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Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications.
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Queratinócitos , Serina-Treonina Quinases TOR , Adulto , Humanos , Temperatura , Queratinócitos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células-Tronco/metabolismo , Alvo Mecanístico do Complexo 1 de RapamicinaRESUMO
Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.
Assuntos
Carboxipeptidases , Ariloxifosforamidatos , Masculino , Humanos , Fluorescência , Carboxipeptidases/metabolismo , Hidrolases , Aminoácidos , Corantes Fluorescentes/químicaRESUMO
Senescent cells promote cancer development and progression through chronic inflammation caused by a senescence-associated secretory phenotype (SASP). Although various senotherapeutic strategies targeting senescent cells have been developed for the prevention and treatment of cancers, technology for the in vivo detection and evaluation of senescent cell accumulation has not yet been established. Here, we identified activatable fluorescent probes targeting dipeptidylpeptidase-4 (DPP4) as an effective probe for detecting senescent cells through an enzymatic activity-based screening of fluorescent probes. We also determined that these probes were highly, selectively, and rapidly activated in senescent cells during live cell imaging. Furthermore, we successfully visualized senescent cells in the organs of mice using DPP4-targeted probes. These results are expected to lead to the development of a diagnostic technology for noninvasively detecting senescent cells in vivo and could play a role in the application of DPP4 prodrugs for senotherapy.
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OBJECTIVES: Although the risk of complications due to postoperative pancreatic fistula (POPF) have been evaluated based on the amylase level in drained ascitic fluid, this method has much room for improvement regarding diagnostic accuracy and facility of the measurement. This study aimed to investigate the clinical value of measuring pancreatic chymotrypsin activity for rapid and accurate prediction of POPF after pancreaticoduodenectomy. METHODS: In 52 consecutive patients undergoing pancreaticoduodenectomy, the chymotrypsin activity in pancreatic juice was measured by calculating the increase in fluorescence intensity during the first 5 min after activation with an enzyme-activatable fluorophore. The predictive value for clinically relevant POPF (CR-POPF) was compared between this technique and the conventional method based on the amylase level. RESULTS: According to receiver operating characteristic analyses, pancreatic chymotrypsin activity on postoperative day (POD) 3 measured with a multiplate reader had the highest predictive value for CR-POPF (area under the curve [AUC], 0.752; P < 0.001), yielding 77.8 % sensitivity and 68.8 % specificity. The AUC and sensitivity/specificity of the amylase level in ascitic fluid on POD 3 were 0.695 (P = 0.053) and 77.8 %/41.2 %, respectively. Multivariable analysis identified high pancreatic chymotrypsin activity on POD 3 as an independent risk factor for CR-POPF. Measurement of pancreatic chymotrypsin activity with a prototype portable fluorescence photometer could significantly predict CR-POPF (AUC, 0.731; P = 0.010). CONCLUSION: Measurement of pancreatic chymotrypsin activity enabled accurate and rapid prediction of CR-POPF after pancreaticoduodenectomy. This can help surgeons to implement appropriate drain management at the patient's bedside without delay.
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Quimotripsina , Fístula Pancreática , Humanos , Fístula Pancreática/diagnóstico , Fístula Pancreática/etiologia , Fístula Pancreática/cirurgia , Pâncreas/cirurgia , Pancreaticoduodenectomia/efeitos adversos , Fatores de Risco , Complicações Pós-Operatórias/etiologia , Drenagem/métodos , Amilases , Estudos RetrospectivosRESUMO
9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging.
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Análise Espectral Raman , Estrutura Molecular , Vibração , Nitrilas/química , Nitrilas/síntese químicaRESUMO
Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.
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Controle Comportamental , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Luz , Neurônios/fisiologia , Neurônios/efeitos da radiação , Animais , Fluorescência , Interneurônios/fisiologia , Regiões Promotoras Genéticas/genética , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismoRESUMO
Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect ß-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.
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Análise Espectral Raman , gama-Glutamiltransferase , Animais , Células CultivadasRESUMO
Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV both in vitro and in fission yeast. We introduced SynPCB2.1, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB2.1. These tools not only enable the easy use of multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms. This article has an associated First Person interview with the first author of the paper.
