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2.
J Cell Sci ; 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34806750

RESUMO

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 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 in vitro and in fission yeast. We introduced SynPCB, 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 SynPCB. These tools not only enable the easy use of the 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.

3.
Front Oncol ; 11: 714527, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34490111

RESUMO

Introduction: Radical resection is the only curative treatment for pancreatic cancer, which is a life-threatening disease. However, it is often not easy to accurately identify the extent of the tumor before and during surgery. Here we describe the development of a novel method to detect pancreatic tumors using a tumor-specific enzyme-activatable fluorescence probe. Methods: Tumor and non-tumor lysate or small specimen collected from the resected specimen were selected to serve as the most appropriate fluorescence probe to distinguish cancer tissues from noncancerous tissues. The selected probe was sprayed onto the cut surface of the resected specimen of cancer tissue to acquire a fluorescence image. Next, we evaluated the ability of the probe to detect the tumor and calculated the tumor-to-background ratio (TBR) by comparing the fluorescence image with the pathological extent of the tumor. Finally, we searched for a tumor-specific enzyme that optimally activates the selected probe. Results: Using a library comprising 309 unique fluorescence probes, we selected GP-HMRG as the most appropriate activatable fluorescence probe. We obtained eight fluorescence images of resected specimens, among which four approximated the pathological findings of the tumor, which achieved the highest TBR. Finally, dipeptidyl-peptidase IV (DPP-IV) or a DPP-IV-like enzyme was identified as the target enzyme. Conclusion: This novel method may enable rapid and real-time visualization of pancreatic cancer through the enzymatic activities of cancer tissues.

4.
Sci Rep ; 11(1): 17946, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504174

RESUMO

Fluorescence imaging of tumours facilitates rapid intraoperative diagnosis. Thus far, a promising activatable fluorescence probe for hepatocellular carcinoma (HCC) has not been developed. Herein, the utility of the fluorescence imaging of HCC using a ß-galactosidase (ß-Gal)-activatable fluorescence probe SPiDER-ßGal was examined. ß-Gal activity was measured in cryopreserved tissues from 68 patients. Live cell imaging of HCC cell lines and imaging of tumour-bearing model mice were performed using SPiDER-ßGal. Furthermore, fluorescence imaging was performed in 27 freshly resected human HCC specimens. In cryopreserved samples, ß-Gal activity was significantly higher in tumour tissues than in non-tumour tissues. Fluorescence was observed in HCC cell lines. In mouse models, tumours displayed stronger fluorescence than normal liver tissue. In freshly resected specimens, fluorescence intensity in the tumour was significantly higher than that in non-tumour liver specimens as early as 2 min after spraying. Receiver operating characteristic curves were generated to determine the diagnostic value of SPiDER-ßGal 10 min after its spraying; an area under the curve of 0.864, sensitivity of 85.2%, and specificity of 74.1% were observed for SPiDER-ßGal. SPiDER-ßGal is useful for the rapid fluorescence imaging of HCC. Fluorescence imaging guided by SPiDER-ßGal would help surgeons detect tumours rapidly and achieve complete liver resection.


Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/enzimologia , Corantes Fluorescentes/metabolismo , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/enzimologia , Imagem Óptica/métodos , beta-Galactosidase/metabolismo , Idoso , Animais , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Feminino , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Sensibilidade e Especificidade
5.
Bioorg Med Chem ; 44: 116281, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34216983

RESUMO

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.

6.
Sci Rep ; 11(1): 10664, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021168

RESUMO

Diagnosis of peritoneal metastasis in gastric cancer (GC) is essential for determining appropriate therapeutic strategies and avoiding non-essential laparotomy or gastrectomy. Recently, a variety of activatable fluorescence probes that can detect enzyme activities have been developed for cancer imaging. The aim of this study was to identify the key enzyme involved in peritoneal metastasis in GC. The enzymatic activity of gamma-glutamyl transpeptidase, dipeptidyl peptidase IV, and ß-galactosidase (ß-Gal) was assessed in lysates prepared from preserved human GC (n = 89) and normal peritoneal (NP; n = 20) samples. ß-Gal activity was significantly higher in the human GC samples than in NP samples, whereas no differences were observed in the activities of the other enzymes. Therefore, we used SPiDER-ßGal, a fluorescent probe that can be activated by ß-Gal, for imaging GC cell lines, peritoneal metastasis in a mouse model, and fresh human resected GC samples (n = 13). All cell lines showed fluorescence after applying SPiDER-ßGal, and metastatic nodules in the mice gradually developed high fluorescence that could be visualized with SPiDER-ßGal. The human GC samples showed significantly higher fluorescence than NP samples. ß-Gal is a useful target enzyme for fluorescence imaging of peritoneal metastasis in GC.


