Update on Nox function, site of action and regulation in Botrytis cinerea

Additional file 1: Table S1. Strains used in this work

Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect

Pro-inflammatory TNF-α and IFN-γ Promote Tumor Growth and Metastasis via Induction of MACC1

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Early stage CRC patients have a good prognosis. If distant metastasis occurs, the 5-year survival drops below 10%. Despite treatment success over the last decades, treatment options for metastatic disease are still limited. Therefore, novel targets are needed to foster therapy of advanced stage CRC patients and hinder progression of early stage patients into metastasis. A novel target is the crucial oncogene Metastasis-Associated in Colon Cancer 1 (MACC1) involved in molecular pathogenesis of CRC metastasis. MACC1 induces cell proliferation and motility, supports cellular survival and rewires metabolism resulting in increased metastasis in vivo. MACC1 is a prognostic biomarker not only for CRC but for more than 20 solid cancer entities. Inflammation plays a pivotal role in tumorigenesis, tumor progression and metastasis. For CRC, inflammatory bowel disease and ulcerative colitis are important inflammation associated risk factors. Certain cytokines, such as TNF-α and IFN-γ, are key factors in determining the contribution of the inflammatory process to CRC. Knowledge of the connection between inflammation and MACC1 driven tumors remains unclear. Gene expression analysis of CRC cells after cytokine stimulation was analyzed by qRT-PCR and Western blot. Cellular motility was assessed by Boyden chamber assays. MACC1 promoter activity after stimulation with pro-inflammatory cytokines was measured using promoter-luciferase constructs. To investigate signal transduction from receptor to effector molecules, blocking experiments using neutralizing antibodies and knockdown experiments were performed. Following TNF-α stimulation, MACC1 and c-Jun expression were significantly increased at the mRNA and protein level. Knockdown of c-Jun reduced MACC1 inducibility following TNF-α stimulation. TNF-α promoted MACC1-induced cell migration that was reverted following MACC1 knockdown. Moreover, MACC1 and c-Jun expression were downregulated by blocking TNFR1, but not TNFR2. Knock down of the NF-κB subunit, p65, reduced basal MACC1 and c-Jun mRNA expression levels. Adalimumab, a clinically approved monoclonal anti-TNF-α antibody, hindered MACC1 induction. The present study highlights that TNF-α regulates the induction of MACC1 via the NF-κB subunit p65 and the transcription factor c-Jun in CRC cells. This finding unravels a novel signaling pathway upstream of MACC1 and provides a potential therapeutic target for the treatment of CRC patients with an associated inflammation

Store-operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses. Using mass cytometry (CyTOF) to analyze the immune cell composition in the lamina propria (LP) of patients with ulcerative colitis (UC) and Crohn's disease (CD), we observed an enrichment of CD(4+) effector T cells producing IL-17A and TNF, CD(8+) T cells producing IFNγ, T regulatory (Treg) cells, and innate lymphoid cells (ILC). The function of these immune cells is regulated by store-operated Ca(2+) entry (SOCE), which results from the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI and STIM proteins. We observed that the pharmacologic inhibition of SOCE attenuated the production of proinflammatory cytokines including IL-2, IL-4, IL-6, IL-17A, TNF, and IFNγ by human colonic T cells and ILCs, reduced the production of IL-6 by B cells and the production of IFNγ by myeloid cells, but had no effect on the viability, differentiation, and function of intestinal epithelial cells. T cell-specific deletion of CRAC channel genes in mice showed that Orai1, Stim1, and Stim2-deficient T cells have quantitatively distinct defects in SOCE, which correlate with gradually more pronounced impairment of cytokine production by Th1 and Th17 cells and the severity of IBD. Moreover, the pharmacologic inhibition of SOCE with a selective CRAC channel inhibitor attenuated IBD severity and colitogenic T cell function in mice. Our data indicate that SOCE inhibition may be a suitable new approach for the treatment of IBD

The NADPH oxidase complexes in Botrytis cinerea: evidence for a close association with the ER and the tetraspanin Pls1.

NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ΔbcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination

Correction: The NADPH Oxidase Complexes in Botrytis cinerea: Evidence for a Close Association with the ER and the Tetraspanin Pls1.

[This corrects the article on p. e55879 in vol. 8.]

Continuous Glucose Monitoring and Physical Activity

Continuous glucose monitoring (CGM) use has several potential positive effects on diabetes management. These benefits are, e.g., increased time in range (TIR), optimized therapy, and developed documentation. Physical activity is a recommended intervention tool in diabetes management, especially for people with type 2 diabetes (T2D). The benefits of physical activity for people with diabetes can be seen as an improvement of glycemic control, glycemic variability, and the reduction of insulin resistance. In relation to the physical activity of people with T2D, the benefits of CGM use can even be increased, and CGM can be a helpful tool to prevent adverse events due to physical activity of people with diabetes, such as hypoglycemic events and nocturnal hypoglycemia after sports. This narrative review aims to provide solid recommendations for the use of CGM in everyday life physical activities based on the noted benefits and to give a general overview of the guidelines on physical activity and CGM use for people with diabetes

Deep impact? Is mercury in dab (Limanda limanda) a marker for dumped munition? Results from munition dump site Kolberger Heide (Baltic Sea)

Dumped munitions contain various harmful substances which can affect marine biota like fish. One of them is mercury (Hg), included in the common explosive primer Hg fulminate. There is still a lack of knowledge whether dumped munitions impact the Hg concentrations in the Baltic Sea environment. This study aims to answer the question if dab caught at the dump site Kolberger Heide show higher Hg concentrations released from munition sources and whether Hg in fish is a usable marker for munition exposure. Therefore, a total of 251 individual dab (Limanda limanda) were analysed including 99 fish from the dump site. In fish from the Kolberger Heide, no elevated Hg concentrations were found compared to reference sites when age-dependent bioaccumulation of mercury was considered. Therefore we conclude that Hg in fish is no suitable indicator for exposure to munition dumping, e.g. in the frame of possible future monitoring studies as Hg exposure originating from dumped munition is only a small contributor to overall Hg exposure of fish