Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Fine-Tuning of CD8(+) T Cell Mitochondrial Metabolism by the Respiratory Chain Repressor MCJ Dictates Protection to Influenza Virus.

Mitochondrial respiration is regulated in CD8(+) T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8(+) T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8(+) T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8(+) T cell metabolism during the contraction phase. Consequently, memory CD8(+) T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8(+) T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8(+) T cell responses.

Nitroreductase-Triggered Fluorophore Labeling of Cells and Tissues under Hypoxia.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho-quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

Strategies to convert organic fluorophores into red/near-infrared emitting analogues and their utilization in bioimaging probes.

Organic fluorophores aided by current microscopy imaging modalities are essential for studying biological systems. Recently, red/near-infrared emitting fluorophores have attracted great research efforts, as they enable bioimaging applications with reduced autofluorescence interference and light scattering, two significant obstacles for deep-tissue imaging, as well as reduced photodamage and photobleaching. Herein, we analyzed the current strategies to convert key organic fluorophores bearing xanthene, coumarin, and naphthalene cores into longer wavelength-emitting derivatives by focussing on their effectiveness and limitations. Together, we introduced typical examples of how such fluorophores can be used to develop molecular probes for biological analytes, along with key sensing features. Finally, we listed several critical issues to be considered in developing new fluorophores.

Dissecting the Crosstalk between Bisulfite and Hypochlorous Acid in the Reaction-Based Fluorescence Detection with Dicyanovinyl-Based Probes.

Fluorescent probes bearing a reactive moiety of 1,1-dicyanovinyl are known to detect several biological species including bisulfite and hypochlorous acid, which, however, possess a selectivity issue among those analytes. Structural modifications of the reactive group for optimal steric and electron effects based on theoretical calculations led us to address the selectivity issue, offering new reactive moieties that provide complete analyte selectivity, including that between bisulfite and hypochlorous acid, in cells as well as in solution.

A General Strategy Toward pH-Resistant Phenolic Fluorophores for High-Fidelity Sensing and Bioimaging Applications.

Aryl alcohol-type or phenolic fluorophores offer diverse opportunities for developing bioimaging agents and fluorescence probes. Due to the inherently acidic hydroxyl functionality, phenolic fluorophores provide pH-dependent emission signals. Therefore, except for developing pH probes, the pH-dependent nature of phenolic fluorophores should be considered in bioimaging applications but has been neglected. Here we show that a simple structural remedy converts conventional phenolic fluorophores into pH-resistant derivatives, which also offer "medium-resistant" emission properties. The structural modification involves a single-step introduction of a hydrogen-bonding acceptor such as morpholine nearby the phenolic hydroxyl group, which also leads to emission bathochromic shift, increased Stokes shift, enhanced photo-stability and stronger emission for several dyes. The strategy greatly expands the current fluorophores' repertoire for reliable bioimaging applications, as demonstrated here with ratiometric imaging of cells and tissues.

A Small-Molecule Fluorescence Probe for Nuclear ATP.

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.

Rationally Designed Two-Photon Ratiometric Elastase Probe for Investigating Inflammatory Bowel Disease.

Elastase, a serine protease, plays important roles in our body in food digestion and defence against pathogens. Particularly, the elastase present in neutrophils is directly associated with inflammatory bowel disease (IBD). Through a rational approach, we have developed a fluorescent elastase probe that has multiple advantages for biological applications including two-photon ratiometric imaging capability. Using the probe, which is capable of detecting intracellular elastase activity associated with inflammation, we have investigated elastase level changes in various mouse organs under an IBD condition for the first time. The results reveal notably higher elastase levels in the liver and duodenum of the healthy mice than in the other investigated organs. Under the IBD condition, we observed significant elastase level changes in the liver, duodenum, colon, and lung. The downregulation of elastase in the liver under the IBD condition suggests migration of neutrophils into the upregulated organs. The notable upregulation of elastase in the duodenum is explained by self-production of elastase, in addition to the neutrophil migration from the liver. We have observed little elastase level changes in selected organs of immune-deficient mice raised under the normal and IBD conditions, which supports the neutrophil migration as the reason for perturbed elastase activity in the healthy mice. The results also suggest an important role of the liver in maintaining the immune response associated with the inflammation-induced elastase level changes. The probe offers an ideal tool for mapping neutrophil migration in body. Further understanding of the elastase-associated biology and diagnosis of IBD by monitoring affected organs are anticipated using the probe.

A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.

Fluorophore Labeling of Proteins: a Versatile Trigger-Release-Conjugation Platform Based on the Quinone Methide Chemistry.

In situ conjugation of fluorescent molecules to biomolecules such as proteins under spatiotemporal control offers a powerful means for studying biological systems. For that purpose, the o-quinone methide chemistry involving a sequence of the trigger-release-conjugation (TRC) process provides a versatile conjugation method. We have developed a new TRC platform bearing a quaternary ammonium salt for the release process, which can be structurally modified and readily synthesized from commonly used aryl alcohol-type organic fluorophores under environmentally benign conditions. We show that different aryl alcohol fluorophores containing the o-(morpholinium)methyl group for the release process allow efficient fluorophore labeling of proteins under both light- and chemical-triggering conditions. The bioconjugation in cells as well as in tissues was further demonstrated with an o-(morpholinium)methyl analogue containing a triggering group sensitive to reactive oxygen species. The new TRC system thus provides a versatile and unique platform for in situ fluorophore labeling of proteins in biological systems under spatiotemporal control.

