Red Fluorescent Protein Expression in Transgenic Founder of Angelfish (Pterophyllum sp) Driven by Zebrafish Myosin Light Chain 2 Promoter

Angelfish (Pterophyllum sp.) are attractive fish popular with aquarists. The introduction of fluorescent protein genes into angelfish has been reported, but specific techniques have not been revealed. This study aimed to develop a strategy to produce red fluorescent protein (RFP) transgenic angelfish driven by the myosin light chain 2 (mylz2) promoter from zebrafish. A 1999 bp Mylz2 promoter fragment was isolated from zebrafish muscle genomic DNA. This promoter fragment was then cloned into the plasmid pDsred2-1 open-loop at restriction enzyme SacI and AgeI sites to create the final transgene construct pMylz2-RFP. Angelfish embryos at one cell stage were microinjected with approximately 100 pg of the plasmid pMylz2-RFP. From 524 microinjected embryos, 16 successfully hatched, while 12 showed red fluorescence signals. Two larvae survived to 2 months of age. They showed significant red fluorescence expression in the muscles, suggesting that the angelfish could be used as potential transgenic founders to evaluate the next generation of stable red fluorescence expression transgenic fish.

Photoluminescence mechanism of carbon dots: triggering high-color-purity red fluorescence emission through edge amino protonation

Due to complex structure and surface functionalities, photoluminescence mechanisms of Carbon Dots are unknown, and it is challenging to synthesize Carbon Dots to achieve the desired optical properties. Herein, Carbon Dots simultaneously exhibiting high-color-purity (FWHM~24 nm) long wavelength one-photon fluorescence emission at 620 nm and NIR induced two-photon fluorescence emission at 630 and 680 nm are prepared by edge amino protonation treatment. Systematic analysis reveals that the protonation of 2,3-diaminophenazine changes the molecular state of Carbon Dots, decreases the photon transition band gap, and triggers red fluorescence emission with the dramatically narrowed peak width. As the oxidation products of reactant o-phenylendiamine, the emergence of 2,3-diaminophenazine as a photoluminescence determiner suggests that fluorophore products of precursor conversion are viable determinants of the desired luminescence properties of Carbon Dots. This work shows a new way for predicting and controlling photoluminescence properties of Carbon Dots, and may guide the development of tunable Carbon Dots for a broad range of applications.

164 Potent tumor organoid infiltration and killing by PBMC-derived effector cells

BackgroundAlloplex Biotherapeutics has developed a novel autologous cellular therapy for cancer that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand a heterogeneous population of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). The 2-week manufacturing process from PBMCs consistently results 300-fold expansion of NK cells, CD8+ T cells, gamma/delta T cells, NKT cells and some CD4+ T cells, collectively called SUPLEXA therapeutic cells. SUPLEXA cells will be delivered back to cancer patients via intravenous administrations on a weekly schedule as an autologous adoptive cellular immunotherapy for cancer. In this study, we tested SUPLEXA cells developed from normal healthy volunteer PBMCs for their ability to infiltrate and kill patient-derived tumor organoids (PDO) as a pre-clinical assessment for potency against 2 different types of tumor organoids.MethodsTumor organoids derived from colorectal cancer (CRC) or non-small cell lung carcinoma (NSCLC) patients were labeled with cell-trace red dye and plated at equal density in a 96-well plate. After 3 days culture, SUPLEXA cells were thawed (82.8% viable), labeled with cell-trace violet dye, and added to PDO at 1:2 serial diluted numbers ranging from 2 million to 7,800 cells per well. Fluorescent images were captured at 24 hours after adding SUPLEXA cells to PDO models to measure PDO size, tumor infiltration, and PDO killing.ResultsAdding SUPLEXA cells to PDO from CRC and NSCLC resulted in significant infiltration and killing of organoids by 24 hours as shown by the fluorescent images and the organoid size plot for the CRC PDO model (figure 1). Significant reduction in PDO size was observed by adding 31,240 SUPLEXA cells. Similar results were observed with the NSCLC PDO model with significant reduction in PDO size by adding 15,600 SUPLEXA cells. Obvious organoid infiltration was observed in both PDO models and organoid fluorescence was significantly reduced by addition of SUPLEXA cells in both PDO models to suggest that SUPLEXA cells were able to reduce tumor burden (figure 2).Abstract 164 Figure 1CRC organoid infiltration and killing by SUPLEXA. A representative fluorescent image of CRC organoid killing with addition of increasing SUPLEXA cell numbers and a plot showing statistical analysis of 6 replicate wells for changes in CRC organoid size in relation to SUPLEXA cell number additionsAbstract 164 Figure 2Dose-dependent killing in CRC and NSCLC PDO models. CRC and NSCLC organoids were detected by total red fluorescence at 24 hours after adding the indicated numbers of SUPLEXA cells. Loss of red fluorescence after adding SUPLEXA is a measure of overall tumor cell killing/burden in organoids. Data is plotted as mean ± SEM for n=6 replicates per group.ConclusionsSUPLEXA cells infiltrated and killed tumor cells in patient-derived organoids within 24 hours of culture at low cell concentrations indicating potent tumor killing activity. The observed activity in both colorectal and lung cancer organoid models support broad anti-tumor killing activity by SUPLEXA. These results provide further evidence that PBMCs from cancer patients can be activated and expanded by our approach as a novel autologous cellular immunotherapy for cancer.

Aflibercept clearance through the drainage system in a rat model

Background As intravitreal anti-VEGF injections became the mainstay of treatment for many retinal diseases, the cause of a secondary sustained elevated intraocular pressure is still unclear. The aim of our study was to study the clearance of Aflibercept from the anterior chamber angle, in a rat model, to test if an aggregation exists. Methods Choroidal neovascular lesions (CNV) were induced in the right eye of 12 brown Norway rats, using indirect laser ophthalmoscope. Intravitreal Aflibercept injection (0.12 mg/3 µl) was performed 3 days after CNV induction. Rats were euthanized at predetermine time intervals of 3, 6, 24 and 48 h post injection, with immediate enucleation for histological analysis with H&E and immunofluorescence staining. Aflibercept molecules were stained with red fluorescence thanks to the formation of the immune complex Aflibercept-Rabbit anti human IgG-Anti rabbit antibodies-Cy3. Results Immediately after the injection, a strong fluorescence signal was detected, indicating the presence of Aflibercept in the iridocorneal angle. At 3- and 6-h interval a strong signal of Aflibercept was still seen. Six hours post injection, the signal was highly concentrated in Schlemm’s canal. In the 2 eyes harvested 24 h post Aflibercept injection, red fluorescence signal intensity was decreased in one eye, occupying mainly intra scleral venous plexuses, and absent in the other eye. At 48 h there was no fluorescence signal, confirming complete clearance of Aflibercept. Conclusions In our rat model, a complete clearance of Aflibercept from the anterior chamber angle, was seen 48 h after the injection. This finding refutes the theory of possible connection between IOP elevation and mechanical obstruction. Evacuation time of Aflibercept through the angle is of the same magnitude as that of Bevacizumab in the same rat model.