Journal articles: 'IRFP720'

1

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.

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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.

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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.

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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.

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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.

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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.

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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.

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Chan, Xin Hui Derryn, Ghayathri Balasundaram, Amalina Binte Ebrahim Attia, Julian L. Goggi, Boominathan Ramasamy, Weiping Han, Malini Olivo, and Shigeki Sugii. "Multimodal imaging approach to monitor browning of adipose tissue in vivo." Journal of Lipid Research 59, no. 6 (April 13, 2018): 1071–78. http://dx.doi.org/10.1194/jlr.d083410.

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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.

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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.

10

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.

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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.

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Stepanenko, Olesya, Olga Stepanenko, Irina Kuznetsova, and Konstantin Turoverov. "The Pathways of the iRFP713 Unfolding Induced by Different Denaturants." International Journal of Molecular Sciences 19, no. 9 (September 15, 2018): 2776. http://dx.doi.org/10.3390/ijms19092776.

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Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome RpBphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH+ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH+ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter A, was found to have a different residual structure in the denaturants used.

12

Stepanenko, Olesya V., Olga V. Stepanenko, Irina M. Kuznetsova, and Konstantin K. Turoverov. "The unfolding of iRFP713 in a crowded milieu." PeerJ 7 (April 8, 2019): e6707. http://dx.doi.org/10.7717/peerj.6707.

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The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein.

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Alam, Mohammad Wajih, Khan A. Wahid, Md Fahmid Islam, Wendy Bernhard, Clarence R. Geyer, and Franco J. Vizeacoumar. "A Low-Cost and Portable Smart Instrumentation for Detecting Colorectal Cancer Cells." Applied Sciences 9, no. 17 (August 26, 2019): 3510. http://dx.doi.org/10.3390/app9173510.

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Fluorescence imaging is a well-known method for monitoring fluorescence emitted from the subject of interest and provides important insights about cell dynamics and molecules in mammalian cells. Currently, many solutions exist for measuring fluorescence, but the application methods are complex and the costs are high. This paper describes the design and development of a low-cost, smart and portable fluorimeter for the detection of colorectal cancer cell expressing IRFP702. A flashlight is used as a light source, which emits light in the visible range and acts as an excitation source, while a photodiode is used as a detector. It also uses a longpass filter to only allow the wavelength of interest to pass from the cultured cell. It eliminates the need of both the dichroic mirror and excitation filter, which makes the developed device low cost, compact and portable as well as lightweight. The custom-built sample chamber is black in color to minimize interference and is printed with a 3D printer to accommodate the detector circuitry. An established colorectal cancer cell line (human colorectal carcinoma (HCT116)) was cultured in the laboratory environment. A near-infrared fluorescent protein IRFP702 was expressed in the colorectal cancer cells that were used to test the proof-of-concept. The fluorescent cancer cells were first tested with a commercial imaging system (Odyssey® CLx) and then with the developed prototype to validate the result in a preclinical setting. The developed fluorimeter is versatile as it can also be used to detect multiple types of cancer cells by simply replacing the filters based on the fluorophore.

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Cook, Zoe T., Nicole L. Brockway, Zachary J. C. Tobias, Joy Pajarla, Isaac S. Boardman, Helen Ippolito, Sylvia Nkombo Nkoula, and Tamily A. Weissman. "Combining near-infrared fluorescence with Brainbow to visualize expression of specific genes within a multicolor context." Molecular Biology of the Cell 30, no. 4 (February 15, 2019): 491–505. http://dx.doi.org/10.1091/mbc.e18-06-0340.

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Fluorescent proteins are a powerful experimental tool, allowing the visualization of gene expression and cellular behaviors in a variety of systems. Multicolor combinations of fluorescent proteins, such as Brainbow, have expanded the range of possible research questions and are useful for distinguishing and tracking cells. The addition of a separately driven color, however, would allow researchers to report expression of a manipulated gene within the multicolor context to investigate mechanistic effects. A far-red or near-infrared protein could be particularly suitable in this context, as these can be distinguished spectrally from Brainbow. We investigated five far-red/near-infrared proteins in zebrafish: TagRFP657, mCardinal, miRFP670, iRFP670, and mIFP. Our results show that both mCardinal and iRFP670 are useful fluorescent proteins for zebrafish expression. We also introduce a new transgenic zebrafish line that expresses Brainbow under the control of the neuroD promoter. We demonstrate that mCardinal can be used to track the expression of a manipulated bone morphogenetic protein receptor within the Brainbow context. The overlay of near-infrared fluorescence onto a Brainbow background defines a clear strategy for future research questions that aim to manipulate or track the effects of specific genes within a population of cells that are delineated using multicolor approaches.

