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1.
Methods Mol Biol ; 2566: 45-52, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152241

RESUMO

As a natural by-product of mitochondrial respiration, reactive oxygen species (ROS) in sperm play a role in promoting fertilization, by intervening in a series of events. Nevertheless, an abnormal and uncounteracted increase in ROS production leads to oxidative stress (OS) which can, ultimately, culminate in cell death. An established relationship between OS and male infertility highlights the importance of an accurate detection method for ROS content that can be easily implemented and reproduced in any andrology lab. More recently, reactive nitrogen species (RNS) production and subsequent nitrosative stress have also been described. Here we describe the use of fluorescent probes, including some that targeted to the mitochondria due to the coupling of a cation (TPP+), in order to assess the levels of different ROS and RNS in human sperm using flow cytometry and/or fluorescent microscopy. This methodology is user friendly and accurate and can be safely applied in research- and/or clinical-based contexts.


Assuntos
Corantes Fluorescentes , Estresse Nitrosativo , Corantes Fluorescentes/metabolismo , Humanos , Masculino , Estresse Oxidativo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo
2.
Methods Mol Biol ; 2559: 95-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36180629

RESUMO

The lack of unambiguous Foxp3+ Treg cell-specific surface markers has prompted the development of various transgenic mouse lines with Foxp3-dependent reporter activity, which involved different fluorochromes and transgenic strategies, including coexpression of multiple transgenes, such as Cre recombinase. Since then, Foxp3 transcriptional reporter has proven to be an indispensable tool to identify and isolate viable Foxp3+ Treg cell populations. However, the physiologic Treg cell pool is functionally heterogeneous and consists of intrathymically (tTreg) and peripherally (pTreg) induced Treg cells, which may confound interpretation of data relying on indiscriminatory Foxp3-fluorochrome reporter expressed in all Treg cells. In this chapter, we describe how the dual Foxp3RFP/GFP reporter can be exploited to discriminate both developmental sublineages based on tTreg cell lineage-specific GFP/Cre recombinase activity, in conjunction with Foxp3-driven RFP expression in all Foxp3+ Treg cells, and provide guidelines for experimental design and implementation. We also elaborate on the possibility to exploit GFP/Cre expression of Foxp3RFP/GFP reporter mice for the manipulation of gene expression (activation and inactivation), such as lineage tracing and in vivo ablation of tTreg cells, while sparing pTreg cells.


Assuntos
Corantes Fluorescentes , Linfócitos T Reguladores , Animais , Linhagem da Célula/genética , Corantes Fluorescentes/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/metabolismo
3.
Methods Mol Biol ; 2572: 55-66, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36161407

RESUMO

Tumor vasculature is the major extrinsic factor that shapes Intra-tumoral heterogeneity (ITH). Non-uniform exposure of microenvironmental cues greatly impacts cancer cell phenotypes leading to ITH, which exacerbates therapy resistance. This raises a need to study the influence of non-uniform perfusion patterns and the resulting heterogeneity that persists within the tumor microenvironment (TME). A method was developed to identify cancer cells based on their proximity to functional blood vessels (BVs) called perfusion-based fluorescent dye labeling of cells (PFDLC). PFDLC works on the principle of perfusion, where a freely diffusible nuclear binding fluorescent dye (Hoechst 33342) is injected intravenously (i.v.) through a tail vein into atumor-bearing mice. The tumors are retrieved post dye perfusion, dissociated into single cells, and sorted based on their dye uptake proportional to their distance from the nearest blood capillary. This method is amenable to multi-omics as well as functional assays.


Assuntos
Corantes Fluorescentes , Neoplasias , Animais , Vasos Sanguíneos/metabolismo , Movimento Celular , Corantes Fluorescentes/metabolismo , Camundongos , Neoplasias/metabolismo , Perfusão , Microambiente Tumoral
4.
Bioorg Chem ; 130: 106199, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36370648

RESUMO

Due to the diverse H2O2 distribution in organelles, fluorescent probes were usually required to be prepared separately, which limited the convenience and practicability. Herein, we reported a flexible strategy to in-situ construct H2O2 fluorescent probes in different organelles. A tetrazine fused probe TP was developed with rapid click reaction capacity and sensitive H2O2 response. When treated with H2O2, the turn-on fluorescence was effectively quenched by the tetrazine part. Only after click reaction with dienophiles, the fluorescence resumed. In application, cells were firstly treated with triphenylphosphorus tagged norbornene (TPP-NB) to label mitochondria, which was followed by the introduction of probe TP to trigger click reaction. The in-situ constructed probe P1 served as a local H2O2 sensor. In a similar way, probe P2 was in-situ constructed in lysosomes via probe TP and morpholine tagged norbornene (MP-NB). With this on-demand modular assembling and double turn-on features, our strategy to construct fluorescent probes presented high flexibility and anti-interference performance, which was expected to inspired more applications in biological studies.


