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Mezzanotte, Laura, Juvita Delancy Iljas, Ivo Que, Alan Chan, Eric Kaijzel, Rob Hoeben, and Clemens Löwik. "Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter." Cell Transplantation 26, no. 12 (December 2017): 1878–89. http://dx.doi.org/10.1177/0963689717739718.
Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging. However, sensitive longitudinal imaging of transplanted stem cells in deep tissue like the brain remains challenging not only due to low light penetration but because of other factors such as low or inferior expression levels of optical reporters in stem cells and stem cell death after transplantation. Here we describe an optimized imaging protocol for sensitive long-term monitoring of bone marrow-derived human mesenchymal stem cells (hMSCs) expressing a novel bioluminescent/near infrared fluorescent (NIRF) fusion reporter transplanted in mouse brain cortex. Lentivirus expressing the luc2-iRFP720 reporter, a fusion between luc2 codon-optimized firefly luciferase (luc2) and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were analyzed for their fluorescent and bioluminescent emission and checked for their differentiation potential. In vivo experiments were performed by transplanting decreasing amounts of luc2-iRFP720 expressing hMSCs in mouse brain, followed by fluorescence and bioluminescence imaging (BLI) starting 1 wk after cell injection when the blood–brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luc2 substrate performances, that is, D-luciferin (D-Luc; 25 μM/kg or 943 μM/kg) or CycLuc1 (25 μM/kg). Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential toward adipocytes, chondrocytes, and osteocytes, suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1 × 105 cells using both fluorescence and BLI. The highest bioluminescent signals (∼1 × 107 photons per second) were achieved 15 min after the injection of D-Luc (943 μM/kg). This allowed us to monitor as low as 1 × 105 hMSCs for the subsequent 7 wk without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.
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Leben, Ruth, Randall L. Lindquist, Anja E. Hauser, Raluca Niesner, and Asylkhan Rakhymzhan. "Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range." International Journal of Molecular Sciences 23, no. 21 (November 2, 2022): 13407. http://dx.doi.org/10.3390/ijms232113407.
Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.
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Wilson, Amy, Kirsty Wilson, Maree Bilandzic, Laura Moffitt, Ming Makanji, Mark Gorrell, Martin Oehler, Adam Rainczuk, Andrew Stephens, and Magdalena Plebanski. "Non-Invasive Fluorescent Monitoring of Ovarian Cancer in an Immunocompetent Mouse Model." Cancers 11, no. 1 (December 31, 2018): 32. http://dx.doi.org/10.3390/cancers11010032.
Ovarian cancers (OCs) are the most lethal gynaecological malignancy, with high levels of relapse and acquired chemo-resistance. Whilst the tumour–immune nexus controls both cancer progression and regression, the lack of an appropriate system to accurately model tumour stage and immune status has hampered the validation of clinically relevant immunotherapies and therapeutic vaccines to date. To address this need, we stably integrated the near-infrared phytochrome iRFP720 at the ROSA26 genomic locus of ID8 mouse OC cells. Intrabursal ovarian implantation into C57BL/6 mice, followed by regular, non-invasive fluorescence imaging, permitted the direct visualization of tumour mass and distribution over the course of progression. Four distinct phases of tumour growth and dissemination were detectable over time that closely mimicked clinical OC progression. Progression-related changes in immune cells also paralleled typical immune profiles observed in human OCs. Specifically, we observed changes in both the CD8+ T cell effector (Teff):regulatory (Treg) ratio, as well as the dendritic cell (DC)-to-myeloid derived suppressor cell (MDSC) ratio over time across multiple immune cell compartments and in peritoneal ascites. Importantly, iRFP720 expression had no detectible influence over immune profiles. This new model permits non-invasive, longitudinal tumour monitoring whilst preserving host–tumour immune interactions, and allows for the pre-clinical assessment of immune profiles throughout disease progression as well as the direct visualization of therapeutic responses. This simple fluorescence-based approach provides a useful new tool for the validation of novel immuno-therapeutics against OC.