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Oswald, Eva, Kanstantsin Lashuk, Johannes Gojo, Dorothee Lenhard, Norman Mack, Sonja Krausert, Daniela Lötsch, et al. "Abstract 1673: Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1673. http://dx.doi.org/10.1158/1538-7445.am2022-1673.

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Abstract Brain and spinal cord tumors are the second most common group of cancers in children. For children who experience relapses of their tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Thus, the need for predictive preclinical platforms explicitly for pediatric brain tumors is an urgent need. In the framework of the ITCC-P4 public-private partnership, we thus far fully established 23 pediatric brain tumor-derived PDX models. A selection of four high grade glioma, one medulloblastoma and one ependymoma models were used to validate a protocol for fluorescence-based optical imaging. Tumor growth characteristics, latency and histopathology were evaluated for the un-transduced and iRFP713-transduced models side by side. In case of iRFP713-transduced PDX models, tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany). All animals were examined for neurological symptoms daily and body weight was examined twice a week. The latency of the tumor models ranged from 29 days to 180 days. The take rate was 100% across all the models with n=12 NSG mice per setting. The transduction did not influence the take rate, but 30 - 40% more donor material was needed due to viability loss during the overnight transduction. It was not possible to determine the transduction efficiency for iRFP713 in the overnight culture as the signal is getting upregulated only 48h - 72h post transduction, e.g., when the cells are already implanted. The signal was stable for up to 180 days in the slowest tumor model ependymoma HN0579. The fastest model, high grade glioma HG0068, reached termination criteria within 24 days. Histopathological examination was strictly correlated with the tumor load determined by optical imaging in situ and ex vivo (organ imaging). The histopathological investigation of the mouse brains displayed no differences in tumor localization, size, and invasiveness between the transduced and the un-transduced lines. The proliferation rate determined by Ki-67 staining was not influenced by the modification of the cells. Further molecular and phenotypic characterization of the transduced vs the un-transduced PDX will increase the utility of this platform for the development of new drugs and the identification of innovative drug targets. Citation Format: Eva Oswald, Kanstantsin Lashuk, Johannes Gojo, Dorothee Lenhard, Norman Mack, Sonja Krausert, Daniela Lötsch, David Jones, Marcel Kool, Till Milde, Walter Berger, Stefan M. Pfister, Julia Schüler. Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1673.

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Baloban, Mikhail, Daria M. Shcherbakova, and Vladislav V. Verkhusha. "Two-Color Imaging using Spectral Variants of iRFP670 and iRFP682 Near-Infrared Fluorescent Proteins." Biophysical Journal 108, no. 2 (January 2015): 624a. http://dx.doi.org/10.1016/j.bpj.2014.11.3394.

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Saragi, Yosua Hamonangan, Abdul Syakur, and Hadha Afrisal. "PERANCANGAN PEMBANGKITAN TEGANGAN TINGGI DC DENGAN TRAFO FLYBACK." Transient: Jurnal Ilmiah Teknik Elektro 9, no. 4 (December 17, 2020): 597–604. http://dx.doi.org/10.14710/transient.v9i4.597-604.

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Penggunaan tegangan tinggi tidak hanya untuk keperluan transmisi, tetapi juga dapat digunakan sebagai pemaanfaatan untuk pembuatan perangkap serangga penggangu bahkan berbahaya dengan cara Short Circuit tegangan tinggi dengan tingkat keamanan yang tinggi. Studi tentang tegangan tinggi yang dimanfaatkan untuk membunuh serangga penggangu sering memberi para insinyur wawasan yang berharga untuk meningkatkan keandalan jangka panjang dari peralatan tegangan tinggi tersebut. Pada umumnya, tegangan tinggi dibangkitkan menggunakan pembangkit tegangan tinggi konvensional yang berukuran besar. Oleh karena itu, diperlukan rangkaian yang lebih sederhana untuk membangkitkan tegangan tinggi, salah satunya menggunakan konverter flyback. Konverter flyback merupakan konverter direct curent to direct current (DC-DC) yang memiliki isolasi di antara masukan dan keluarannya. Komponen utama dari konverter flyback adalah trafo step up dan komponen pensaklaran. Pada Penelitian ini dirancang jendela perangkap dengan memanfaatkan konverter flyback untuk membangkitkan tegangan tinggi. Jenis trafo step up dan komponen pensaklaran yang digunakan adalah trafo flyback dari monitor CRT (Cathode Ray Tube) dan MOSFET IRFP460. Tegangan tinggi hasil pembangkitan konverter flyback kemudian di pasang pada kasa jendela perangkap serangga.

