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1.
PLoS Comput Biol ; 16(9): e1008198, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32931495

RESUMO

Calcium imaging with fluorescent protein sensors is widely used to record activity in neuronal populations. The transform between neural activity and calcium-related fluorescence involves nonlinearities and low-pass filtering, but the effects of the transformation on analyses of neural populations are not well understood. We compared neuronal spikes and fluorescence in matched neural populations in behaving mice. We report multiple discrepancies between analyses performed on the two types of data, including changes in single-neuron selectivity and population decoding. These were only partially resolved by spike inference algorithms applied to fluorescence. To model the relation between spiking and fluorescence we simultaneously recorded spikes and fluorescence from individual neurons. Using these recordings we developed a model transforming spike trains to synthetic-imaging data. The model recapitulated the differences in analyses. Our analysis highlights challenges in relating electrophysiology and imaging data, and suggests forward modeling as an effective way to understand differences between these data.


Assuntos
Cálcio/metabolismo , Fenômenos Eletrofisiológicos/fisiologia , Modelos Neurológicos , Imagem Molecular/métodos , Neurônios , Potenciais de Ação/fisiologia , Animais , Lobo Frontal/citologia , Lobo Frontal/fisiologia , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , Imagem Óptica
2.
Nat Commun ; 11(1): 4052, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792510

RESUMO

Turn-on fluorescence imaging is routinely studied; however, turn-on chemiluminescence has been rarely explored for in vivo imaging. Herein, we report the design and validation of chemiluminescence probe ADLumin-1 as a turn-on probe for amyloid beta (Aß) species. Two-photon imaging indicates that ADLumin-1 can efficiently cross the blood-brain barrier and provides excellent contrast for Aß plaques and cerebral amyloid angiopathy. In vivo brain imaging shows that the chemiluminescence signal of ADLumin-1 from 5-month-old transgenic 5xFAD mice is 1.80-fold higher than that from the age-matched wild-type mice. Moreover, we demonstrate that it is feasible to further dually-amplify signal via chemiluminescence resonance energy transfer (DAS-CRET) using two non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo whole brain imaging. Our results show that DAS-CRET can provide a 2.25-fold margin between 5-month-old 5xFAD mice and wild type mice. We believe that our strategy could be extended to other aggregating-prone proteins.


Assuntos
Peptídeos beta-Amiloides/química , Luminescência , Animais , Medições Luminescentes/métodos , Camundongos , Imagem Molecular/métodos , Imagem Óptica/métodos , Agregados Proteicos
3.
Yakugaku Zasshi ; 140(8): 969-977, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741870

RESUMO

We developed a method of video-rate bioluminescence imaging to visualize proteins secreted from living cells. A protein of interest was fused to Gaussia luciferase (GLase), and the luminescence signals of secreted GLase with coelenterazine (luciferin) were visualized at a video-rate of 30-500 ms/frame by using a water-cooled EM-CCD camera. We established a subclonal rat INS-1E cell line, named iGL cells, stably expressing the fusion protein of insulin and GLase (Insulin-GLase). By stimulation with high glucose, 3D-cultured iGL cells showed synchronized oscillatory secretion of insulin for over 1 h, as similarly observed in an isolated rat pancreatic islet. In 2D-cultured iGL cells, the luminescence images indicated that synchronized insulin secretion was localized in intercellular spaces between cells. Further, the relative amount of insulin secretion from iGL cells was easily determined with a luminometer, and we demonstrated that cell-cell interaction of beta cells is fundamental to increase glucose-stimulated insulin secretion by synchronization. Thus, iGL cells would be valuable for studying oscillatory insulin secretion and evaluating anti-diabetic drugs. Our bioluminescence imaging method with GLase could be generally used for investigating protein secretion in 2D and 3D cell culture systems.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Medições Luminescentes/métodos , Imagem Molecular/métodos , Animais , Comunicação Celular , Linhagem Celular , Células Cultivadas , Humanos , Imidazóis , Luciferases , Pirazinas , Ratos
4.
Yakugaku Zasshi ; 140(8): 979-983, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741871

