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1.
Curr Protoc ; 1(9): e231, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34491623

RESUMO

The use of whole animal models in toxicological studies is essential for understanding the physiological responses caused by chemical exposures. However, such studies can face reproducibility challenges due to unaccounted experimental parameters that can have a marked influence on toxicological outcomes. Zebrafish embryos and larvae are a popular vertebrate animal model for studying cellular, tissue, and organ responses to toxicant exposures. Despite the popularity of this system, standardized protocols that control for the influence of various experimental parameters and culture conditions on the toxicological response in these animals have not been widely adopted, making it difficult to compare findings from different laboratories. Here, we describe a detailed approach for designing and optimizing protocols to assess the impact of chemical exposures on the development and survival of zebrafish embryos and larvae. We first describe our standard procedure to determine two key toxicological thresholds, the maximum tolerable concentration (MTC) and the lethal concentration (LC50 , defined as that in which 50% of larvae die), in response to an exposure that persists from early development through larval maturation. We then describe two protocols to systematically test how key experimental parameters, including genetic background, culture media, animal density, volume, plate material, and developmental stage in which the embryos are exposed, alter the MTC and LC50 . Finally, we provide a step-by-step guide to assess the interaction between two chemicals using this model. These protocols will guide the standardization of toxicological studies using zebrafish and maximize reproducibility. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Zebrafish embryo collection and culture, and establishment of the MTC and LC50 Basic Protocol 2: Evaluation of the impact of culture conditions on toxicant responses of zebrafish embryo and larvae Basic Protocol 3: Identification of the developmental window of sensitivity to toxicant exposure Basic Protocol 4: Testing interaction between multiple toxicants.


Assuntos
Poluentes Químicos da Água , Peixe-Zebra , Animais , Larva , Dose Letal Mediana , Reprodutibilidade dos Testes
2.
BMC Genomics ; 22(1): 661, 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521337

RESUMO

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics' Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models. However, standard pre-processing, including the alignment and cell filtering pipelines might not be ideal for every organism or tissue. Here we applied an alternative strategy, based on the pseudoaligner kallisto, on twenty-two publicly available single cell sequencing datasets from a wide range of tissues of eight organisms and compared the results with the standard 10X Genomics' Cell Ranger pipeline. RESULTS: In most of the tested samples, kallisto produced higher sequencing read alignment rates and total gene detection rates in comparison to Cell Ranger. Although datasets processed with Cell Ranger had higher cell counts, outside of human and mouse datasets, these additional cells were routinely of low quality, containing low gene detection rates. Thorough downstream analysis of one kallisto processed dataset, obtained from the zebrafish pineal gland, revealed clearer clustering, allowing the identification of an additional photoreceptor cell type that previously went undetected. The finding of the new cluster suggests that the photoreceptive pineal gland is essentially a bi-chromatic tissue containing both green and red cone-like photoreceptors and implies that the alignment and pre-processing pipeline can affect the discovery of biologically-relevant cell types. CONCLUSION: While Cell Ranger favors higher cell numbers, using kallisto results in datasets with higher median gene detection per cell. We could demonstrate that cell type identification was not hampered by the lower cell count, but in fact improved as a result of the high gene detection rate and the more stringent filtering. Depending on the acquired dataset, it can be beneficial to favor high quality cells and accept a lower cell count, leading to an improved classification of cell types.


Assuntos
RNA Citoplasmático Pequeno , Análise de Célula Única , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência de RNA , Software , Peixe-Zebra/genética
3.
Nat Commun ; 12(1): 5263, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489457

RESUMO

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Evolução Molecular , Células HEK293 , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Lenalidomida/química , Lenalidomida/farmacologia , Camundongos , Organoides , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
J Hazard Mater ; 416: 125956, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492873

RESUMO

Exposure to a single organochlorine pesticide (OCP) at high concentration and over a short period of exposure constrain our understanding of the contribution of chemical exposure to type 2 diabetes (T2D). A total of 450 male and female zebrafish was exposed to mixtures of five OCPs at 0, 0.05, 0.25, 2.5, and 25 µg/L for 12 weeks. T2D-related hematological parameters (i.e., glucose, insulin, free fatty acid, and triglycerides) and mitochondrial complex I to IV activities were assessed. Metabolomics, proteomics, and transcriptomics were analyzed in female livers, and their data-driven integration was performed. High fasting glucose and low insulin levels were observed only at 0.05 µg/L of the OCP mixture in females, indicating a nonlinear and sexually dependent response. We found that exposure to the OCP mixture inhibited the activities of mitochondrial complexes, especially III and IV. Combining individual and integrated omics analysis, T2D-linked metabolic pathways that regulate mitochondrial function, insulin signaling, and energy homeostasis were altered by the OCP mixture, which explains the observed phenotypic hematological effects. We demonstrated the cause-and-effect relationship between exposures to OCP mixture and T2D using zebrafish model. This study gives an insight into mechanistic research of metabolic diseases caused by chemical exposure using zebrafish.