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Pratiwi, Novy Arizka, Abdul Syakur, and Karnoto Karnoto. "PERANCANGAN PEMBANGKIT TEGANGAN TINGGI IMPULS 11,20 kV DENGAN MENERAPKAN ZERO VOLTAGE SWITCHING (ZVS) PADA KONVERTER FLYBACK." Transmisi 20, no. 1 (January 31, 2018): 8. http://dx.doi.org/10.14710/transmisi.20.1.8-14.

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Tegangan tinggi tidak hanya digunakan untuk keperluan transmisi, tetapi juga untuk penelitian di laboratorium. Pada umumnya, tegangan tinggi dibangkitkan menggunakan pembangkit tegangan tinggi konvensional yang berukuran besar. Oleh karena itu, diperlukan rangkaian yang lebih sederhana untuk membangkitkan tegangan tinggi, misalnya menggunakan konverter flyback. Konverter flyback merupakan konverter DC-DC yang memiliki isolasi di antara masukan dan keluarannya. Komponen utama dari konverter flyback adalah trafo step up dan komponen pensaklaran. Pada penelitian ini dirancang penerapan zero voltage switching (ZVS) pada konverter flyback untuk membangkitkan tegangan tinggi. Trafo step up dan komponen pensaklaran yang digunakan adalah trafo flyback dari monitor CRT (Cathode Ray Tube) dan MOSFET IRFP460. Tegangan tinggi hasil pembangkitan konverter flyback diterapkan pada reaktor ozon. Pada pengujian mode pensaklaran dengan menerapkan ZVS diketahui tegangan drain-source MOSFET (VDS) bernilai nol ketika MOSFET turn off. Pada pengujian tanpa beban dengan variasi duty cycle diperoleh tegangan keluaran sebesar 11,20 kV pada duty cycle 60%. Pengujian tanpa beban dengan variasi frekuensi switching diperoleh tegangan keluaran sebesar 10,80 kV pada frekuensi switching 23 kHz. Pengujian berbeban dengan reaktor ozon sudah dapat menghasilkan ozon dengan konsentrasi 0,02-0,06 ppm pada tegangan operasi 7,4-8,4 kV.

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Richie, Christopher T., Leslie R. Whitaker, Keith W. Whitaker, Julie Necarsulmer, Heather A. Baldwin, Yajun Zhang, Lowella Fortuno, et al. "Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies." Journal of Neuroscience Methods 284 (June 2017): 1–14. http://dx.doi.org/10.1016/j.jneumeth.2017.03.020.

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Garza-Morales, Rodolfo, Beatriz E. Rendon, Mohammad Tariq Malik, Jeannete E. Garza-Cabrales, Anne Aucouturier, Luis G. Bermúdez-Humarán, Kelly M. McMasters, Lacey R. McNally, and Jorge G. Gomez-Gutierrez. "Targeting Melanoma Hypoxia with the Food-Grade Lactic Acid Bacterium Lactococcus Lactis." Cancers 12, no. 2 (February 13, 2020): 438. http://dx.doi.org/10.3390/cancers12020438.

