摘要：Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

摘要：NanoLuc luciferase recently gained popularity due to its small size and superior bioluminescence performance. For in vivo imaging applications, NanoLuc has been limited by its substrate furimazine, which has low solubility and bioavailability. Herein, we compared the performances of recently reported NanoLuc luciferase substrates for in vivo imaging in mice. Two substrates with improved aqueous solubility, hydrofurimazine and fluorofurimazine, were evaluated along with three stabilized O-acetylated furimazine analogues, the hikarazines. All 5 analogues, when tested in vitro, displayed greater signal intensity and reaction duration, in comparison to the standard NanoLuc substrate, furimazine. The two best-performing analogues from the in vitro study were selected for further in vivo testing. The NanoLuc/fluorofurimazine pair demonstrated the highest bioluminescence intensity, post intravenous administration. It was found to be around 9-fold brighter compared to the NanoLuc/furimazine and 11-fold more intense than the NanoLuc/hikarazine-003 pair, with an average of 3-fold higher light emission when the substrate was injected intraperitoneally, in a subcutaneous model. Excitingly, despite the fact that NanoLuc/fluorofurimazine emits mostly blue light, we prove that cells trapped in mice lungs vasculature could be visualised via the NanoLuc/fluorofurimazine pair and compare the results to the AkaLuc/AkaLumine system. Therefore, among the tested analogues, fluorofurimazine enables higher substrate loading and improved optical imaging sensitivity in small animals, upgrading the use of NanoLuc derived bioluminescent systems for deep tissue imaging.

摘要：Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.

摘要：Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, concomitant assessment of drug-associated phenotypes has proven challenging. We have developed cellular target engagement by accumulation of mutant(CeTEAM) as a platform that can seamlessly evaluate drug-target interactions and phenotypicresponses in a single multiparametric experiment. In the presence of binding ligand, accumulationof an initially unstable target protein acts as a biosensor that permits holistic assessment of drugpharmacology under physiological conditions. We demonstrate this proof-of-concept by uncoupling target binding from divergent cellular activities of MTH1 inhibitors, repurposing the R139C variant to dissect complex NUDT15-thiopurine interactions, and profiling the live-cell dynamics of DNA trapping by PARP inhibitors. Further, PARP1-derived drug biosensors facilitated multimodal ex vivo analysis of drug-target engagement and non-invasive tracking of drug binding in live animals. CeTEAM empowers real-time, comprehensive characterization of target engagement by bridging drug binding events and their biological consequences

摘要：Elucidating receptor occupancy (RO) of monoclonal antibodies (mAbs) is a crucial step in characterizing the therapeutic efficacy of mAbs. However, the in vivo assessment of RO, particularly within peripheral tissues, is greatly limited by current technologies. In the present study, we developed a bioluminescence resonance energy transfer (BRET)-based system that leverages the large signal:noise ratio and stringent energy donor-acceptor distance dependency to measure antibody RO in a highly selective and temporal fashion. This versatile and minimally invasive system enables longitudinal monitoring of the in vivo antibody-receptor engagement over several days. As a proof of principle, we quantified cetuximab-epidermal growth factor receptor binding kinetics using this system and assessed cetuximab RO in a tumor xenograft model. Incomplete ROs were observed, even at a supratherapeutic dose of 50 mg/kg, indicating that fractional target accessibility is achieved. The BRETbased imaging approach enables quantification of antibody in vivo RO and provides critical information required to optimize therapeutic mAb efficacy.

摘要：Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.

摘要：Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.

摘要：Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.

摘要：Exosomes are secreted extracellular vesicles with lipid bilayer membranes. They are emerging as a new category of messengers that facilitate cross-talk between cells, tissues, and organs. Thus, a critical demand arises for the development of a sensitive and non-invasive tracking system for endogenous exosomes. We have generated a genetic mouse model that meets this goal. The Nano-luciferase (NanoLuc) reporter was fused with the exosome surface marker CD63 for exosome labeling. The cardiomyocyte-specific αMHC promoter followed by the loxP-STOP-loxP cassette was engineered for temporal and spatial labeling of exosomes originated from cardiomyocytes. The transgenic mouse was bred with a tamoxifen-inducible Cre mouse (Rosa26Cre-ERT2) to achieve inducible expression of CD63NanoLuc reporter. The specific labeling and tissue distribution of endogenous exosomes released from cardiomyocytes were demonstrated by luciferase assay and non-invasive bioluminescent live imaging. This endogenous exosome tracking mouse provides a useful tool for a range of research applications.

摘要：The lack of techniques to trace brain cell behavior in vivo hampers the ability to monitor status of cells in a living brain. Extracellular vesicles (EVs), nanosized membrane-surrounded vesicles, released by virtually all brain cells might be able to report their status in easily accessible biofluids, such as blood. EVs communicate among tissues using lipids, saccharides, proteins, and nucleic acid cargo that reflect the state and composition of their source cells. Currently, identifying the origin of brain-derived EVs has been challenging, as they consist of a rare population diluted in an overwhelming number of blood and peripheral tissue-derived EVs. Here, we developed a sensitive platform to select out pre-labelled brain-derived EVs in blood as a platform to study the molecular fingerprints of brain cells. This proof-of-principle study used a transducible construct tagging tetraspanin (TSN) CD63, a membrane-spanning hallmark of EVs equipped with affinity, bioluminescent, and fluorescent tags to increase detection sensitivity and robustness in capture of EVs secreted from pre-labelled cells into biofluids. Our platform enables unprecedented efficient isolation of neural EVs from the blood. These EVs derived from pre-labelled mouse brain cells or engrafted human neuronal progenitor cells (hNPCs) were submitted to multiplex analyses, including transcript and protein levels, in compliance with the multibiomolecule EV carriers. Overall, our novel strategy to track brain-derived EVs in a complex biofluid opens up new avenues to study EVs released from pre-labelled cells in near and distal compartments into the biofluid source.