Development of elliptic core-shell nanoparticles with fluorinated surfactants for 19F MRI.

Perfluorocarbon-encapsulated silica nanoparticles possess attractive features such as biological inertness and favorable colloidal properties for bioimaging with fluorine magnetic resonance imaging (19F MRI). Herein, a series of elliptic shaped silica nanoparticles with perfluorocarbon liquid perfluoro-15-crown-5 ether as core (PFCE@SiO2) were synthesized using fluorinated surfactants N-(perfluorononylmethyl)-N,N,N-trimethylammonium chloride (C10-TAC) and N-(perfluoroheptylmethyl)-N,N,N-trimethylammonium chloride (C8-TAC). The nanoparticles are characterized to obtain elliptic core-shell structures. PFCE@SiO2 showed strong 19F NMR signals of the encapsulated PFCE, indicating the potential as a highly sensitive 19F MRI probe. These elliptic PFCE@SiO2 nanoparticles provide a new option of 19F MRI probe with a morphology different from conventional nanospheres.

Trifunctional fatty acid derivatives: the impact of diazirine placement.

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

Trifunctional fatty acid derivatives demonstrate the impact of diazirine placement.

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation.

Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis. Herein, we report a design strategy for probes that suppress undesired organelle accumulation using a bioisostere for intracellular protein imaging in living cells. Our design allows the protein labeling probes to possess both membrane permeability and suppress non-specific accumulation and has been shown to use several protein labeling systems, such as PYP-tag and Halo tag systems. We further developed a fluorogenic PYP-tag labeling probe for intracellular proteins and used it to visualize multiple localizations of target proteins in the intracellular system. Our strategy offers a versatile design for undesired accumulation-suppressed probes with cationic dye scaffolds and provides a valuable tool for intracellular protein imaging.

Hybrid Small-Molecule/Protein Fluorescent Probes.

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid probes are constructed by diverse site-specific chemical protein labeling approaches through chemical reactions to exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemical reactions. The hybrid small-molecule/protein fluorescent probes are employed for imaging protein trafficking, conformational changes, and bioanalytes surrounding proteins. In addition, fluorescent hybrid probes facilitate visualization of protein dynamics at the single-molecule level and the defined structure with super-resolution imaging. In this review, we discuss development and the bioimaging applications of fluorescent probes based on small-molecule/protein hybrids.

No-wash fluorogenic labeling of proteins for reversible photoswitching in live cells.

Photoswitchable fluorescent molecules (PSFMs) are positioned as valuable tools for biomolecule localization tracking and super-resolution imaging technologies due to their unique ability to reversibly control fluorescence intensity upon light irradiation. Despite the high demand for PSFMs that are suitable for live-cell imaging, no general method has been reported that enables reversible fluorescence control on proteins of interest in living cells. Herein, we have established a platform to realize reversible fluorescence switching in living cells by adapting a protein labeling system. We have developed a new PSFM, named HTL-Trp-BODIPY-FF, which exhibits strong fluorogenicity upon recognition of Halo-tag protein and reversible fluorescence photoswitching in living cells. This is the first example of a PSFM that can be applicable to a general-purpose Halo-tag protein labeling system for no-wash live-cell imaging.

Recent advancements of fluorescent biosensors using semisynthetic probes.

Fluorescent biosensors are crucial experimental tools for live-cell imaging and the quantification of different biological analytes. Fluorescent protein (FP)-based biosensors are widely used for imaging applications in living systems. However, the use of FP-based biosensors is hindered by their large size, poor photostability, and laborious genetic manipulations required to improve their properties. Recently, semisynthetic fluorescent biosensors have been developed to address the limitations of FP-based biosensors using chemically modified fluorescent probes and self-labeling protein tag/peptide tags or DNA/RNA-based hybrid systems. Semisynthetic biosensors have unique advantages, as they can be easily modified using different probes. Moreover, the self-labeling protein tag, which labels synthetically developed ligands via covalent bonds, has immense potential for biosensor development. This review discusses the recent progress in different types of fluorescent biosensors for metabolites, protein aggregation and degradation, DNA methylation, endocytosis and exocytosis, membrane tension, and cellular viscosity. Here, we explain in detail the design strategy and working principle of these biosensors. The information presented will help the reader to create new biosensors using self-labeling protein tags for various applications.

Rational Design of Hydroxylated Thiazole Orange Photocages for Green Light-Triggered DNA Recombination.