NAD(P)H Quinone Oxidoreductase-1 in Organ and Tumor Tissues: Distinct Activity Levels Observed with a Benzo-rosol-Based Dual-Excitation and Dual-Emission Probe.

NAD(P)H quinone oxidoreductase-1 (NQO1), a protective enzyme against cellular oxidative stress, is expressed abnormally high in solid tumors and thus recognized as a cancer biomarker. To develop a fluorescent NQO1 probe with practicality, we investigated benzo-rosol fluorophores linked with a known self-immolative quinone substrate. Four probe candidates exhibited ratiometric sensing behavior toward the enzyme, satisfying our orbital mismatch stratagem proposed before, under dual-excitation and dual-emission conditions that alleviate the spectral overlap issue commonly observed with the ratiometric probes based on intramolecular charge-transfer change. Among the candidates, two ester-linked compounds exhibited hydrolytic instability to water or an esterase, discouraging us to develop such ester-linked probes. One ether-linked, hydrolytically stable probe provided brighter cellular fluorescence than the other and thus was applied to ratiometric imaging of NQO1 in cells and tissues. We found that the enzyme activity levels are much different in organ tissues: stomach (56), kidney (22), colon (9.8), testis (7.8), bladder (5.6), lung (1.2), and muscle (1.0). Furthermore, a markedly high enzyme level (14.6-fold) was observed in a xenograft tumor tissue compared with that in a normal tissue, which suggests that such an NQO1 probe is promising for cancer diagnosis and for studying the enzyme-associated biology.

Cell-Membrane-Localizing, Two-Photon Probe for Ratiometric Imaging of γ-Glutamyl Transpeptidase in Cancerous Cells and Tissues.

γ-Glutamyl transpeptidase (GGT), a cell surface-bound protease, is associated with various diseases including cancer. The detection of the enzyme activity is an important subject, leading to about 40 activatable fluorescent probes so far. All of them, however, lack the membrane-localizing ability, raising a reliability issue in the quantitative analysis. Disclosed is the first fluorescent probe that senses the cell surface-bound enzyme, which, furthermore, is capable of ratiometric as well as two-photon imaging with desirable features. Ratiometric imaging of cancer cell lines reveals a 6.4-8.4-fold higher GGT levels than those in normal cell lines. A comparison of the enzyme activity in organ tissues of normal and tumor xenograft mice reveals notably different levels of enzyme activity depending on the kind of tissue. Normal tissues exhibited comparable levels of enzyme activity, except the kidney that has significantly higher GGT activity (2.7-4.0-fold) than the other organs. Compared with the normal tissues, considerably higher enzyme activity was observed in the tumor tissues of the thigh (4.0-fold), colon (2.5-fold), lung (3.6-fold), and liver (2.1-fold), but essentially no enhanced activity in the tumor tissues of the spleen, stomach, and pancreas and a comparable level in both the tumor and normal kidney tissues were observed. The probe offers practical means for studying GGT-associated biology in cells and tissues by one- as well as two-photon ratiometric imaging.

An Endeavor in the Reaction-Based Approach to Fluorescent Probes for Biorelevant Analytes: Challenges and Achievements.

The promising features of fluorescence spectroscopy have inspired a quest for fluorescent probes for analysis and monitoring of molecular interactions in biochemical, medical, and environmental sciences. To overcome the competitive supramolecular interactions in aqueous media encountered with conventional molecular-recognition-based probes, the use of reaction-based probes that involve making or breaking of covalent bonds has emerged as a complementary sensing strategy to realize higher selectivity and sensitivity with larger spectroscopic changes. In spite of the enormous efforts, the development of reaction-based fluorescent probes meets with certain challenges in terms of their practical applications, demanding "intelligent design" of probes with an appropriate fluorophore attached to an efficient reactive moiety at the right place. This Account summarizes the results of our efforts made in the development and fine-tuning of reaction-based fluorescent probes toward those goals, classified by the type of analyte (anions, metal cations, and biomolecules) with notes on the challenges and achievements. The reaction-based approach was demonstrated to be powerful for the selective sensing of anions (cyanide and (amino)carboxylates) for the first time, and later it was extended to develop two-photon probes for bisulfite and fluoride ions. The reaction-based approach also enabled selective sensing of noble metal ions such as silver, gold, and palladium along with toxic (methyl)mercury species and paramagnetic copper ions. Furthermore, microscopic imaging and monitoring of biologically relevant species with reaction-based two-photon probes were explored for hydrogen sulfide, hypochlorous acid, formaldehyde, monoamine oxidase enzyme, and ATP.

Synthesis of Near-Infrared-Emitting Benzorhodamines and Their Applications to Bioimaging and Photothermal Therapy.