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Ogawa, Katsuhiro, Kentaro Yamada, Tsuyoshi Etoh, Masahiro Kitagawa, Yoshinori Shirasaka, Kazuko Noguchi, Takeshi Kobayashi, Akira Nishizono, and Masafumi Inomata. "Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720." Journal of Virological Methods 308 (October 2022): 114574. http://dx.doi.org/10.1016/j.jviromet.2022.114574.
Isomura, Minori, Kentaro Yamada, Kazuko Noguchi, and Akira Nishizono. "Near-infrared fluorescent protein iRFP720 is optimal for in vivo fluorescence imaging of rabies virus infection." Journal of General Virology 98, no. 11 (November 1, 2017): 2689–98. http://dx.doi.org/10.1099/jgv.0.000950.
Fukuda, Aya, Shiho Honda, Norie Fujioka, Yuya Sekiguchi, Seiya Mizuno, Yoshihiro Miwa, Fumihiro Sugiyama, Yohei Hayashi, Ken Nishimura, and Koji Hisatake. "Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720." PLOS ONE 14, no. 11 (November 15, 2019): e0225213. http://dx.doi.org/10.1371/journal.pone.0225213.
Hu, Han, Ziyi Zhang, Runyang Wang, Yang Wang, Jing Jin, Linkang Cai, Junhan Yang, et al. "BGC823 Cell Line with the Stable Expression of iRFP720 Retains Its Primary Properties with Promising Fluorescence Imaging Ability." DNA and Cell Biology 39, no. 5 (May 1, 2020): 900–908. http://dx.doi.org/10.1089/dna.2019.5057.
The discovery that white adipocytes can undergo a browning process to become metabolically active beige cells has attracted significant interest in the fight against obesity. However, the study of adipose browning has been impeded by a lack of imaging tools that allow longitudinal and noninvasive monitoring of this process in vivo. Here, we report a preclinical imaging approach to detect development of beige adipocytes during adrenergic stimulation. In this approach, we expressed near-infrared fluorescent protein, iRFP720, driven under an uncoupling protein-1 (Ucp1) promoter in mice by viral transduction, and used multispectral optoacoustic imaging technology with ultrasound tomography (MSOT-US) to assess adipose beiging during adrenergic stimulation. We observed increased photoacoustic signal at 720 nm, coupled with attenuated lipid signals in stimulated animals. As a proof of concept, we validated our approach against hybrid positron emission tomography combined with magnetic resonance (PET/MR) imaging modality, and quantified the extent of adipose browning by MRI-guided segmentation of 2-deoxy-2-18F-fluoro-d-glucose uptake signals. The browning extent detected by MSOT-US and PET/MR are well correlated with Ucp1 induction. Taken together, these systems offer great opportunities for preclinical screening aimed at identifying compounds that promote adipose browning and translation of these discoveries into clinical studies of humans.
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Stepanenko, Olesya V., Olga V. Stepanenko, Olesya G. Shpironok, Alexander V. Fonin, Irina M. Kuznetsova, and Konstantin K. Turoverov. "Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore." International Journal of Molecular Sciences 20, no. 23 (December 2, 2019): 6067. http://dx.doi.org/10.3390/ijms20236067.
Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.
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Степаненко, Ольга В., and Олеся В. Степаненко. "АГРЕГАЦИЯ БЛИЖНЕ-ИНФРАКРАСНОГО ФЛУОРЕСЦЕНТНОГО БЕЛКА iRFP713 ПРИ РАЗВОРАЧИВАНИИ, "Цитология"." Tsitologiya, no. 10 (2018): 842–46. http://dx.doi.org/10.7868/s004137711810017x.