Assuntos
Biomarcadores Tumorais , Neoplasias Peritoneais/diagnóstico , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , beta-Galactosidase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Imunofluorescência , Humanos , Masculino , Imagem Óptica , Prognóstico , Curva ROC , gama-Glutamiltransferase/metabolismo
7.
Clin Cancer Res ; 27(14): 3936-3947, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031057

RESUMO

PURPOSE: Five-aminolevulinic acid (5-ALA) is widely used as an intraoperative fluorescent probe for radical resection of high-grade glioma, and thus aids in extending progression-free survival of patients. However, there exist some cases where 5-ALA fails to fluoresce. In some other cases, it may undergo fluorescence quenching but cannot be orally readministered during surgery. This study aimed to develop a novel hydroxymethyl rhodamine green (HMRG)-based fluorescence labeling system that can be repeatedly administered as a topical spray during surgery for the detection of glioblastoma. EXPERIMENTAL DESIGN: We performed a three-stage probe screening using tumor lysates and fresh tumor tissues with our probe library consisting of a variety of HMRG probes with different dipeptides. We then performed proteome and transcript expression analyses to detect candidate enzymes responsible for cleaving the probe. Moreover, in vitro and ex vivo studies using U87 glioblastoma cell line were conducted to validate the findings. RESULTS: The probe screening identified proline-arginine-HMRG (PR-HMRG) as the optimal probe that distinguished tumors from peritumoral tissues. Proteome analysis identified calpain-1 (CAPN1) to be responsible for cleaving the probe. CAPN1 was highly expressed in tumor tissues which reacted to the PR-HMRG probe. Knockdown of this enzyme suppressed fluorescence intensity in U87 glioblastoma cells. In situ assay using a mouse U87 xenograft model demonstrated marked contrast of fluorescence with the probe between the tumor and peritumoral tissues. CONCLUSIONS: The novel fluorescent probe PR-HMRG is effective in detecting glioblastoma when applied topically. Further investigations are warranted to assess the efficacy and safety of its clinical use.

8.
Chem Commun (Camb) ; 57(47): 5802-5805, 2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-33999073

RESUMO

We have designed and developed non-fluorescent, cell-permeable photoactivatable fluorophores, photoactivatable SPiDERs (paSPiDERs), which exhibit fluorescence activation upon light irradiation, accompanied by the generation of a quinone methide intermediate that binds covalently to intracellular proteins. The fluorescence signal is durable for 24 hours, resistant to fixation and compatible with immunostaining, and selective cell labeling can be achieved at single-cell resolution.


Assuntos
Corantes Fluorescentes/química , Imagem Óptica , Células A549 , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Processos Fotoquímicos
9.
Methods Mol Biol ; 2274: 193-206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050473

RESUMO

Fluorescence (FL)-guided detection of cancer is one of the most promising approaches to achieve intraoperative assessment of surgical margins. Enzymes, such as aminopeptidase, carboxypeptidase, and glycosidase, whose activities are increased in cancer, have attracted great interest as imaging targets for rapid and sensitive visualization of cancerous tissues with fluorescent probes. Activatable probes, which are initially nonfluorescent but become strongly fluorescent upon rapid one-step cleavage of their substrate moiety by the target enzyme, are especially promising for practical clinical application during surgical or endoscopic procedures due to the highly amplified FL change generated by enzyme-catalyzed turnover at lesion sites. Here, we describe robust protocols for using activatable fluorescent probes targeting cancer-associated enzyme activities to visualize cultured cancer cells, metastatic cancer in a mouse model, and cancerous lesions in surgical specimens from patients.


Assuntos
Aminopeptidases/metabolismo , Carboxipeptidases/metabolismo , Diagnóstico por Imagem/métodos , Corantes Fluorescentes/química , Neoplasias/patologia , Neoplasias Peritoneais/secundário , Animais , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Neoplasias/diagnóstico por imagem , Neoplasias/enzimologia , Neoplasias Peritoneais/diagnóstico por imagem , Neoplasias Peritoneais/enzimologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Anal Chem ; 93(7): 3470-3476, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33566568

RESUMO

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.


Assuntos
Carboxipeptidases , Peptídeos , Fluorescência , Lisina
11.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33542099

RESUMO

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.


Assuntos
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/metabolismo
12.
Angew Chem Int Ed Engl ; 60(4): 2125-2129, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33096584

RESUMO

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 in vivo cancer imaging for a prolonged period and was also compatible with fixation and immunostaining of the cancer tissue.


Assuntos
Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos , gama-Glutamiltransferase/metabolismo , Animais , Xenoenxertos , Humanos , Camundongos , Espectrometria de Fluorescência/métodos
13.
Molecules ; 25(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339370

RESUMO

The use of fluorescent probes in a multitude of applications is still an expanding field. This review covers the recent progress made in small molecular, spirocyclic xanthene-based probes containing different heteroatoms (e.g., oxygen, silicon, carbon) in position 10'. After a short introduction, we will focus on applications like the interaction of probes with enzymes and targeted labeling of organelles and proteins, detection of small molecules, as well as their use in therapeutics or diagnostics and super-resolution microscopy. Furthermore, the last part will summarize recent advances in the synthesis and understanding of their structure-behavior relationship including novel computational approaches.