RESUMO

Monoamine neurotransmitters are released by specialized neurons that regulate behavioral and cognitive functions. Although localization of monoaminergic neurons in the brain is well known, the distribution, concentration, and kinetics of monoamines remain unclear. We used mass spectrometry imaging (MSI) for simultaneous and quantitative imaging of endogenous monoamines to generate a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels. We observed several nuclei rich in both 5-HT and a catecholamine (DA or NE). Additionally, we analyzed de novo monoamine synthesis or fluctuations in those nuclei. We propose that MSI is a useful tool to gain deeper understanding of associations among the localization, levels, and turnover of monoamines in different brain areas and their role in inducing behavioral changes.


Assuntos
Monoaminas Biogênicas/análise , Monoaminas Biogênicas/metabolismo , Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Espectrometria de Massas/métodos , Imagem Molecular/métodos , Neurotransmissores/metabolismo , Animais , Dopamina/análise , Dopamina/metabolismo , Camundongos , Neurônios/metabolismo , Neurotransmissores/fisiologia , Norepinefrina/análise , Norepinefrina/metabolismo , Serotonina/análise , Serotonina/metabolismo
5.
Nat Protoc ; 15(9): 3088-3104, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807908

RESUMO

Endocytosis is a fundamental process occurring in all eukaryotic cells. Live cell imaging of endocytosis has helped to decipher many of its mechanisms and regulations. With the pulsed-pH (ppH) protocol, one can detect the formation of individual endocytic vesicles (EVs) with an unmatched temporal resolution of 2 s. The ppH protocol makes use of cargo protein (e.g., the transferrin receptor) coupled to a pH-sensitive fluorescent protein, such as superecliptic pHluorin (SEP), which is brightly fluorescent at pH 7.4 but not fluorescent at pH <6.0. If the SEP moiety is at the surface, its fluorescence will decrease when cells are exposed to a low pH (5.5) buffer. If the SEP moiety has been internalized, SEP will remain fluorescent even during application of the low pH buffer. Fast perfusion enables the complete exchange of low and high pH extracellular solutions every 2 s, defining the temporal resolution of the technique. Unlike other imaging-based endocytosis assays, the ppH protocol detects EVs without a priori hypotheses on the dynamics of vesicle formation. Here, we explain how the ppH protocol quantifies the endocytic activity of living cells and the recruitment of associated proteins in real time. We provide a step-by-step procedure for expression of the reporter proteins with transient transfection, live cell image acquisition with synchronized pH changes and automated analysis. The whole protocol can be performed in 2 d to provide quantitative information on the endocytic process being studied.


Assuntos
Imagem Molecular/métodos , Vesículas Transportadoras/metabolismo , Animais , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Análise Espaço-Temporal
6.
Nat Protoc ; 15(9): 3105-3128, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32826993

RESUMO

Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc-/-, il2rgα-/- immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc-/-, il2rgα-/- model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.


Assuntos
Transformação Celular Neoplásica , Imagem Molecular/métodos , Análise de Célula Única/métodos , Peixe-Zebra/imunologia , Animais , Linhagem Celular Tumoral , Humanos
7.
Proc Natl Acad Sci U S A ; 117(26): 14779-14789, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32561645

RESUMO

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.