В настоящей работе мы исследовали процессы разворачивания-сворачивания белка iRFP713, принадлежащего к классу ближне-инфракрасных флуоресцентных маркеров (NIR FPs), широко используемых для исследования молекулярных процессов отдельной клетки и визуализации тканей и органов целого организма в реальном масштабе времени. Разворачивание iRFP713 в апоформе (в отсутствие хромоформа) и в холоформе (в комплексе с биливердином) под действием сильного химического денатуранта гуанидинтиоцианата (GTC) начинается с диссоциации димера белка на мономеры. Агрегация iRFP713 при использовании GTC обусловлена совпадением области концентраций денатуранта, в которой происходит образование промежуточного состояния белка, с областью концентраций денатуранта, оптимальной для нейтрализации заряда поверхности белка.
Tong, Xinlin. "Mechanisms of action of Dipeptidyl Peptidase 9 in liver cancer." Thesis, The University of Sydney, 2020. https://hdl.handle.net/2123/24732.
Dipeptidyl peptidase 9 (DPP9) is an emerging cancer associated protease, which has unclear roles in hepatocellular carcinoma (HCC). DPP9 has important roles in DNA Double-Strand Break Repair (DSBR), inflammasome and immunoregulation, metabolism, and disease pathogenesis. Here we used hepatocyte-specific gene knock-out and enzymatic deficiency strategies to characterise the roles of DPP9 in the context of DNA Double-Strand Breaks (DSBs) and HCC.
DPP9 gki (DPP9 enzyme-negative gene-knock-in; DPP9S729A/S729A) Mouse Embryonic Fibroblasts (MEFs) had been derived from DPP9S729A/S729A mice. Compared to DPP9 wild-type (wt) MEFs, DPP9 enzymatically deficient MEFs were hypersensitive to DSBs. DPP9 localisation was not changed following DSBs regardless of its enzyme activity. The chromatin-associated DPP9 did not quantitatively change following DSBs.
DPP9 hepatocyte-specific depleted male C57Bl/6J mice and littermate control mice were treated with Diethylnitrosamine (DEN) and Thioacetamide (TAA), and were provided with an atherogenic High Fat Diet (HFD) until the age of 28 weeks. The hepatocyte-specific DPP9 depletion was not lethal. In this model, mice with hepatocyte-specific DPP9 depletion had reduced mass of the liver and subcutaneous adipose tissue, and lower fasting plasma glucose. These mice developed similar levels of HCC, inflammation and steatosis as the control group by histology assessment. These mice with hepatocyte-specific DPP9 depletion showed increased levels of active caspase-1 and of the autophagy marker beclin1. These mice did not have significantly different levels of the DSB marker γH2AX.
Following the success of a near-infrared fluorescent protein (iRFP720) based ovarian cancer monitoring system in our collaborative laboratory, the iRFP720 fluorescent cellular tag integration was also successfully performed on a mouse liver cancer cell line (Hepa 1-6). Such fluorescent liver cancer cell line could be used in the future for the development of an orthoptic HCC model.
In conclusion, DPP9 in the hepatocytes does not drive cancer formation. Increased caspase-1 activation and autophagy regulation was observed in mice with hepatocyte-specific DPP9 depletion. DPP9 might have a role in glucose metabolism regulation, which needs further investigation. DPP9 could be therapeutic target in the liver via intrahepatic regulation of inflammasome activation, autophagy and metabolism.
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Wang, Hui [Verfasser], Martin [Akademischer Betreuer] Klingenspor, Thomas [Gutachter] Skurk, and Martin [Gutachter] Klingenspor. "Phenotypic characterization of a novel Ucp1-LUC-iRFP713 reporter mouse model for visualization and quantification of brown and beige fat recruitment / Hui Wang ; Gutachter: Thomas Skurk, Martin Klingenspor ; Betreuer: Martin Klingenspor." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1211725154/34.
Lashuk, Kanstantsin, Jacqueline Bersano, Dorothee Lenhard, Eva Oswald, Kerstin Klingner, and Julia Schüler. "Abstract 2806: Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-2806.