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Melanoma is the most aggressive form of skin cancer. Hypoxia is a feature of the tumor microenvironment that reduces efficacy of immuno- and chemotherapies, resulting in poor clinical outcomes. Lactococcus lactis is a facultative anaerobic gram-positive lactic acid bacterium (LAB) that is Generally Recognized as Safe (GRAS). Recently, the use of LAB as a delivery vehicle has emerged as an alternative strategy to deliver therapeutic molecules; therefore, we investigated whether L. lactis can target and localize within melanoma hypoxic niches. To simulate hypoxic conditions in vitro, melanoma cells A2058, A375 and MeWo were cultured in a chamber with a gas mixture of 5% CO2, 94% N2 and 1% O2. Among the cell lines tested, MeWo cells displayed greater survival rates when compared to A2058 and A375 cells. Co-cultures of L. lactis expressing GFP or mCherry and MeWo cells revealed that L. lactis efficiently express the transgenes under hypoxic conditions. Moreover, multispectral optoacoustic tomography (MSOT), and near infrared (NIR) imaging of tumor-bearing BALB/c mice revealed that the intravenous injection of either L. lactis expressing β-galactosidase (β-gal) or infrared fluorescent protein (IRFP713) results in the establishment of the recombinant bacteria within tumor hypoxic niches. Overall, our data suggest that L. lactis represents an alternative strategy to target and deliver therapeutic molecules into the tumor hypoxic microenvironment.

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Sita, Timothy L., Fotini M. Kouri, Lisa A. Hurley, Timothy J. Merkel, Alexandra Chalastanis, Jasmine L. May, Serena T. Ghelfi, et al. "Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo." Proceedings of the National Academy of Sciences 114, no. 16 (April 3, 2017): 4129–34. http://dx.doi.org/10.1073/pnas.1702736114.

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RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood–brain barrier-/blood–tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.

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Fehér, Anita, Andrea Schnúr, Suchitra Muenthaisong, Tamás Bellák, Ferhan Ayaydin, György Várady, Elisabeth Kemter, Eckhard Wolf, and András Dinnyés. "Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter." Scientific Reports 12, no. 1 (June 14, 2022). http://dx.doi.org/10.1038/s41598-022-12956-1.

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AbstractStem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

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Comenge, Joan, Jack Sharkey, Oihane Fragueiro, Bettina Wilm, Mathias Brust, Patricia Murray, Raphael Levy, and Antonius Plagge. "Multimodal cell tracking from systemic administration to tumour growth by combining gold nanorods and reporter genes." eLife 7 (June 27, 2018). http://dx.doi.org/10.7554/elife.33140.

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Understanding the fate of exogenous cells after implantation is important for clinical applications. Preclinical studies allow imaging of cell location and survival. Labelling with nanoparticles enables high sensitivity detection, but cell division and cell death cause signal dilution and false positives. By contrast, genetic reporter signals are amplified by cell division. Here, we characterise lentivirus-based bi-cistronic reporter gene vectors and silica-coated gold nanorods (GNRs) as synergistic tools for cell labelling and tracking. Co-expression of the bioluminescence reporter luciferase and the optoacoustic reporter near-infrared fluorescent protein iRFP720 enabled cell tracking over time in mice. Multispectral optoacoustic tomography (MSOT) showed immediate biodistribution of GNR-labelled cells after intracardiac injection and successive clearance of GNRs (day 1–15) with high resolution, while optoacoustic iRFP720 detection indicated tumour growth (day 10–40). This multimodal cell tracking approach could be applied widely for cancer and regenerative medicine research to monitor short- and long-term biodistribution, tumour formation and metastasis.

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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." SSRN Electronic Journal, 2022. http://dx.doi.org/10.2139/ssrn.4088788.

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"A Novel Near-infrared Fluorescent Protein, iRFP720, Facilitates Transcriptional Profiling of Prostate Cancer Bone Metastasis in Mice." Anticancer Research 37, no. 6 (May 29, 2017). http://dx.doi.org/10.21873/anticanres.11655.

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Silva-Bea, Sergio, Mónica Francisco-Tomé, Jorge J. Cabrera-Alvargonzález, Carmen Potel, Maximiliano Álvarez, Sonia Pérez, Benito Regueiro, and Maria P. Cabral. "In vivo monitoring of Lactiplantibacillus plantarum in the nasal and vaginal mucosa using infrared fluorescence." Applied Microbiology and Biotechnology, August 24, 2022. http://dx.doi.org/10.1007/s00253-022-12121-8.