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Schizosaccharomyces , Humanos , Proteínas Luminescentes/genética , Ficobilinas , Ficocianina , Schizosaccharomyces/genéticaRESUMO
Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.
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Proteínas de Arabidopsis/metabolismo , Corantes Fluorescentes/farmacocinética , Células Vegetais/química , Arabidopsis/citologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Endocitose , Corantes Fluorescentes/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/química , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Rodaminas/química , Rodaminas/farmacocinética , Plântula , Imagem com Lapso de Tempo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
Acidification of intracellular vesicles, such as endosomes and lysosomes, is a key pathway for regulating the function of internal proteins. Most conventional methods of measuring pH are not satisfactory for quantifying the pH inside these vesicles. Here, we investigated the molecular requirements for a fluorescence probe to measure the intravesicular acidic pH in living cells by means of fluorescence lifetime imaging microscopy (FLIM). The developed probe, m-DiMeNAF488, exhibits a pH-dependent equilibrium between highly fluorescent and moderately fluorescent forms, which has distinct and detectable fluorescence lifetimes of 4.36 and 0.58 ns, respectively. The pKa(τ) value of m-DiMeNAF488 was determined to be 4.58, which would be favorable for evaluating the pH in the acidic vesicles. We were able to monitor the pH changes in phagosomes during phagocytosis by means of FLIM using m-DiMeNAF488. This probe is expected to be a useful tool for investigating acidic pH-regulated biological phenomena.
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Lisossomos , Imagem Óptica , Ácidos/análise , Endossomos , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/química , Microscopia de Fluorescência/métodosRESUMO
We previously showed that spraying the fluorescent probe gGlu-HMRG (γ-glutamyl hydroxymethyl rhodamine green) can visualize even tiny tumors on the mesentery and peritoneal wall of tumor-bearing mice. However, during surgery, repeated spraying is necessary to detect tumors located deep within organs. Here, we examine whether deeply located tumors can be stained by intravenous administration of this probe. In mice bearing subcutaneous tumors, intravenous administration of gGlu-HMRG resulted in a rapid and specific increase of fluorescence in the tumor, which was visible to the naked eye within 5 min, and the maximum fluorescence intensity ratio of tumor to normal tissue (T/N = 4.3) was reached at 30 min. In mice bearing lung tumors, the T/N ratio reached approximately 20 at 30 min after administration, and deeply located tumors were clearly visualized. These results suggest that intravenous administration of gGlu-HMRG may be a useful technique in fluorescence-guided surgery of tumors.
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Corantes Fluorescentes , Neoplasias , Administração Intravenosa , Animais , Linhagem Celular Tumoral , Camundongos , Neoplasias/patologia , Rodaminas , gama-GlutamiltransferaseRESUMO
Basic carboxypeptidases (basic CPs) cleave the C-terminal basic amino acid of peptides, and their activity is upregulated in some types of cancers. Therefore, detecting the activity of basic CPs in living cells would be important not only for studying the physiological functions of these enzymes but also for visualization of cancerous tissues. Here, we report two fluorescein diacetate (FDA)-based activatable fluorescence probes, named 5ArgAF-FDA and 5LysAF-FDA, in which the substrate amino acid arginine or lysine is conjugated to the benzene moiety via an azoformyl linker. In live-cell fluorescence imaging of CPM, one of the seven basic CPs, 5ArgAF-FDA showed a larger intracellular fluorescence increase than did 5LysAF-FDA within a few minutes. This increase was inhibited by coincubation with 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), an inhibitor of basic CPs. When 5ArgAF-FDA was applied to a coculture of two breast cancer cell lines with different CPM activities, the fluorescence increase in individual cells was correlated with the expression level of CPM, suggesting that 5ArgAF-FDA has the ability to distinguish cell lines having different levels of CPM activity, owing to its high intracellular retention. We believe these probes will be useful for imaging cancers with upregulated basic CP activity.
