma under hypoxic conditions

<p>Abstract</p> <p>Background</p> <p>Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions <it>in vitro </it>in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined <it>in vivo </it>in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined <it>in vitro </it>in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level <it>in vitro </it>in GaMG and on protein level in U251, U373 and GaMG cells.</p> <p>Results</p> <p>We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied <it>in vitro</it>. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation <it>in vivo</it>.</p> <p>Conclusion</p> <p>GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.</p

Huddle Up: Using Mediation to Help Settle the National Football League Labor Dispute

In a patient transferred from Togo to Cologne, Germany, Lassa fever was diagnosed 12 days post mortem. Sixty-two contacts in Cologne were categorised according to the level of exposure, and gradual infection control measures were applied. No clinical signs of Lassa virus infection or Lassa specific antibodies were observed in the 62 contacts. Thirty-three individuals had direct contact to blood, other body fluids or tissue of the patients. Notably, with standard precautions, no transmission occurred between the index patient and healthcare workers. However, one secondary infection occurred in an undertaker exposed to the corpse in Rhineland-Palatinate, who was treated on the isolation unit at the University Hospital of Frankfurt. After German authorities raised an alert regarding the imported Lassa fever case, an American healthcare worker who had cared for the index patient in Togo, and who presented with diarrhoea, vomiting and fever, was placed in isolation and medevacked to the United States. The event and the transmission of Lassa virus infection outside of Africa underlines the need for early diagnosis and use of adequate personal protection equipment (PPE), when highly contagious infections cannot be excluded. It also demonstrates that larger outbreaks can be prevented by infection control measures, including standard PPE

Sensitivity and specificity of loss of heterozygosity analysis for the classification of rare germline variants in BRCA1/2: results of the observational AGO-TR1 study (NCT02222883)

Variant-specific loss of heterozygosity (LOH) analyses may be useful to classify BRCA1/2 germline variants of unknown significance (VUS). The sensitivity and specificity of this approach, however, remains unknown. We performed comparative next-generation sequencing analyses of the BRCA1/2 genes using blood-derived and tumour-derived DNA of 488 patients with ovarian cancer enrolled in the observational AGO-TR1 trial (NCT02222883). Overall, 94 pathogenic, 90 benign and 24 VUS were identified in the germline. A significantly increased variant fraction (VF) of a germline variant in the tumour indicates loss of the wild-type allele; a decreased VF indicates loss of the variant allele. We demonstrate that significantly increased VFs predict pathogenicity with high sensitivity (0.84, 95% CI 0.77 to 0.91), poor specificity (0.63, 95% CI 0.53 to 0.73) and poor positive predictive value (PPV; 0.71, 95% CI 0.62 to 0.79). Significantly decreased VFs predict benignity with low sensitivity (0.26, 95% CI 0.17 to 0.35), high specificity (1.0, 95% CI 0.96 to 1.00) and PPV (1.0, 95% CI 0.85 to 1.00). Variant classification based on significantly increased VFs results in an unacceptable proportion of false-positive results. A significantly decreased VF in the tumour may be exploited as a reliable predictor for benignity, with no false-negative result observed. When applying the latter approach, VUS identified in four patients can now be considered benign. Trial registration number NCT02222883

A novel mode of operation of SLC22A11: Membrane insertion of estrone sulfate versus translocation of uric acid and glutamate

Estrone sulfate alias estrone-3-sulfate (E3S) is considerably larger and much more hydrophobic than typical substrates of SLC22 transporters. It is puzzling that many otherwise unrelated transporters have been reported to transport E3S. Here we scrutinized the mechanism of transport of E3S by SLC22A11 (alias OAT4), by direct comparison with uric acid (UA), an important physiological substrate. Heterologous expression of SLC22A11 in human 293 cells gave rise to a huge unidirectional efflux of glutamate (Glu) and aspartate, as determined by LC-MS/MS. The uptake of E3S was 20-fold faster than the uptake of UA. Yet, the outward transport of Glu was inhibited by extracellular E3S, but not by UA. The release of E3S after preloading was trans-stimulated by extracellular dehydroepiandrosterone sulfate (DHEAS), but neither by UA nor 6-carboxyfluorescein (6CF). The equilibrium accumulation of E3S was enhanced 3-fold by replacement of chloride with gluconate, but the opposite effect was observed for UA. These results establish that SLC22A11 provides entirely different transport mechanisms for E3S and UA. Therefore, E3S must not be used as a substitute for UA to assay the function of SLC22A11. In equilibrium accumulation experiments, the transporter-mediated uptake was a linear function of the concentration of UA and 6CF. By contrast, in the same concentration range the graph for E3S was hyperbolic. This suggests that SLC22A11 inserts E3S into a small volume with limited capacity, the plasma membrane. Our data support the notion that the reverse process, extraction from the membrane, is also catalyzed by the carrier. (C) 2017 Elsevier Inc. All rights reserved

Technological Evaluation of Algae-Based Fillers for Polymer 3D Printing

One approach to reducing the environmental footprint of conventional polymers is to compound them with bio-based fillers. Plant-based materials have already been successfully used as polymer fillers. In this context, algae-based fillers received minor attention. Due to their unique growth efficiency and ability to capture large amounts of CO2, the use of algae-based fillers could have economic and ecologic advantages. In this work, a possible use of algae as a sustainable filler for filament materials was technologically evaluated. In practical investigations, conventional polyethylene-terephthalate-glycol (PETG) was mixed with the microalgae spirulina platensis and chlorella vulgaris and extruded to 3D printing filaments. Based on printed test specimens and material samples, the printability, mechanical, and thermal properties of the composite were determined. Filaments with a homogeneous distribution of algae particles and stable diameters up to a filler content of 30 wt.% could be produced. All filaments had good printability and adequate moisture sensitivity for higher algae contents. For 30 wt.% the tensile strength of the produced filaments decreases from 54 MPa to 24 MPa, the flexural strength decreases from 87 MPa to 69 MPa, and the material operating temperature decreases slightly from 70 °C to 66 °C. The addition of smaller amounts of algae results in minor changes regarding the overall performance. The properties of the material were comparable to those of other natural fillers such as wood, bamboo or cork. The main objective of adding bio-based materials to polymeric matrices can be achieved. Our results suggest that algae-based filaments can be produced as a more sustainable and low-cost material