Assuntos
Diabetes Mellitus Tipo 2 , Hidrocarbonetos Clorados , Praguicidas , Animais , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/genética , Feminino , Insulina , Masculino , Praguicidas/análise , Praguicidas/toxicidade , Peixe-Zebra
5.
J Hazard Mater ; 416: 125969, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492880

RESUMO

In the present study the effects of sublethal concentrations of polystyrene microplastics (PS-MPs) on zebrafish were evaluated at multiple levels, related to fish activity and oxidative stress, metabolic changes and contraction parameters in the heart tissue. Zebrafish were fed for 21 days food enriched with PS-MPs (particle sizes 3-12 µm) and a battery of stress indices like DNA damage, lipid peroxidation, autophagy, ubiquitin levels, caspases activation, metabolite adjustments, frequency and force of ventricular contraction were measured in fish heart, parallel to fish swimming velocity. In particular, exposure to PS-MPs caused significant decrease in heart function and swimming competence, while enhanced levels of oxidative stress indices and metabolic adjustments were observed in the heart of challenged species. Among stress indices, DNA damage was more vulnerable to the effect of PS-MPs. Our results provide evidence on the multiplicity of the PS-MPs effects on cellular function, physiology and metabolic pathways and heart rate of adult fish and subsequent effects on fish activity and fish fitness thus enlightening MPs characterization as a potent environmental pollutant.


Assuntos
Microplásticos , Poluentes Químicos da Água , Animais , Estresse Oxidativo , Plásticos , Poliestirenos/metabolismo , Poliestirenos/toxicidade , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo
6.
J Hazard Mater ; 416: 126142, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492931

RESUMO

2-Methylisoborneol (2-MIB), a natural odorous substance, is widely distributed in water environment, but there is a paucity of information concerning its systemic toxicity. Herein, we investigated the effects of 2-MIB exposure on developmental parameters, locomotive behavior, oxidative stress, apoptosis and transcriptome of zebrafish. Zebrafish embryos exposed to different concentrations (0, 0.5, 5 and 42.8 µg/L) of 2-MIB showed no changes in mortality, hatchability, and malformation rate, but the body length of zebrafish larvae was significantly increased in a dose-dependent manner, and accompanied by the changes of growth hormone/insulin-like growth factor (GH/IGF) axis and the hypothalamic-pituitary-thyroid (HPT) axis genes. Moreover, the swimming activity of zebrafish larvae increased, which may be due to the increase of acetylcholinesterase (AChE) activity. Meanwhile, 2-MIB caused oxidative stress and apoptosis in zebrafish larvae by altering the NF-E2-related factor 2 (Nrf2) and mitochondrial signaling pathways, respectively. Transcriptome sequencing assay showed that the phototransduction signaling pathway was significantly enriched, and most of the genes in this pathway exhibited enhanced expression after exposure to 2-MIB. These findings provide an important reference for risk assessment and early warning to 2-MIB exposure.


Assuntos
Poluentes Químicos da Água , Peixe-Zebra , Animais , Embrião não Mamífero , Larva/genética , Transcriptoma , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética
7.
BMC Res Notes ; 14(1): 359, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526111

RESUMO

OBJECTIVE: Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). RESULTS: The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.


Assuntos
Colágeno Tipo XI , Peixe-Zebra , Animais , Anticorpos , Proteínas da Matriz Extracelular , Peptídeos , Coelhos
8.
Nanoscale ; 13(34): 14297-14303, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34473172

RESUMO

The application of perovskite quantum dots (PQDs) in biomedical fields such as bioimaging and biosensing has been limited owing to their instability in the physiological environment. Herein, PQDs are innovatively encapsulated into nano-micelles composed of a polyethylene glycol-polycaprolactone (PEG-PCL) block copolymer, which allows the preparation of biocompatible PQDs (bio-PQDs) with excellent water resistance. Due to the benefits of extraordinary water resistance and biocompatibility, these bio-PQDs are capable of real-time and long-term quantitatively monitoring the H2S level in living cells as well as in zebrafish.