Assuntos
Corantes Fluorescentes , Peróxido de Hidrogênio , Humanos , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Células HeLa , Lisossomos/metabolismo , Mitocôndrias , Norbornanos/metabolismo
5.
Methods Mol Biol ; 2582: 23-29, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370341

RESUMO

The method of labeling proteins of interest with fluorescent dyes that can specifically stain organelles in living cells provides a tool for investigating various cellular processes under a microscope. Visualization (imaging) of the cells using fluorescence has many advantages, including the ability to stain multiple cell organelles and intracellular proteins simultaneously and discriminately, and is used in many research fields. In this chapter, we describe the observation of cell organelles using fluorescence staining to analyze the functions of CCN family proteins involved in various cellular events.


Assuntos
Proteínas de Sinalização Intercelular CCN , Imagem Óptica , Microscopia de Fluorescência/métodos , Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/metabolismo , Corantes Fluorescentes/metabolismo , Organelas/metabolismo , Proteínas/metabolismo
6.
ACS Chem Biol ; 17(11): 3218-3228, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36318872

RESUMO

Ectodomain shedding is a form of limited proteolysis in which a protease cleaves a transmembrane protein, releasing the extracellular domain from the cell surface. Cells use this process to regulate a wide variety of biological events. Typically, immunological detection methods are employed for the analysis of ectodomains secreted into the cultured media. In this paper, we describe a new strategy using an affinity-based protein-labeling fluorescent probe to study ectodomain shedding. We analyzed the ectodomain shedding of cell surface carbonic anhydrases (CAIX and CAXII), which are important biomarkers for tumor hypoxia. Using both chemical and genetic approaches, we identified that the ADAM17 metalloprotease is responsible for the shedding of carbonic anhydrases. Compared to current immunological methods, this protein-labeling approach not only detects ectodomain released into the culture media but also allows real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, thereby providing a more detailed insight into the mechanism of ectodomain shedding as well as protein lifetime on the cell surface.


Assuntos
Anidrases Carbônicas , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismo , Metaloproteases/metabolismo
7.
Int J Mol Sci ; 23(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36232401

RESUMO

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.


Assuntos
Endocanabinoides , Corantes Fluorescentes , Animais , Ácidos Araquidônicos , Linhagem Celular , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Corantes Fluorescentes/metabolismo , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Alcamidas Poli-Insaturadas , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
8.
Acta Neuropathol Commun ; 10(1): 153, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36307888

RESUMO

Protein misfolding is a prominent pathological hallmark of neurodegenerative disorders, including Alzheimer's disease (AD). Studies have shown that the diversity of ß sheet-rich protein deposits (such as amyloid ß plaques and neurofibrillary tangles), present across different brain regions, might underlie different disease phenotypes and only certain types of aggregates might be associated with cognitive decline. Conformationally sensitive fluorescent amyloid probes have the ability to report different structures of protein aggregates by virtue of their shifting emission spectra. Here we defined the binding affinity of the fluorescent amyloid probes BSB and MCAAD to disease-relevant protein aggregates, and combined the two probes to examine formalin-fixed paraffin-embedded mouse and human brain samples. Coupled with quantitative spectral phasor analysis, the dual-probe staining approach revealed remarkable heterogeneity of protein aggregates across the samples. Distinct emission spectra were consistent with certain types of deposits present in the mouse and human brain sections. The sensitivity of this staining, imaging and analysis approach outperformed conventional immunohistochemistry with the detected spectral differences between the greater parenchyma of cognitively normal and AD cases indicating a subtle yet widespread proteopathy associated with disease. Our method offers more sensitive, objective, and quantitative examination of protein misfolding pathology using conventional tissue sections.