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Carboxipeptidases , Peptídeos , Fluorescência , LisinaRESUMO
Quinone methide (QM) species have been included in the design of various functional molecules. In this review, we present a comprehensive overview of bioanalytical tools based on QM chemistry. In the first part, we focus on self-immolative linkers that have been incorporated into functional molecules such as prodrugs and fluorescent probes. In the latter half, we outline how the highly electrophilic property of QMs, enabling them to react rapidly with neighboring nucleophiles, has been applied to develop inhibitors or labeling probes for enzymes, as well as self-immobilizing fluorogenic probes with high spatial resolution. This review systematically summarizes the versatile QM toolbox available for investigating biological processes.
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Fosfatase Alcalina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Indolquinonas/farmacologia , beta-Galactosidase/antagonistas & inibidores , Fosfatase Alcalina/metabolismo , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Indolquinonas/síntese química , Indolquinonas/química , Estrutura Molecular , beta-Galactosidase/metabolismoRESUMO
γ-Glutamyltranspeptidase (GGT) is overexpressed in several types of cancer. Existing GGT-targeting fluorescence probes can image these cancers, but the fluorescent hydrolysis product leaks from the target cancer cells during prolonged incubation or fixation. Here, we present a functionalized fluorescence probe for GGT, 4-CH2 F-HMDiEtR-gGlu, which is designed to generate an azaquinone methide intermediate during activation by GGT; this intermediate reacts with intracellular nucleophiles to generate a fluorescent adduct that is trapped inside the cells, without loss of the target enzyme activity. Application of the probe to patient-derived xenograft (PDX) mice enabled inâ vivo cancer imaging for a prolonged period and was also compatible with fixation and immunostaining of the cancer tissue.
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Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos , gama-Glutamiltransferase/metabolismo , Animais , Xenoenxertos , Humanos , Camundongos , Espectrometria de Fluorescência/métodosRESUMO
Raman probes based on alkyne or nitrile tags hold promise for highly multiplexed imaging. However, sensing of enzyme activities with Raman probes is difficult because few mechanisms are available to modulate the vibrational response. Here we present a general strategy to prepare activatable Raman probes that show enhanced Raman signals due to electronic preresonance (EPR) upon reaction with enzymes under physiological conditions. We identified a xanthene derivative bearing a nitrile group at position 9 (9CN-JCP) as a suitable scaffold dye, and synthesized four types of activatable Raman probes, which are targeted to different enzymes (three aminopeptidases and a glycosidase) and tuned to different vibrational frequencies by isotope editing of the nitrile group. We validated the activation of the Raman signals of these probes by the target enzymes and succeeded in simultaneous imaging of the four enzyme activities in live cells. Different cell lines showed different patterns of these enzyme activities.
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Aminopeptidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Sondas Moleculares/química , Análise Espectral Raman/métodos , Aminopeptidases/química , Linhagem Celular Tumoral , Glicosídeo Hidrolases/química , Humanos , Marcação por Isótopo , Microscopia de Fluorescência , Nitrilas/química , Especificidade por SubstratoRESUMO
Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equilibrium (between the fluorescent dissociated form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-infrared (NIR) and green ranges, we succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.
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Corantes Fluorescentes/química , Glutationa/química , Imagem Óptica , Xantenos/química , Animais , Células Cultivadas , Chlorocebus aethiops , Estrutura Molecular , Células VeroRESUMO
Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-l-leucyl-l-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase cytotoxicity assay, retinal sections, and electroretinograms. Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N-methyl-d-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a noninvasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.
