Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer and . Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes . Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses - at least in models of triple-negative breast cancer

Whole-body imaging using Amrose v2 in FMT.

<p>Amrose v2 expressing DT40 cells imaged with a Fluorescence Molecular Tomography (FMT) system which allows for tomographic reconstruction of the tube using 670 nm excitation and 710 nm emission.</p

Whole-body imaging comparing Amrose v1 to mRaspberry.

<p>Fluorescence images of FPs samples in the esophagous using 2 different sets of excitation/emission wavelengths (590/610 and 635/670) overlaid on black and white images. Pixel values are in arbitrary units. Colorbar displays the transparency function. Bar chart – Comparison of accumulated FP intensity over a region of interest (dotted square) at the peak wavelength. Procedure defined unit  =  p.d.u.</p

Comparing absorption and excitation.

<p>Normalized absorption and excitation spectra of Amrose v1, eqFP615 and eqFP650 taken from <i>E.coli</i> produced purified protein using pRSETa vector (Invitrogen).</p

Comparison of Amrose variants in the zebrafish model.

<p>Fig. 4a. Amrose v0–v4 variants, as well as HcRed, mCherry and mPlum, visualized in zebrafish 24 hours after 100 ng/µl mRNA injection using 633 nm excitation and scanning the emission spectrum. The crosshairs indicate where the measurement took place. Fig. 4b. Intensity as recorded in zebrafish (crosshairs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107069#pone-0107069-g004" target="_blank">Fig. 4a</a> show where measurement took place) at an excitation wavelength of 633 nm and scanned for emission at 11 nm intervals from 654 nm to 794 nm. Procedure defined unit  =  p.d.u.</p

Spectral properties of purified protein in PBS.

<p>r  =  reported.</p><p>Spectral properties of purified protein in PBS.</p