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Abstract Lactic acid bacteria (LAB) of the genus Lactiplantibacillus have been explored as potential mucosal vaccine vectors due to their ability to elicit an immune response against expressed foreign antigens and to their safety. However, tools for monitoring LAB distribution and persistence at the mucosal surfaces are needed. Here, we characterize Lactiplantibacillus plantarum bacteria expressing the infrared fluorescent protein IRFP713 for exploring their in vivo distribution in the mucosa and potential use as a mucosal vaccine vector. This bacterial species is commonly used as a vaginal probiotic and was recently found to have a niche in the human nose. Three different fluorescent L. plantarum strains were obtained using the nisin-inducible pNZRK-IRFP713 plasmid which contains the nisRK genes, showing stable and constitutive expression of IRFP713 in vitro. One of these strains was further monitored in BALB/c mice using near-infrared fluorescence, indicating successful colonization of the nasal and vaginal mucosae for up to 72 h. This study thus provides a tool for the in vivo spatiotemporal monitoring of lactiplantibacilli, allowing non-invasive bacterial detection in these mucosal sites. Key points • Stable and constitutive expression of the IRFP713 protein was obtained in different L. plantarum strains. • IRFP713+ L. plantarum 3.12.1 was monitored in vivo using near-infrared fluorescence. • Residence times observed after intranasal and vaginal inoculation were 24–72 h. Graphical abstract

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Wei, Likun, Meiniang Wang, Haitao Xiang, Yuan Jiang, Jinhua Gong, Dan Su, M. A. R. Al Azad, et al. "Bamboo Shark as a Small Animal Model for Single Domain Antibody Production." Frontiers in Bioengineering and Biotechnology 9 (December 8, 2021). http://dx.doi.org/10.3389/fbioe.2021.792111.

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The development of shark single domain antibodies (sdAbs) is hindered by the high cost and tediousness of large-sized shark farming. Here, we demonstrated white-spotted bamboo sharks (Chiloscyllium plagiosum) being cultivated commercially as a promising small animal model to produce sdAbs. We found that immunoglobulin new antigen receptor (IgNAR) presented in bamboo shark genome, transcriptome, and plasma. Four complete IgNAR clusters including variable domains (vNARs) were discovered in the germline, and the Variable–Joining pair from IgNAR1 cluster was dominant from immune repertoires in blood. Bamboo sharks developed effective immune responses upon green fluorescent protein (GFP), near-infrared fluorescent protein iRFP713, and Freund’s adjuvant immunization revealed by elevated lymphocyte counts and antigen specific IgNAR. Before and after immunization, the complementarity determining region 3 (CDR3) of IgNAR were the major determinant of IgNAR diversity revealed by 400-bp deep sequencing. To prove that bamboo sharks could produce high-affinity IgNAR, we isolated anti-GFP and anti-iRFP713 vNARs with up to 0.3 and 3.8 nM affinities, respectively, from immunized sharks. Moreover, we constructed biparatopic vNARs with the highest known affinities (20.7 pM) to GFP and validated the functions of anti-GFP vNARs as intrabodies in mammalian cells. Taken together, our study will accelerate the discovery and development of bamboo shark sdAbs for biomedical industry at low cost and easy operation.

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Hock, Andreas K., Eric C. Cheung, Timothy J. Humpton, Tiziana Monteverde, Viola Paulus-Hock, Pearl Lee, Ewan McGhee, et al. "Development of an inducible mouse model of iRFP713 to track recombinase activity and tumour development in vivo." Scientific Reports 7, no. 1 (May 12, 2017). http://dx.doi.org/10.1038/s41598-017-01741-0.

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Humpton, Timothy J., Andreas K. Hock, Christos Kiourtis, Marco De Donatis, Frédéric Fercoq, Colin Nixon, Sheila Bryson, et al. "A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo." Science Signaling 15, no. 720 (February 8, 2022). http://dx.doi.org/10.1126/scisignal.abd9099.

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Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are suboptimal in vivo because of poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled noninvasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol- or CCl 4 -induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

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Wang, Hui, Monja Willershäuser, Yongguo Li, Tobias Fromme, Katharina Schnabl, Andrea Bast-Habersbrunner, Samira Ramisch, Sabine Mocek, and Martin Klingenspor. "Uncoupling protein 1 expression does not protect mice from diet-induced obesity." American Journal of Physiology-Endocrinology and Metabolism, November 30, 2020. http://dx.doi.org/10.1152/ajpendo.00285.2020.