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Calpaína/metabolismo , Carbamatos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Corantes Fluorescentes/química , Retina/enzimologia , Rodaminas/química , Animais , Calpaína/análise , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Fármacos Neuroprotetores/farmacologia , Imagem Óptica , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacosRESUMO
BACKGROUND: It is still difficult to detect and diagnose early adenocarcinoma of the esophagogastric junction (EGJ) using conventional endoscopy or image-enhanced endoscopy. A glutamylprolyl hydroxymethyl rhodamine green (EP-HMRG) fluorescent probe that can be enzymatically activated to become fluorescent after the cleavage of a dipeptidyl peptidase (DPP)-IV-specific sequence has been developed and is reported to be useful for the detection of squamous cell carcinoma of the head and neck, and esophagus; however, there is a lack of studies that focuses on detecting EGJ adenocarcinoma by fluorescence molecular imaging. Therefore, we investigated the visualization of early EGJ adenocarcinoma by applying EP-HMRG and using clinical samples resected by endoscopic submucosal dissection (ESD). METHODS: Fluorescence imaging with EP-HMRG was performed in 21 clinical samples resected by ESD, and the fluorescence intensity of the tumor and non-tumor regions of interest was prospectively measured. Immunohistochemistry was also performed to determine the expression of DPP-IV. RESULTS: Fluorescence imaging of the clinical samples showed that the tumor lesions were visualized within a few minutes after the application of EP-HMRG, with a sensitivity, specificity, and accuracy of 85.7, 85.7, and 85.7%, respectively. However, tumors with a background of intestinal metaplasia did not have a sufficient contrast-to-background ratio since complete intestinal metaplasia also expresses DPP-IV. Immunohistochemistry measurements revealed that all fluorescent tumor lesions expressed DPP-IV. CONCLUSIONS: Fluorescence imaging with EP-HMRG could be useful for the detection of early EGJ adenocarcinoma lesions that do not have a background of intestinal metaplasia.
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Adenocarcinoma/diagnóstico , Dipeptidil Peptidase 4/metabolismo , Neoplasias Esofágicas/diagnóstico , Junção Esofagogástrica/patologia , Corantes Fluorescentes/farmacologia , Rodaminas/farmacologia , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Neoplasias Esofágicas/metabolismo , Junção Esofagogástrica/metabolismo , Feminino , Corantes Fluorescentes/química , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Molecular , Imagem Óptica , Estudos Prospectivos , Rodaminas/química , Sensibilidade e EspecificidadeRESUMO
Sialidases catalyse the hydrolysis of terminal sialic acid residues of various glycoconjugates and visualising sialidase activity is important for understanding its function in the biological and pathological context. Upon developing a novel fluorescence probe for sialidase with improved fluorescence characteristics based on our previously reported fluorophore, HMRef, an inherent instability of sialic acid conjugates was found to both reduce selectivity and sensitivity. We aimed at increasing the stability of the probes by incorporating a self-immolative spacer with a higher pKa between the sialic acid residue and HMRef to develop HMRef-S-Neu5Ac, which shows superior stability allowing for the specific detection of sialidase.
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Corantes Fluorescentes/uso terapêutico , Ácido N-Acetilneuramínico/uso terapêutico , Humanos , Ácido N-Acetilneuramínico/farmacologiaRESUMO
As osteoclasts have the central roles in normal bone remodeling, it is ideal to regulate only the osteoclasts performing pathological bone destruction without affecting normal osteoclasts. Based on a hypothesis that pathological osteoclasts form under the pathological microenvironment of the bone tissues, we here set up optimum culture conditions to examine the entity of pathologically activated osteoclasts (PAOCs). Through searching various inflammatory cytokines and their combinations, we found the highest resorbing activity of osteoclasts when osteoclasts were formed in the presence of M-CSF, receptor activator of NF-κB ligand, and IL-1ß. We have postulated that these osteoclasts are PAOCs. Analysis using confocal laser microscopy revealed that PAOCs showed extremely high proton secretion detected by the acid-sensitive fluorescence probe Rh-PM and bone resorption activity compared with normal osteoclasts. PAOCs showed unique morphology bearing high thickness and high motility with motile cellular processes in comparison with normal osteoclasts. We further examined the expression of Kindlin-3 and Talin-1, essential molecules for activating integrin ß-chains. Although normal osteoclasts express high levels of Kindlin-3 and Talin-1, expression of these molecules was markedly suppressed in PAOCs, suggesting the abnormality in the adhesion property. When whole membrane surface of mature osteoclasts was biotinylated and analyzed, the IL-1ß-induced cell surface protein was detected. PAOCs could form a subpopulation of osteoclasts possibly different from normal osteoclasts. PAOC-specific molecules could be an ideal target for regulating pathological bone destruction.