Photostable and near-infrared (NIR)-emitting organic fluorophores with large Stokes shifts are in great demand for long-term bioimaging at deeper depths with minimal autofluorescence and self-quenching. Herein, a new class of benzorhodamines and their analogues that are photostable and emit in the NIR region (up to 785 nm) with large Stokes shifts (>120 nm) is reported. The synthesis involves condensation of 7-alkylamino-2-naphthols with 2-[4-(dimethylamino)-2-hydroxybenzoyl]benzoic acid, which leads to bent-shaped benzorhodamines that emit orange fluorescence (≈600 nm); however, introduction of steric hindrance near the condensation site switched the regioselectivity, to provide a linear benzorhodamine system for the first time. The linear benzorhodamine derivatives provide bright fluorescence images in cells and in tissue. A carboxy-benzorhodamine was applied for photothermal therapy of cancer cells and xenograft cancer mice.

Ratiometric Imaging of γ-Glutamyl Transpeptidase Unperturbed by pH, Polarity, and Viscosity Changes: A Benzocoumarin-Based Two-Photon Fluorescent Probe.

γ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations; however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity levels between different mouse organ tissues are quantitatively compared with the highest level in the kidney. The probe with practicality holds great promise for studying GGT-associated biological processes directly through ratiometric imaging by two-photon microscopy.

A Benzopyronin-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Lysosomal Bisulfite with Complete Spectral Separation.

Bisulfite (HSO3-), which equilibrates with sulfite (SO32-) and sulfur dioxide (SO2) in aqueous media, can be produced endogenously during oxidation of hydrogen sulfide or sulfur-containing amino acids. Lysosomes, known as the scavengers of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. Herein, we disclose a lysosome-targeting, two-photon excitable, and ratiometric signaling (near-infrared/green) fluorescent probe that detects bisulfite through a fast 1,6-conjugate addition reaction. The probe shows excellent selectivity toward bisulfite over other biologically relevant species. Notably, the probe allows ratiometric fluorescence imaging of lysosomal bisulfite with complete spectral separation under one-photon as well as two-photon excitation conditions.

Modified Isoindolediones as Bright Fluorescent Probes for Cell and Tissue Imaging.

New L-shaped fluorophores possessing five conjugated rings have been synthesized through a four-step procedure involving diketopyrrolopyrrole synthesis and its double N-alkylation, followed by trimethylsilyl bromide-mediated rearrangement to thieno[2,3-f]isoindole-5,8-dione and an intramolecular Friedel-Crafts reaction. In comparison with the parent isoindolediones and π-expanded diketopyrrolopyrroles, these new dyes show red-shifted absorption and emission (up to ≈630 nm). Their structural rigidity is responsible for both the observed small Stokes shifts and large fluorescence quantum yields. Tissue imaging studies revealed that these new dyes show advantageous features including minimal autofluorescence interference and pronounced solvent-sensitive emission. Interestingly, there is a fundamental difference between a dye possessing an amino group and its analog bearing an N-alkyl substituent. The former dye under two-photon excitation at 900 nm gives bright images whereas its N-alkylated counterpart does not. A new type of membrane localization has been discovered by an N-alkylated isoindoledione possessing a benzofuryl substituent. In spite of the fact that the fluorescence quantum yield of this dye in a range of solvents is rather low, it does stain cell membranes exclusively. This new mode of cellular staining opens the door towards further development of membrane staining dyes.

A Ratiometric Two-Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In†Cellulo Observation of Lysosomal Membrane Fusion Processes.

Vesicles exchange their contents through membrane fusion processes, kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss-and-run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.

Ratiometric Imaging of Tissue by Two-Photon Microscopy: Observation of a High Level of Formaldehyde around Mouse Intestinal Crypts.

Ratiometric imaging by two-photon microscopy can offer a viable tool for the relative quantification of biological analytes inside tissue with minimal influence from environmental factors that affect fluorescence signal. We demonstrate the ratiometric imaging of formaldehyde at the suborgan level using a two-photon fluorescent probe, which involves pixel-to-pixel ratiometric data transformation. This study reveals for the first time a high level of formaldehyde around the crypts of mouse small intestine, implicating its possible protective role along with the released antimicrobials from the Paneth cells.

Fluorescent Labeling of Protein Using Blue-Emitting 8-Amino-BODIPY Derivatives.

8-Amino-BODIPY (boron-dipyrromethane) dyes show bright blue fluorescence. Disclosed here are synthesis and characterization of the photophysical properties of a series of functionalized 8-Amino-BODIPY (BP1-4) for protein labeling. The compact structure and solvent-insensitive absorption property of the dye are desirable features for protein labeling. For the model protein, bovine serum albumin (BSA), the labeling proceeds under mild condition via amide bond formation or thiol-ene conjugation with maintaining the bright blue fluorescence. The chromatography and mass spectroscopy analysis clearly support the labeling of the BODIPY dye on the BSA. The protein labeling with blue-emitting BODIPY would be applicable for studying protein dynamics and fluorescence resonance energy transfer (FRET) with intrinsic biomolecules.