Assuntos
Doença de Alzheimer , Amiloidose , Animais , Humanos , Camundongos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/patologia , Proteínas tau/metabolismo , Agregados Proteicos , Espectrometria de Fluorescência , Placa Amiloide/patologia , Amiloide/metabolismo , Encéfalo/patologia , Amiloidose/patologia , Corantes Fluorescentes/metabolismo
9.
Anal Chem ; 94(44): 15261-15269, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36282989

RESUMO

Lewy pathologies, which mainly consist of insoluble α-synuclein (α-syn) aggregates, are the hallmarks of Parkinson's disease and many other neurodegenerative diseases termed "synucleinopathies". Detection of Lewy pathologies with optical methods is of interest for preclinical studies, while the α-syn fluorescent probe is still in great demand. By rational design, we obtained a series of D-π-A-based trisubstituted alkenes with acceptable optical properties and high binding affinities to α-syn fibrils. Among these probes, FPQXN and TQXN-2 exhibited high binding affinities (6 and 8 nM, respectively), significant fluorescence enhancements (17.2- and 26.6-fold, respectively), and satisfying quantum yields (36.5% and 10.4%, respectively), which met the need for the in vitro neuropathological staining of Lewy pathologies in the PD brain sections. In addition, TQXN-2 showed great potential in fluorescent discrimination of Lewy pathologies and Aß plaques. Our research provides flexible tools for in vitro detection of α-syn aggregates and offers new structural frameworks for the further development of α-syn fluorescent probes.


Assuntos
Corantes Fluorescentes , Doença de Parkinson , Humanos , Corantes Fluorescentes/metabolismo , Alcenos/metabolismo , alfa-Sinucleína/química , Doença de Parkinson/metabolismo , Placa Amiloide/metabolismo , Encéfalo/metabolismo
10.
J Mater Chem B ; 10(43): 8875-8882, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36226623

RESUMO

A single fluorescent probe (SF-probe) that can simultaneously and discriminatively visualize two organelles is a powerful tool to investigate their interaction in cellular processes. However, it is still challenging to develop this unique type of fluorescent probe due to the lack of a feasible design strategy. Herein, we proposed a dual-targeting group strategy to construct SF-probes by integrating two different organelle-targeting groups into the same fluorophore. A versatile fluorophore and two nonintrusive organelle-targeting groups are elements of this strategy. In view of only a few SF-probes having been developed for the simultaneous and discriminative imaging of lipid droplets (LDs) and endoplasmic reticulum (ER), as a proof of concept, a SF-probe, LDER, was designed and synthesized by introducing an LD targeting group and an ER targeting group onto the 1,8-naphthalimide fluorophore. Owing to the specific structure of the fluorophore, both targeting groups at two terminals of 1,8-naphthalimide can fully play their respective roles without mutual interference. Furthermore, the ability of the two groups to target their respective targets is comparable, enabling LDER to bind LDs and ER evenly. Meanwhile, LDER is very susceptible to polarity, which is advantageous for the discriminative imaging of LDs and ER. In addition, the interaction between LDs and ER was investigated.


Assuntos
Corantes Fluorescentes , Gotículas Lipídicas , Gotículas Lipídicas/metabolismo , Corantes Fluorescentes/metabolismo , Naftalimidas/metabolismo , Retículo Endoplasmático/metabolismo
11.
J Med Chem ; 65(21): 14527-14538, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36283122

RESUMO

Neurofibrillary tangles (NFTs), which are composed of abnormally hyperphosphorylated Tau, are one of the main pathologic hallmarks of Alzheimer's disease and other tauopathies. The fluorescent imaging probes currently used to target NFTs cannot distinguish them well from ß-amyloid plaques, thus limiting their utility to diagnose diseases. Here, we developed a fused cycloheptatriene-BODIPY derivative (TNIR7-1A) that displays properties favorable for near-infrared (NIR) imaging with high affinity and specificity to NFTs in vitro. In addition, TNIR7-1A effectively penetrated the blood-brain barrier and clearly distinguished tauopathy in transgenic mice (rTg4510) from control mice using NIR fluorescence imaging in vivo. The sensitivity and specificity of TNIR7-1A for NFTs were confirmed ex vivo by fluorescence staining of the tauopathy mouse model, while molecular docking studies indicated that TNIR7-1A bound to NFTs through hydrophobic interactions. These results suggest that TNIR7-1A can act as a high-performance probe to detect NFTs in vitro and in vivo selectively.


Assuntos
Doença de Alzheimer , Tauopatias , Animais , Camundongos , Proteínas tau/metabolismo , Simulação de Acoplamento Molecular , Emaranhados Neurofibrilares/metabolismo , Doença de Alzheimer/metabolismo , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Encéfalo/metabolismo
12.
Cell Chem Biol ; 29(10): 1505-1516.e7, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36270233

RESUMO

Proteases are attractive targets for infectious disease diagnostics. Peptide-based sensors that are cleaved by pathogen proteases can provide a rapid readout of infection. However, identifying peptide substrates specific to a targeted pathogen is a significant challenge. Here, we demonstrate that a structured propeptide domain from a bacterial protease can be repurposed as a protease-activated biosensor of the cholera pathogen Vibrio cholerae. We found that the peptidase inhibitor I9 domain of the secreted V. cholerae protease IvaP is rapidly degraded by V. cholerae, but not by other intestinal bacteria. By conjugating the I9 domain to an environment-sensitive fluorophore, we developed a fluorescent probe that enables the species-specific detection of V. cholerae in mixed bacterial cultures without nonspecific cleavage by other bacteria or intestinal cells. Our findings demonstrate that the IvaP propeptide is sufficient to impart selectivity to a cleavage-based V. cholerae biosensor, suggesting I9 domains could potentially be harnessed for diagnostic applications.