Assuntos
Contagem de Células Sanguíneas/métodos , Microscopia Ultravioleta/métodos , Imagem Molecular/métodos , Contagem de Células Sanguíneas/instrumentação , Células Sanguíneas/classificação , Células Sanguíneas/citologia , Desenho de Equipamento , Humanos , Microscopia Ultravioleta/instrumentação , Imagem Molecular/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito
8.
Cancer Sci ; 111(9): 3164-3173, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32589345

RESUMO

Structural DNA nanotechnology enables DNA to be used as nanomaterials for novel nanostructure construction with unprecedented functionalities. Artificial DNA nanostructures can be designed and generated with precisely controlled features, resulting in its utility in bionanotechnological and biomedical applications. A tetrahedral DNA nanostructure (TDN), the most popular DNA nanostructure, with high stability and simple synthesis procedure, is a promising candidate as nanocarriers in drug delivery and bioimaging platforms, particularly in precision medicine as well as diagnosis for cancer therapy. Recent evidence collectively indicated that TDN successfully enhanced cancer therapeutic efficiency both in vitro and in vivo. Here, we summarize the development of TDN and highlight various aspects of TDN applications in cancer therapy based on previous reports, including anticancer drug loading, photodynamic therapy, therapeutic oligonucleotides, bioimaging platforms, and other molecules and discuss a perspective in opportunities and challenges for future TDN-based nanomedicine.


Assuntos
DNA/química , Sistemas de Liberação de Medicamentos , Imagem Molecular , Nanoestruturas , Neoplasias/diagnóstico , Neoplasias/terapia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Portadores de Fármacos , Desenho de Fármacos , Humanos , Imagem Molecular/métodos , Terapia de Alvo Molecular , Nanomedicina/métodos , Nanotecnologia/métodos , Neoplasias/genética , Relação Estrutura-Atividade
9.
Anticancer Res ; 40(6): 3571-3577, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487660

RESUMO

BACKGROUND/AIM: We evaluated urinary levels of porphyrin metabolites, such as uroporphyrin (UP) and coproporphyrin (CP), after 5-Aminolevulinic acid (ALA) administration in patients with or without pancreatic cancer (PaC). PATIENTS AND METHODS: Sixty-seven subjects with PaC, 11 with pancreatitis, and 9 with normal pancreas (NP) were enrolled. Urine samples from all subjects were collected prior to ALA administration and at more than 4 hours after ALA administration. We measured the urinary levels of UP and CP by high-performance liquid chromatography analysis. RESULTS: The PaC group showed significantly higher UP levels compared to NP groups (104.9 nmol/g Cre vs. 53.4 nmol/g Cre, p=0.014). Moreover, PaC patients with long-term survival had significantly lower urinary levels of UP at diagnosis (98.8 nmol/gCre) than the short-term survival group (125.2 nmol/gCre) (p=0.042). CONCLUSION: The urinary levels of UP after ALA administration might serve as a promising biomarker for diagnosis and prognosis prediction of PaC.


Assuntos
Ácidos Levulínicos , Luz , Imagem Molecular , Neoplasias Pancreáticas/diagnóstico , Fármacos Fotossensibilizantes , Idoso , Biomarcadores , Biomarcadores Tumorais , Detecção Precoce de Câncer , Feminino , Humanos , Ácidos Levulínicos/metabolismo , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Imagem Molecular/métodos , Imagem Molecular/normas , Neoplasias Pancreáticas/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Porfirinas , Sensibilidade e Especificidade
10.
Nat Methods ; 17(5): 531-540, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371980

RESUMO

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.


Assuntos
Encéfalo/metabolismo , Cartilagem/metabolismo , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Placa Amiloide/metabolismo , Imagem Individual de Molécula/métodos , Animais , Encéfalo/patologia , Cartilagem/patologia , Núcleo Celular/metabolismo , Células Cultivadas , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Mitocôndrias/metabolismo , Imagem Molecular/métodos , Poro Nuclear/metabolismo , Placa Amiloide/patologia
11.
Lancet Haematol ; 7(6): e479-e489, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32470439

RESUMO

Mature lymphoproliferative diseases are a heterogeneous group of neoplasms arising from different stages of B-cell and T-cell development. With improved understanding of the molecular processes in lymphoma and novel treatment options, arises a growing need for the molecular characterisation of tumours. Molecular imaging with single-photon-emission CT and PET using specific radionuclide tracers can provide whole-body information to investigate cancer biology, to evaluate phenotypic heterogeneity, to identify resistance to targeted therapy, and to assess the biodistribution of drugs in patients. In this Review, we evaluate the existing literature on molecular imaging in lymphoma, other than 18F-fluordeoxyglucose molecular imaging. The aim is to examine the contribution of molecular imaging to the understanding of the biology of lymphoma and to discuss potential implications for the diagnostics and therapy of this disease. Finally, we discuss possible applications for molecular imaging of patients with lymphoma in the clinical context.