Assuntos
Pontos Quânticos , Animais , Compostos de Cálcio , Óxidos , Pontos Quânticos/toxicidade , Titânio , Água , Peixe-Zebra
9.
BMC Genomics ; 22(1): 658, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517816

RESUMO

BACKGROUND: Zebrafish is a popular animal model used for high-throughput screening of chemical hazards, however, investigations of transcriptomic mechanisms of toxicity are still needed. Here, our goal was to identify genes and biological pathways that Aryl Hydrocarbon Receptor 2 (AHR2) Activators and flame retardant chemicals (FRCs) alter in developing zebrafish. Taking advantage of a compendium of phenotypically-anchored RNA sequencing data collected from 48-h post fertilization (hpf) zebrafish, we inferred a co-expression network that grouped genes based on their transcriptional response. RESULTS: Genes responding to the FRCs and AHR2 Activators localized to distinct regions of the network, with FRCs inducing a broader response related to neurobehavior. AHR2 Activators centered in one region related to chemical stress responses. We also discovered several highly co-expressed genes in this module, including cyp1a, and we subsequently show that these genes are definitively within the AHR2 signaling pathway. Systematic removal of the two chemical types from the data, and analysis of network changes identified neurogenesis associated with FRCs, and regulation of vascular development associated with both chemical classes. We also identified highly connected genes responding specifically to each class that are potential biomarkers of exposure. CONCLUSIONS: Overall, we created the first zebrafish chemical-specific gene co-expression network illuminating how chemicals alter the transcriptome relative to each other. In addition to our conclusions regarding FRCs and AHR2 Activators, our network can be leveraged by other studies investigating chemical mechanisms of toxicity.


Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transcriptoma , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
10.
Analyst ; 146(18): 5517-5527, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34515714

RESUMO

Reactive sulfur species (RSS) play pivotal roles in various pathological and physiological processes. There exists an intricate relevance in generation and metabolism among these substances. Although they are nucleophilic, there are still some differences in their reactivity. There are many methods to detect them by using reactive fluorescent probes, but the systematic study of their reactivity is still lacking. In our study, we designed a multiple reaction site fluorescent probe based on benzene conjugated benzopyrylium and NBD. The study revealed that besides both biothiols and hydrogen sulfide, sulfur dioxide (SO2) can cleave the ether bond. There are two reaction forms for GSH with low reactivity: cutting the ether bond and adding the conjugated double bond of benzopyrylium. Nevertheless, Cys/Hcy with higher activity can further rearrange with NBD after cutting the ether bond. In addition, SO2 can not only cleave the ether bond, but also continue to add the conjugated double bond of benzopyrylium. The above processes lead to multicolor emission of the probe, thus realizing the characteristic analysis of different sulfides. Thus the probe can be used for the detection of sulfide in mitochondria, and further for the imaging of sulfide in cells and zebrafish. This effective analysis method will provide a broad application prospect for practical applications.


Assuntos
Cisteína , Sulfeto de Hidrogênio , Animais , Corantes Fluorescentes , Glutationa , Células HeLa , Homocisteína , Humanos , Sulfetos , Peixe-Zebra
11.
Analyst ; 146(17): 5264-5270, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34337624

RESUMO

Peroxynitrite (ONOO-) is a highly reactive substance, and plays an essential part in maintaining cellular homeostasis. It is crucial to monitor the ONOO- level in cells in normal and abnormal states. We introduced a p-dimethylaminophenylether-based fluorescent probe PDPE-PN, which could be synthesized readily. The new probe had prominent sensitivity and specificity, and a fast response towards ONOO-. The spectral performance of probe PDPE-PN was outstanding and the limit of detection was 69 nM. Probe PDPE-PN with low toxicity was applied to detect endogenous/exogenous ONOO- in RAW 264.7 macrophages and zebrafish. Importantly, successful application of the new receptor opens up new ideas for the design of ONOO- probes.


Assuntos
Corantes Fluorescentes , Peixe-Zebra , Animais , Corantes Fluorescentes/toxicidade , Macrófagos , Ácido Peroxinitroso/toxicidade
12.
Chemosphere ; 282: 131124, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34374342

