Transcriptome sequencing revealed differences in the response of renal cancer cells to hypoxia and CoCl2 treatment [version 1; referees: 2 approved]

Human cancer cells are subjected to hypoxic conditions in many tumours. Hypoxia causes alterations in the glycolytic pathway activation through stabilization of hypoxia-inducible factor 1. Currently, two approaches are commonly used to model hypoxia: an alternative to generating low-oxygen conditions in an incubator, cells can be treated with CoCl2. We performed RNA-seq experiments to study transcriptomes of human Caki-1 cells under real hypoxia and after CoCl2 treatment. Despite causing transcriptional changes of a much higher order of magnitude for the genes in the hypoxia regulation pathway, CoCl2 treatment fails to induce alterations in the glycolysis / gluconeogenesis pathway. Moreover, CoCl2 caused aberrant activation of other oxidoreductases in glycine, serine and threonine metabolism pathways

Optimized Method for Preparation of IgG-Binding Bacterial Magnetic Nanoparticles

<div><p>In this study, the optimized method for designing IgG-binding magnetosomes based on integration of IgG-binding fusion proteins into magnetosome membrane <i>in vitro</i> is presented. Fusion proteins Mbb and Mistbb consisting of magnetosome membrane protein MamC and membrane associating protein Mistic from <i>Bacillus subtilis</i> as anchors and BB-domains of <i>Staphylococcus aureus</i> protein A as IgG-binding region were used. With Response Surface Methodology (RSM) the highest level of proteins integration into magnetosome membrane was achieved under the following parameters: pH 8.78, without adding NaCl and 55 s of vortexing for Mbb; pH 9.48, 323 mM NaCl and 55 s of vortexing for Mistbb. Modified magnetosomes with Mbb and Mistbb displayed on their surface demonstrated comparable levels of IgG-binding activity, suggesting that both proteins could be efficiently used as anchor molecules. We also demonstrated that such modified magnetosomes are stable in PBS buffer during at least two weeks. IgG-binding magnetosomes obtained by this approach could serve as a multifunctional platform for displaying various types of antibodies.</p></div

Coomassie stained SDS-PAGE of purified, heterologously expressed proteins Mbb and Mistbb.

<p>M – protein molecular weight marker.</p

The results of IgG-binding activities of BMP-Mistbb and BMP-Mbb comparison by ELISA.

<p>Experiment was performed in triplicate.</p

AFM images and topographic cross sections along the lines of IgG (A); BMP-Mbb incubated with IgG (B); BMP-Mistbb incubated with IgG (C); intact magnetosomes incubated with IgG (D).

<p>AFM images and topographic cross sections along the lines of IgG (A); BMP-Mbb incubated with IgG (B); BMP-Mistbb incubated with IgG (C); intact magnetosomes incubated with IgG (D).</p

AFM images and topographic cross sections along the lines of intact magnetosomes (A); magnetosomes with integrated Mbb (B) and Mistbb (C) into the magnetosome membrane.

<p>AFM images and topographic cross sections along the lines of intact magnetosomes (A); magnetosomes with integrated Mbb (B) and Mistbb (C) into the magnetosome membrane.</p

The matrix of the BBD experiment for optimization of proteins integration and the corresponding experimental data.

<p>The matrix of the BBD experiment for optimization of proteins integration and the corresponding experimental data.</p

The level of variables for the Box-Behnken design.

<p>The level of variables for the Box-Behnken design.</p

The results of IgG-binding activities of Mistbb and Mbb comparison by ELISA.

<p>The results of IgG-binding activities of Mistbb and Mbb comparison by ELISA.</p

Immunoglobulin-binding ability of Mbb and Mistbb integrated into magnetosome membrane.

<p>Dilution 1∶1 corresponds to 1 µg of antibody per 1 ml. Experiment was performed in triplicate.</p