Assuntos
Fluordesoxiglucose F18/metabolismo , Linfoma/diagnóstico por imagem , Imagem Molecular/métodos , Biomarcadores Tumorais/metabolismo , Ensaios Clínicos como Assunto , Humanos , Imunoterapia/métodos , Linfoma/terapia , Transtornos Linfoproliferativos/patologia , Estadiamento de Neoplasias/métodos , Tomografia por Emissão de Pósitrons/métodos , Medicina de Precisão/métodos , Radioimunoterapia/métodos , Radioisótopos/metabolismo , Distribuição Tecidual/efeitos dos fármacos
12.
Yakugaku Zasshi ; 140(5): 633-640, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32378663

RESUMO

Because active-targeted liposomes are very complex formulations, quality characteristics of functional lipids have not been defined yet, and this is a major obstacle in clinical application of active targeted liposomes. We have developed high functionality and quality (HFQ) lipids, which define quality characteristics of functional lipids for clinical drug delivery system (DDS) applications. Because HFQ lipids are designed to enable facile and rapid functionalization of DDS carrier by simple and one-step mixing, we are expanding applications for not only liposomes but also exosomes and cells. Recently, we developed multi-color deep imaging by tissue clearing for analysis of spatial distribution of DDS in various tissues. Nanocarriers are usually non-uniformly distributed in solid tumors because of their heterogeneity. Especially, in refractory cancer such as pancreatic cancer, the presence of collagen and blood vessels greatly affects intra-tumor distribution of DDS carrier. Therefore information on spatial relations between the tissue structure and DDS carrier is important to regulate precisely intra-tumor distribution of DDS carrier. Recently, our group has established multi-color deep imaging to analyze spatial distribution of stromal collagen, liposomes, and blood vessels in pancreatic tumor tissue. In this review, we present recent research in developing HFQ lipids. Moreover, current status of research on DDS for pancreatic cancer treatment is reviewed.


Assuntos
Portadores de Fármacos , Sistemas de Liberação de Medicamentos/métodos , Desenvolvimento de Medicamentos , Lipossomos , Imagem Molecular/métodos , Nanopartículas , Distribuição Tecidual , Humanos , Neoplasias Pancreáticas/metabolismo
13.
Pediatr Blood Cancer ; 67(7): e28407, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32426927

RESUMO

Little is known about pseudoprogression in brain tumours other than gliomas. A 9-year-old male child with a pineal teratoma/germinoma underwent surgical resection followed by adjuvant chemo-radiotherapy. The magnetic resonance imaging scan 4 months post-radiotherapy showed a contrast-enhancing lesion within the surgical cavity suspicious of recurrence. These radiological findings subsequently resolved without any specific intervention. The child continues in remission 2 years post-treatment. This case illustrates the occurrence of pseudoprogression post-radiotherapy in intracranial GCT and highlights an unmet need for greater implementation of functional imaging techniques in paediatric neuro-oncology to avoid undue discontinuation of effective treatments or inappropriate enrolment in clinical trials.