The precise control of DNA recombination enables the cell- or time-dependent regulation of gene expression in studies of gene function. Caged estrogen receptor ligands combined with a Cre-ERT2/loxP system are useful tools for light-triggered DNA recombination. However, the photolysis of most caged compounds requires ultraviolet or blue light, which is toxic and displays low tissue penetration. Although a cyanine-based photo-responsive protecting group (PPG) can release estrogen receptor ligands with longer-wavelength light, its low photolytic efficiency requires long illumination times. We developed a caged estrogen receptor ligand with improved green light-responsive PPGs. The rational modification of Hydroxylated Thiazole Orange (HTO) photocages using electron-donating groups (EDGs), such as dimethoxy (DiMeO)-substituted HTO, resulted in high photolytic efficiency (up to ÏµΦ ≈320 M-1  cm-1 ). Theoretical calculations demonstrated that the enhanced photolytic efficiencies were derived from the increased intramolecular charge transfer by EDGs upon excitation. The efficient uncaging of estrogen receptor ligands enabled the control of gene recombination in a ligand-dependent Cre-ERT2/loxP system in live cells.

Variational autoencoder-based chemical latent space for large molecular structures with 3D complexity.

The structural diversity of chemical libraries, which are systematic collections of compounds that have potential to bind to biomolecules, can be represented by chemical latent space. A chemical latent space is a projection of a compound structure into a mathematical space based on several molecular features, and it can express structural diversity within a compound library in order to explore a broader chemical space and generate novel compound structures for drug candidates. In this study, we developed a deep-learning method, called NP-VAE (Natural Product-oriented Variational Autoencoder), based on variational autoencoder for managing hard-to-analyze datasets from DrugBank and large molecular structures such as natural compounds with chirality, an essential factor in the 3D complexity of compounds. NP-VAE was successful in constructing the chemical latent space from large-sized compounds that were unable to be handled in existing methods, achieving higher reconstruction accuracy, and demonstrating stable performance as a generative model across various indices. Furthermore, by exploring the acquired latent space, we succeeded in comprehensively analyzing a compound library containing natural compounds and generating novel compound structures with optimized functions.

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19 F†MR Imaging.

19 F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as 19 F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel 19 F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in 19 F MRI imaging. Importantly, our 19 F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells. In this study, we have created an unnatural ligand with an unprecedented protein-tag labeling property in living cells and overcome the above-mentioned problem. Of special interest is that some fluorescent probes with the ligand can selectively and efficiently label intracellular proteins without binding to cell-surface proteins, even if the proteins are present on the cell membrane. We also developed a cell-membrane impermeable fluorescent probe that selectively labels cell-surface proteins without labeling of intracellular proteins. These localization-selective properties enabled us to visually discriminate two kinetically distinct glucose transporter 4 (GLUT4) molecules that show different multiple subcellular localization and translocation dynamics in live cells. Taking advantage of the probes, we revealed that N-glycosylation of GLUT4 influences intracellular localization. Furthermore, we were able to visually distinguish active GLUT4 molecules that underwent membrane translocation at least twice within an hour from those that remained intracellularly, discovering previously unrecognized dynamic behaviors of GLUT4. This technology provides not only a valuable tool for study on multiple localization and dynamics of proteins but also important information on diseases caused by protein translocation dysfunction.

Persistent Fluorescence Switching of a Probe Using a Photochromic Quencher with High Photostability Assisted by Protein-Surface Modification.

Photoswitchable fluorescent molecules (PSFMs) are widely applicable in the life sciences for super-resolution imaging. Owing to the large and hydrophobic molecular structures of PSFMs that may aggregate in a biological medium, the development of synthetic PSFMs with persistent reversible photoswitching is challenging. Here, we established a protein-surface-assisted photoswitching strategy that allows for persistent reversible fluorescence photoswitching of a PSFM in an aqueous solution. As a first step, we applied the photochromic chromophore furylfulgimide (FF) as a photoswitchable fluorescence quencher and developed a Förster resonance energy transfer-based PSFM, named FF-TMR. Most importantly, the protein-surface modification strategy allows FF-TMR to exhibit persistent reversible photoswitching performance in an aqueous solution. In fixed cells, the fluorescence intensity of FF-TMR bound to antitubulin antibody was repetitively modulated. The protein-surface-assisted photoswitching strategy will be a useful platform to broaden the utility of functionalized synthetic chromophores enabling persistent fluorescence switching that inherits their high resistance to light irradiation.

Long-term imaging of intranuclear Mg2+ dynamics during mitosis using a localized fluorescent probe.

A novel fluorescent Mg2+ probe was developed based on a small molecule-protein hybrid. This probe enables subcellular targeting, long-term imaging, and high selectivity for Mg2+ over Ca2+. Using ratiometric fluorescence microscopy with a co-localized standard fluorophore, the variations in intranuclear Mg2+ concentrations during mitosis could be visualized.