Assuntos
Técnicas Biossensoriais , Vibrio cholerae , Vibrio cholerae/metabolismo , Corantes Fluorescentes/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo
13.
Nat Commun ; 13(1): 5841, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36192426

RESUMO

Cell-based assays can monitor virus infection at a single-cell level with high sensitivity and cost-efficiency. For this purpose, it is crucial to develop molecular probes that respond selectively to physiological changes in live cells. We report stimuli-responsive light-emitters built on a T-shaped benzimidazole platform, and consecutive borylation reactions to produce a library of homologs displaying systematic changes in fluorescence quantum yield and environmental sensitivity. We find that certain fluorophores localize selectively at the endoplasmic reticulum, and interact with proteins involved in the stress signaling pathways. Notably, the mono-borylated compound responds selectively to the stress conditions by enhancing fluorescence, and detects avian influenza virus infection at the single-cell level. Our findings demonstrate the unprecedented practical utility of the stress-responsive molecular probes to differentiate cellular states for early diagnosis.


Assuntos
Vírus da Influenza A , Influenza Aviária , Animais , Benzimidazóis , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Vírus da Influenza A/fisiologia , Influenza Aviária/diagnóstico , Influenza Aviária/metabolismo , Sondas Moleculares/metabolismo
14.
Int Heart J ; 63(5): 904-914, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36184550

RESUMO

Forsythiaside, one of the main bioactive components of Chinese medicine Lian Qiao, exerts antioxidant, anti-bacterial, and anti-inflammatory effects. To date, the mechanism of Forsythiaside in cardiomyocyte injury remains unclear. However, the antioxidant effects of Forsythiaside on cardiac cells are currently unknown. This study investigated the effect and mechanism of Forsythiaside on oxidative stress in H9c2 cardiomyocytes. H9c2 cells were treated with H2O2 and Forsythiaside and then transfected with small-interfering RNA against nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability, apoptosis, accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential were measured using methyl thiazolyl tetrazolium (MTT), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123, respectively. The levels of oxidative stress-related markers were determined using their respective detection kits. Furthermore, the levels of apoptosis- and Nrf2 pathway-related molecules were determined via Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Forsythiaside had no obvious toxicity on H9c2 cells. H2O2 suppressed the viability, and reduced the levels of mitochondrial membrane potential, B-cell lymphoma-2 (Bcl-2), glutathione peroxidase (GSH-Px) and catalase (CAT) and superoxide dismutase (SOD), while promoted apoptosis, ROS accumulation, and elevated the levels of cleaved caspase 3, BCL2-Associated X (Bax) and malondialdehyde (MDA) in H9c2 cells. Contrarily, Forsythiaside reversed the aforementioned effects. H2O2 advanced the levels of cytoplasm Nrf2, heme oxygenase-1 (HO-1), and nucleus Nrf2 in H9c2 cells, whereas Forsythiaside enhanced these effects. SiNrf2 reversed the functions of H2O2 or Forsythiaside in cell viability, MDA, SOD, GSH-Px, CAT, Nrf2, and HO-1 in H9c2 cells, whereas Forsythiaside reversed the aforementioned effects of siNrf2. In sum, Forsythiaside protected H9c2 cells from oxidative stress and apoptosis induced by H2O2 by activating the Nrf2/HO-1 pathway.


Assuntos
Heme Oxigenase-1 , Fator 2 Relacionado a NF-E2 , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Apoptose , Caspase 3/metabolismo , Catalase/metabolismo , Catalase/farmacologia , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidilexotransferase/farmacologia , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Glutationa Peroxidase/metabolismo , Glicosídeos , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/farmacologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Malondialdeído/metabolismo , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodamina 123/metabolismo , Rodamina 123/farmacologia , Transdução de Sinais , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/metabolismo
15.
Commun Biol ; 5(1): 1100, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253454

RESUMO

Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. Classical demixing algorithms tend to give suboptimal results in terms of localization precision and correction of chromatic errors. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision and drift correction, compensation of chromatic distortions, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic errors at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.