Assuntos
Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Neoplasias Embrionárias de Células Germinativas/patologia , Pinealoma/patologia , Radioterapia Adjuvante/métodos , Teratoma/patologia , Criança , Progressão da Doença , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas/diagnóstico por imagem , Neoplasias Embrionárias de Células Germinativas/radioterapia , Pinealoma/diagnóstico por imagem , Pinealoma/radioterapia , Prognóstico , Teratoma/diagnóstico por imagem , Teratoma/radioterapia
14.
Proc Natl Acad Sci U S A ; 117(23): 12991-12999, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439710

RESUMO

Malignant melanoma has one of the highest mortality rates of any cancer because of its aggressive nature and high metastatic potential. Clinical staging of the disease at the time of diagnosis is very important for the prognosis and outcome of melanoma treatment. In this study, we designed and synthesized the 18F-labeled pyridine-based benzamide derivatives N-(2-(dimethylamino)ethyl)-5-[18F]fluoropicolinamide ([18F]DMPY2) and N-(2-(dimethylamino)ethyl)-6-[18F]fluoronicotinamide ([18F]DMPY3) to detect primary and metastatic melanoma at an early stage and evaluated their performance in this task. [18F]DMPY2 and [18F]DMPY3 were synthesized by direct radiofluorination of the bromo precursor, and radiochemical yields were ∼15-20%. Cell uptakes of [18F]DMPY2 and [18F]DMPY3 were >103-fold and 18-fold higher, respectively, in B16F10 (mouse melanoma) cells than in negative control cells. Biodistribution studies revealed strong tumor uptake and retention of [18F]DMPY2 (24.8% injected dose per gram of tissue [ID/g] at 60 min) and [18F]DMPY3 (11.7%ID/g at 60 min) in B16F10 xenografts. MicroPET imaging of both agents demonstrated strong tumoral uptake/retention and rapid washout, resulting in excellent tumor-to-background contrast in B16F10 xenografts. In particular, [18F]DMPY2 clearly visualized almost all metastatic lesions in lung and lymph nodes, with excellent image quality. [18F]DMPY2 demonstrated a significantly higher tumor-to-liver ratio than [18F]fluorodeoxyglucose ([18F]FDG) and the previously reported benzamide tracers N-[2-(diethylamino)-ethyl]-5-[18F]fluoropicolinamide ([18F]P3BZA) and N-[2-(diethylamino)-ethyl]-4-[18F]fluorobenzamide ([18F]FBZA) in B16F10-bearing or SK-MEL-3 (human melanoma)-bearing mice. In conclusion, [18F]DMPY2 might have strong potential for the diagnosis of early stage primary and metastatic melanoma using positron emission tomography (PET).


Assuntos
Melanoma/diagnóstico por imagem , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Neoplasias Cutâneas/diagnóstico por imagem , Animais , Linhagem Celular Tumoral , Radioisótopos de Flúor/administração & dosagem , Humanos , Camundongos , Ácidos Picolínicos/administração & dosagem , Compostos Radiofarmacêuticos/química , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Phys Rev Lett ; 124(19): 198104, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32469536

RESUMO

The localization of point sources in optical microscopy enables nm-precision imaging of single-molecules and biological dynamics. We report a new method of localization microscopy using twin Airy beams that yields precise 3D localization with the key advantages of extended depth range, higher optical throughput, and potential for imaging higher emitter densities than are possible using other techniques. A precision of better than 30 nm was achieved over a depth range in excess of 7 µm using a 60×, 1.4 NA objective. An illustrative application to extended-depth-range blood-flow imaging in a live zebrafish is also demonstrated.


Assuntos
Imageamento Tridimensional/métodos , Microscopia/métodos , Animais , Cloaca/irrigação sanguínea , Imageamento Tridimensional/instrumentação , Microscopia/instrumentação , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Fluxo Sanguíneo Regional , Peixe-Zebra
16.
J Cancer Res Clin Oncol ; 146(8): 1941-1951, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32447486