Assuntos
Corantes Fluorescentes/química , Compostos de Espiro/química , Xantenos/química , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Animais , Humanos , Microscopia de Fluorescência , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico , Relação Estrutura-Atividade
14.
ACS Cent Sci ; 6(12): 2217-2227, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33376783

RESUMO

Accurate detection of breast tumors and discrimination of tumor from normal tissues during breast-conserving surgery are essential to reduce the risk of misdiagnosis or recurrence. However, existing probes show substantial background signals in normal breast tissues. In this study, we focus on glycosidase activities in breast tumors. We synthesized a series of 12 fluorescent probes and performed imaging-based evaluation on surgically resected human breast specimens. Among them, the α-mannosidase-reactive fluorescent probe HMRef-αMan detected breast cancer with 90% sensitivity and 100% specificity. We identified α-mannosidase 2C1 as the target enzyme and confirmed its overexpression in various breast tumors. We found that fibroadenoma, the most common benign breast lesion in young woman, tends to have higher α-mannosidase 2C1 activity than malignant cancer. Combined application of green-emitting HMRef-αMan and a red-emitting γ-glutamyltranspeptidase probe enabled efficient dual-color, dual-target optical discrimination of malignant and benign tumors.

15.
J Am Chem Soc ; 142(49): 20701-20707, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33225696

RESUMO

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.


Assuntos
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 Substrato
16.
Chem Commun (Camb) ; 56(86): 13173-13176, 2020 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-33020769

RESUMO

Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells.

17.
World J Surg ; 44(12): 4245-4253, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32909125

RESUMO

BACKGROUND: Bile leakage is the most common postoperative complication associated with hepatobiliary and pancreatic surgery. Until now, however, a rapid, accurate diagnostic method for monitoring intraoperative and postoperative bile leakage had not been established. METHOD: Bilirubin levels in drained abdominal fluids collected from 23 patients who had undergone hepatectomy (n = 22) or liver transplantation (n = 1) were measured using a microplate reader with excitation/emission wavelengths of 497/527 nm after applying 5 µM of UnaG to the samples. UnaG was also sprayed directly on hepatic raw surfaces in swine hepatectomy models to identify bile leaks by fluorescence imaging. RESULTS: The bilirubin levels measured by UnaG fluorescence imaging showed favorable correlations with the results of the conventional light-absorptiometric methods (indirect bilirubin: rs = 0.939, p < 0.001; direct bilirubin: rs = 0.929, p < 0.001). Approximate time required for bilirubin measurements with UnaG was 15 min, whereas it took about 40 min with the conventional method at a hospital laboratory. Following administration of UnaG on hepatic surfaces, the fluorescence imaging identified bile leaks not only on the resected specimens but also in the abdominal cavity of the swine hepatectomy models. CONCLUSION: Fluorescence imaging techniques using UnaG may enable real-time identification of bile leaks during hepatectomy and on-site rapid diagnosis of bile leaks after surgery.


Assuntos
Bile , Bilirrubina , Animais , Drenagem , Hepatectomia/efeitos adversos , Humanos , Fígado , Complicações Pós-Operatórias/diagnóstico , Suínos
18.
Plant Cell ; 32(10): 3081-3094, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32763980

RESUMO

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.


Assuntos
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)/metabolismo
19.
Bioconjug Chem ; 31(9): 2241-2251, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32840357

RESUMO

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.


Assuntos
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 fármacos
20.
Gen Thorac Cardiovasc Surg ; 68(12): 1418-1424, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32488832

RESUMO

OBJECTIVE: ɤ-glutamyltranspeptidase is an enzyme expressed in various malignancies including lung cancer. It rapidly activates non-fluorescent ɤ-glutamyl hydroxymethyl rhodamine green to highly fluorescent hydroxymethyl rhodamine green. The resultant tumor fluorescence is therefore an indicator of cellular ɤ-glutamyltranspeptidase activity. We have explored the use of ɤ-glutamyl hydroxymethyl rhodamine green as an intraoperative imaging tool for visualizing cancers. Herein, we evaluated the potential of the tumor fluorescence as a postoperative prognostic indicator. METHODS: We included patients with non-small cell lung cancer who had undergone radical resection from 2012 to 2014 in the study. We assessed the fluorescence intensity of the resected tumor and normal lung tissue by ex vivo imaging using ɤ-glutamyl hydroxymethyl rhodamine green. RESULTS: Sixty-seven patients were eligible for the study (adenocarcinomas, n = 44; squamous cell carcinoma, n = 14; other histologies, n = 8). The pathological stages were I, II, III, and IV in 39, 15, 12, and 1 patient, respectively. Based on the fluorescence of the tumor tissue, the patients were divided into high fluorescence (n = 33) and low fluorescence (n = 34) groups. The 5-year overall survival rate was significantly higher in the high fluorescence group (72.7%) compared to the low fluorescence group (47.1%, P = 0.025). Similarly, pathological stage I patients of the high fluorescence group had higher 5-year overall survival (85.7% vs. 44.4%, P = 0.009) and recurrence-free survival (76.2% vs. 44.4% P = 0.044) rates compared to those of the low fluorescence group. CONCLUSIONS: ɤ-glutamyl hydroxymethyl rhodamine green fluorescence is a good postoperative prognostic indicator in patients with non-small cell lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Fluorescência , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/cirurgia , Prognóstico , Rodaminas
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