Assuntos
Microscopia , Poro Nuclear , Algoritmos , Corantes Fluorescentes/metabolismo , Microscopia/métodos , Poro Nuclear/metabolismo , Imagem Individual de Molécula/métodos
16.
PLoS One ; 17(10): e0276107, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36256604

RESUMO

The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aß) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aß plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-Aß VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and Aß plaques at concentrations as low as 0.22 nM. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-induced hypothermia was lower than the concentration required to produce in situ labeling. These findings reveal an important dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aß-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.


Assuntos
Doença de Alzheimer , Camelídeos Americanos , Hipotermia , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Neurotensina/metabolismo , Doença de Alzheimer/metabolismo , Meios de Contraste/metabolismo , Hipotermia/metabolismo , Corantes Fluorescentes/metabolismo , Encéfalo/metabolismo , Peptídeos beta-Amiloides/metabolismo , Placa Amiloide/patologia , Camundongos Transgênicos , Modelos Animais de Doenças , Transcitose , Peso Corporal
17.
J Immunol Methods ; 510: 113348, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36058258

RESUMO

Multi-color flow cytometry is the standard approach for immunophenotyping clinical samples. With the recent advances in cancer immunotherapy, myeloid-derived suppressor cells (MDSC), immature myeloid-lineage cells in cancer patient blood and the tumor microenvironment, are highlighted as an important immune cell population that correlates with prognosis and therapeutic efficacy. In contrast to their clear functions and existence, immunophenotyping of MDSC is not consistent among investigators due to surface antigens overlapping with many normal hematopoietic lineage cell populations. We performed a clinical study and analyzed more than 1000 breast cancer patients blood samples to quantitate MDSC during breast cancer progression. In this methodology manuscript, we described detailed procedures for study design, sample logistics and handling, staining and flow cytometric analysis. This protocol used a 7-color fluorochrome-conjugated antibody panel to analyze polymorphonuclear (PMN)- and monocytic (M)-MDSC subsets simultaneously. The interim analysis results of this study showed that both PMN and M-MDSC populations are increased in patients with bone metastasis compared with patients with visceral organ metastasis. In conclusion, this work provides a versatile, comprehensive, and practical protocol to measure MDSC in patient blood samples.


Assuntos
Neoplasias da Mama , Células Supressoras Mieloides , Antígenos de Superfície , Neoplasias da Mama/patologia , Feminino , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Humanos , Imunofenotipagem , Microambiente Tumoral
18.
J Am Chem Soc ; 144(38): 17651-17660, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36121306

RESUMO

Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with important roles in regulating organelle function and stress. Because of its high reactivity, targeted delivery of H2S using small molecule H2S donors has garnered significant interest to minimize off-target effects. Although mitochondrially targeted H2S donors, such as AP39, have been reported previously and exhibit significantly higher potency than nontargeted donors, the expansion of targeted H2S delivery to other subcellular organelles remains largely absent. To fill this key unmet need, we report a library of organelle-targeted H2S donors that localize H2S delivery to specific subcellular organelles, including the Golgi apparatus, lysosome, endoplasmic reticulum, and mitochondria. We measured H2S production in vitro from each donor, confirmed the localization of H2S delivery using organelle-specific H2S responsive fluorescent probes, and demonstrated enhanced potency of these targeted H2S donors in providing protection against organelle-specific stress. We anticipate this class of targeted H2S donors will enable future studies of subcellular roles of H2S and the pathways by which H2S alleviates subcellular organelle stress.


Assuntos
Sulfeto de Hidrogênio , Corantes Fluorescentes/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Transdução de Sinais
19.
Int J Mol Sci ; 23(18)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36142507

RESUMO

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.


Assuntos
Corantes Fluorescentes , Proteínas de Neoplasias , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Corantes Fluorescentes/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Preparações Farmacêuticas
20.
Chem Commun (Camb) ; 58(76): 10727-10730, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36069370

RESUMO

The occurrence of endoplasmic reticulum (ER) stress is the main cause of a variety of biological processes that are closely related to numerous diseases. The homeostasis of the ER microenvironment can be disrupted under ER stress. In this research, by linking a pentafluorophenyl to the green fluorescent protein chromophore, we have developed a new ER-targeting fluorescent probe (GE-Y) for measuring changes of intracellular ER viscosity caused by ER stress. Importantly, an increase in ER viscosity was observed using GE-Y in cells undergoing autophagy. As such, our research provides an ideal tool for studying ER stress and autophagy.


Assuntos
Retículo Endoplasmático , Corantes Fluorescentes , Autofagia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Viscosidade
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