RESUMO

PURPOSE: Currently, the routine screening program has insufficient capacity for the early diagnosis of lung cancer. Therefore, a type of chitosan-molecular beacon (CS-MB) probe was developed to recognize the miR-155-5p and image the lung cancer cells for the early diagnosis. METHODS: Based on the molecular beacon (MB) technology and nanotechnology, the CS-MB probe was synthesized self-assembly. There are four types of cells-three kinds of animal models and one type of histopathological sections of human lung cancer were utilized as models, including A549, SPC-A1, H446 lung cancer cells, tumor-initiating cells (TICs), subcutaneous and lung xenografts mice, and lox-stop-lox(LSL) K-ras G12D transgenic mice. The transgenic mice dynamically displayed the process from normal lung tissues to atypical hyperplasia, adenoma, carcinoma in situ, and adenocarcinoma. The different miR-155-5p expression levels in these cells and models were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The CS-MB probe was used to recognize the miR-155-5p and image the lung cancer cells by confocal microscopy in vitro and by living imaging system in vivo. RESULTS: The CS-MB probe could be used to recognize the miR-155-5p and image the lung cancer cells significantly in these cells and models. The fluorescence intensity trends detected by the CS-MB probe were similar to the expression levels trends of miR-155 tested by qRT-PCR. Moreover, the fluorescence intensity showed an increasing trend with the tumor progression in the transgenic mice model, and the occurrence and development of lung cancer were dynamically monitored by the differen fluorescence intensity. In addition, the miR-155-5p in human lung cancer tissues could be detected by the miR-155-5p MB. CONCLUSION: Both in vivo and in vitro experiments demonstrated that the CS-MB probe could be utilized to recognize the miR-155-5p and image the lung cancer cells. It provided a novel experimental and theoretical basis for the early diagnosis of the disease. Also, the histopathological sections of human lung cancer research laid the foundation for subsequent preclinical studies. In addition, different MBs could be designed to detect other miRNAs for the early diagnosis of other tumors.


Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , MicroRNAs/análise , Células A549 , Animais , Quitosana/química , Detecção Precoce de Câncer/métodos , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , MicroRNAs/biossíntese , MicroRNAs/genética , Imagem Molecular/métodos , Sondas Moleculares/química , Nanotecnologia
17.
Nat Commun ; 11(1): 2399, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404879

RESUMO

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.


Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Vasodilatadores/metabolismo , Animais , Biotina/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Células HEK293 , Humanos , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Neurotransmissores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Reprodutibilidade dos Testes , Vasodilatadores/química
18.
PLoS Biol ; 18(4): e3000665, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275651

RESUMO

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.


Assuntos
Sistemas CRISPR-Cas , Epitopos/genética , Edição de Genes/métodos , Neurônios/imunologia , Proteínas/genética , Animais , Células Cultivadas , Dependovirus/genética , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Vetores Genéticos , Genoma , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos Transgênicos , Microscopia de Fluorescência , Imagem Molecular/métodos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Proteínas/imunologia , Proteínas/metabolismo , Ratos Wistar , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Análise Espaço-Temporal
19.
Org Biomol Chem ; 18(16): 3104-3116, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32253415

RESUMO

Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range (1-40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labelling procedure to insert fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq µmol-1. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2 and EphB4-overexpressing A375 human melanoma cells, whereas [18F]2 did not accumulate at these cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors with PET.


Assuntos
Radioisótopos de Flúor/química , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/síntese química , Receptores da Família Eph/análise , Xantinas/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Efrina-A2/análise , Humanos , Melanoma/diagnóstico por imagem , Melanoma/patologia , Imagem Molecular/métodos , Ratos , Receptor EphB4/análise , Receptores da Família Eph/antagonistas & inibidores
20.
J Vis Exp ; (156)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32150168

RESUMO

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.


Assuntos
Cálcio/metabolismo , Hibridização in Situ Fluorescente/métodos , Imagem Molecular/métodos , Neurogênese , Neurônios/citologia , Células-Tronco/citologia , Xenopus laevis/crescimento & desenvolvimento , Animais , Neurônios/metabolismo , Células-Tronco/metabolismo , Xenopus laevis/metabolismo
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