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We studied the metabolic phenotype of a novel Ucp1-LUC-iRFP713 knock-in reporter gene mouse model originally generated to monitor endogenous Ucp1 gene expression. Both reporter mice and reporter cells reliably reflected Ucp1 gene expression in vivo and in vitro. We here report an unexpected reduction in UCP1 content in homozygous knock-in (KI) reporter mice. As a result, the thermogenic capacity of KI mice stimulated by norepinephrine was largely blunted, making them more sensitive to an acute cold exposure. In return, these reporter mice with reduced UCP1 expression enabled us to investigate the physiological role of UCP1 in the prevention of weight gain. We observed no substantial differences in body mass across the three genotypes, irrespective of the type of diet or the ambient temperature, possibly due to the insufficient UCP1 activation. Indeed, activation of UCP1 by daily injection of the selective β3-adrenergic receptor agonist CL316,243 resulted in significantly greater reduction of body weight in WT mice than in KI mice. Taken together, we conclude that the intact expression of UCP1 is essential for cold-induced thermogenesis but the presence of UCP1 per se does not protect mice from diet-induced obesity.

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Chen, Muxiong, Zhe Feng, Xiaoxiao Fan, Jun Sun, Weihang Geng, Tianxiang Wu, Jinghao Sheng, Jun Qian, and Zhengping Xu. "Long-term monitoring of intravital biological processes using fluorescent protein-assisted NIR-II imaging." Nature Communications 13, no. 1 (November 4, 2022). http://dx.doi.org/10.1038/s41467-022-34274-w.

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AbstractHigh spatial resolution, low background, and deep tissue penetration have made near-infrared II (NIR-II) fluorescence imaging one of the most critical tools for in vivo observation and measurement. However, the relatively short retention time and potential toxicity of synthetic NIR-II fluorophores limit their long-term application. Here, we report the use of infrared fluorescent proteins (iRFPs) as in vitro and in vivo NIR-II probes permitting prolonged continuous imaging (up to 15 months). As a representative example, iRFP713 is knocked into the mouse genome to generate a transgenic model to allow temporal and/or spatial expression control of the probe. To demonstrate its feasibility in a genuine diagnostic context, we adopt two liver regeneration models and successfully track the process for a week. The performance and monitoring efficacy are comparable to those of μCT and superior to those of indocyanine green dye. We are also able to effectively observe the pancreas, despite its deep location, under both physiological and pathological conditions. These results indicate that the iRFP-assisted NIR-II fluorescence system is suitable for monitoring various tissues and in vivo biological processes, providing a powerful noninvasive long-term imaging platform.

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Adam, Grizly, Leonardus Heru Pratomo, and Arifin Wibisono. "Desain dan Implementasi PLC Outseal untuk Menggerakan Motor DC dengan Berbagai Variasi Kecepatan." Seminar Nasional Teknik Elektro, Informatika dan Sistem Informasi 1, no. 1 (August 29, 2022). http://dx.doi.org/10.35842/sintaks.v1i1.24.

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Programmable Logic Controller (PLC) sebuah unit pengendali pusat pada industri, terdiri dari dari Central Processing Unit (CPU), memori serta modul Input / Output untuk mengatur data input / output. PLC Outseal merupakan inovasi baru dengan memanfaatkan Arduino sebagai CPU dan dikembangkan dengan menambahkan pin input, output, input ADC dan berbagai fungsi yang dapat digunakan sebagaimana mestinya PLC berfungsi. PLC Outseal dikendalikan dengan program melalui Outseal Studio berupa ladder diagram dengan berbagai fungsi di dalamnya seperti fungsi pembangkit Pulse Width Modulation (PWM), fungsi logika, fungsi pengatur waktu dan masih banyak lagi. Pada makalah ini akan dikaji suatu pemanfaatan PLC Outseal untuk menggerakkan kecepatan motor dengan menggunakan beberapa variasi kecepatan berbasis PWM. Suatu motor DC dengan tegangan sebesar 180VDC digunakan dalam penelitian ini, sehingga dibutuhkan suatu konverter DC ke DC dua kuadran untuk menggerakkannya. Konverter DC-DC ini diimplementasi dengan menggunakan dua buah MOSFET jenis IRFP460 sebagai saklar daya. Sedangkan bagian penggerak saklar menggunakan TLP250 yang dikombinasikan dengan IR2111 untuk membentuk komplemen masing-masing saklar daya. Berdasarkan hasil ujicoba yang dilakukan di laboratorium PLC Outseal dapat digunakan sebagai pengendali kecepatan motor DC dengan berbagai variasi kecepatan dengan merubah PWM yang diatur melalui setpoint

Journal articles on the topic 'IRFP720'

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