JAK inhibition ameliorates bone destruction by simultaneously targeting mature osteoclasts and their precursors.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by chronic inflammation and resultant cartilage/bone destruction because of aberrantly activated osteoclasts. Recently, novel treatments with several Janus kinase (JAK) inhibitors have been shown to successfully ameliorate arthritis-related inflammation and bone erosion, although their mechanisms of action for limiting bone destruction remain unclear. Here, we examined the effects of a JAK inhibitor on mature osteoclasts and their precursors by intravital multiphoton imaging. METHODS: Inflammatory bone destruction was induced by local injection of lipopolysaccharides into transgenic mice carrying reporters for mature osteoclasts or their precursors. Mice were treated with the JAK inhibitor, ABT-317, which selectively inhibits the activation of JAK1, and then subjected to intravital imaging with multiphoton microscopy. We also used RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanism underlying the effects of the JAK inhibitor on osteoclasts. RESULTS: The JAK inhibitor, ABT-317, suppressed bone resorption by blocking the function of mature osteoclasts and by targeting the migratory behaviors of osteoclast precursors to the bone surface. Further exhaustive RNA-Seq analysis demonstrated that Ccr1 expression on osteoclast precursors was suppressed in the JAK inhibitor-treated mice; the CCR1 antagonist, J-113863, altered the migratory behaviors of osteoclast precursors, which led to the inhibition of bone destruction under inflammatory conditions. CONCLUSIONS: This is the first study to determine the pharmacological actions by which a JAK inhibitor blocks bone destruction under inflammatory conditions; this inhibition is beneficial because of its dual effects on both mature osteoclasts and immature osteoclast precursors.

Development of a Versatile Protein Labeling Tool for Live-Cell Imaging Using Fluorescent ß-Lactamase Inhibitors.

To understand the function of protein in live cells, real-time monitoring of protein dynamics and sensing of their surrounding environment are important methods. Fluorescent labeling tools are thus needed that possess fast labeling kinetics, high efficiency, and long-term stability. We developed a versatile chemical protein-labeling tool based on fluorophore-conjugated diazabicyclooctane ß-lactamase inhibitors (BLIs) and wild-type TEM-1 ß-lactamase protein tag. The fluorescent probes efficiently formed a stable carbamoylated complex with ß-lactamase, and the labeled proteins were visualized over a long period of time in live cells. Moreover, use of an α-fluorinated carboxylate ester-based BLI prodrug enabled the probe to permeate cell membranes and stably label intracellular proteins after unexpected spontaneous ester hydrolysis. Lastly, combining the labeling tool with a pH-activatable fluorescent probe allowed visual monitoring of lysosomal protein translocation during autophagy.

Hybrid, dual visible and near-infrared fluorescence emission of (6,5) single-walled carbon nanotubes modified with fluorescein through aryl diazonium salt chemistry.

The aryl diazonium salt chemistry offers enhancement of near-infrared (NIR) emission of single-walled carbon nanotubes (SWCNTs), although, the attachment of functional molecules which could bring hybrid properties through the process is underdeveloped. In this work, we utilize aryl diazonium salt of fluorescein to createsp3defects on (6,5) SWCNTs. We study the influence of pH on the grafting process identifying that pH 5-6 is necessary for a successful reaction. The fluorescein-modified (6,5) SWCNTs (F-(6,5) SWCNTs) exhibit red-shiftedE11* emission in the NIR region attributed to luminescentsp3defects, but also visible (Vis) fluorescence at 515 nm from surface-attached fluorescein molecules. The fluorescence in both Vis and NIR regions of F-(6,5) SWCNTs exhibit strong pH-dependency associated with the dissociation of fluorescein molecules with an indication of photoinduced-electron transfer quenching the Vis emission of fluorescein dianion. The F-(6,5) SWCNTs could potentially be used for dual-channel medical imaging as indicated by our preliminary experiments. We hope that our research will encourage new, bold modifications of SWCNTs with functional molecules introducing new, unique hybrid properties.

Efficient visible/NIR light-driven uncaging of hydroxylated thiazole orange-based caged compounds in aqueous media.

In photoactivation strategies with bioactive molecules, one-photon visible or two-photon near-infrared light-sensitive caged compounds are desirable tools for biological applications because they offer reduced phototoxicity and deep tissue penetration. However, visible-light-sensitive photoremovable protecting groups (PPGs) reported so far have displayed high hydrophobicity and low uncaging cross sections (ÎµΦ < 50) in aqueous media, which can obstruct the control of bioactivity with high spatial and temporal precision. In this study, we developed hydroxylated thiazole orange (HTO) derivatives as visible-light-sensitive PPGs with high uncaging cross sections (ÎµΦ ≈ 370) in aqueous solution. In addition, 2PE photolysis reactions of HTO-caged glutamate were achieved using a NIR laser (940 nm). Moreover, HTO-caged glutamate can activate N-methyl-d-aspartic acid receptors in Xenopus oocytes and mammalian cells with green-light illumination, thus allowing optical control of biological functions.

An "OFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems.

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling.

Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetylh-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The ß-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

Anti-Siglec-15 antibody suppresses bone resorption by inhibiting osteoclast multinucleation without attenuating bone formation.

Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.