RESUMO

Environmental exposure to nanoplastics is inevitable as the application of nanoplastics in our daily life is more and more extensively. So, the adverse effects of nanoplastics on human health are also gaining greater concerns. However, the subsequent toxicological response to nanoplastics, especially on cardiovascular damage was still largely unknown. In this regard, the evaluation of cardiovascular effects of nanoplastics was performed in zebrafish embryos. The results indicated that the no observed adverse effect level (NOAEL) of nanoplastics is 50 µg/mL. The pericardial toxicity and hemodynamic changes were assessed by Albino (melanin allele) mutant zebrafish line. Severe pericardial edema was observed in zebrafish embryos after exposure to nanoplastics. At the concentration higher than NOAEL, nanoplastics significantly decreased the cardiac output (CO) and blood flow velocity. The fluorescence images manifested that the nanoplastics could inhibit the subintestinal angiogenesis of transgenic zebrafish embryos line Tg (fli-1: EGFP), which might disturb the cardiovascular formation and development. The resulting vascular endothelial dysfunction and hypercoagulable state of circulating blood further accelerated thrombosis. Reactive oxidative stress (ROS) and systemic inflammation were also found in Wild AB and Tg (mpo: GFP) zebrafish embryos, respectively. We also found many neutrophils recruiting in the tail vein where the zebrafish embryo thrombosis occurred. Our data suggested that nanoplastics could trigger the cardiovascular toxicity in zebrafish embryos, which could provide an essential clue for the safety assessment of nanoplastics.


Assuntos
Nanopartículas , Peixe-Zebra , Animais , Embrião não Mamífero , Humanos , Microplásticos , Pericárdio , Polietileno
13.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445067

RESUMO

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left-right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.


Assuntos
Cílios/patologia , Transtornos da Motilidade Ciliar/patologia , Peixe-Zebra , Animais , Transtornos da Motilidade Ciliar/fisiopatologia , Modelos Animais de Doenças , Humanos , Microscopia Eletrônica de Transmissão , Peixe-Zebra/fisiologia
14.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445098

RESUMO

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed "Gran1"). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/química , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Peptídeos/química , Peptídeos/imunologia , Tuberculose/microbiologia , Peixe-Zebra
15.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445620

RESUMO

The clinical success of cisplatin, carboplatin, and oxaliplatin has sparked the interest of medicinal inorganic chemistry to synthesize and study compounds with non-platinum metal centers. Despite Ru(II)-polypyridyl complexes being widely studied and well established for their antitumor properties, there are not enough in vivo studies to establish the potentiality of this type of compound. Therefore, we report to the best of our knowledge the first in vivo study of Ru(II)-polypyridyl complexes against breast cancer with promising results. In order to conduct our study, we used MCF7 zebrafish xenografts and ruthenium complexes [Ru(bipy)2(C12H8N6-N,N)][CF3SO3]2Ru1 and [{Ru(bipy)2}2(µ-C12H8N6-N,N)][CF3SO3]4Ru2, which were recently developed by our group. Ru1 and Ru2 reduced the tumor size by an average of 30% without causing significant signs of lethality when administered at low doses of 1.25 mg·L-1. Moreover, the in vitro selectivity results were confirmed in vivo against MCF7 breast cancer cells. Surprisingly, this work suggests that both the mono- and the dinuclear Ru(II)-polypyridyl compounds have in vivo potential against breast cancer, since there were no significant differences between both treatments, highlighting Ru1 and Ru2 as promising chemotherapy agents in breast cancer therapy.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos de Rutênio/química , Compostos de Rutênio/farmacologia , Animais , Apoptose , Neoplasias da Mama/patologia , Proliferação de Células , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Técnicas In Vitro , Estrutura Molecular , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
16.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445647

RESUMO

Unveiling the molecular features in the heart is essential for the study of heart diseases. Non-cardiomyocytes (nonCMs) play critical roles in providing structural and mechanical support to the working myocardium. There is an increasing amount of single-cell RNA-sequencing (scRNA-seq) data characterizing the transcriptomic profiles of nonCM cells. However, no tool allows researchers to easily access the information. Thus, in this study, we develop an open-access web portal, ExpressHeart, to visualize scRNA-seq data of nonCMs from five laboratories encompassing three species. ExpressHeart enables comprehensive visualization of major cell types and subtypes in each study; visualizes gene expression in each cell type/subtype in various ways; and facilitates identifying cell-type-specific and species-specific marker genes. ExpressHeart also provides an interface to directly combine information across datasets, for example, generating lists of high confidence DEGs by taking the intersection across different datasets. Moreover, ExpressHeart performs comparisons across datasets. We show that some homolog genes (e.g., Mmp14 in mice and mmp14b in zebrafish) are expressed in different cell types between mice and zebrafish, suggesting different functions across species. We expect ExpressHeart to serve as a valuable portal for investigators, shedding light on the roles of genes on heart development in nonCM cells.


Assuntos
Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Ventrículos do Coração/metabolismo , Internet , Macrófagos/metabolismo , Pericitos/metabolismo , Transcriptoma , Algoritmos , Animais , Perfilação da Expressão Gênica , Humanos , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Software , Peixe-Zebra
17.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445435

RESUMO

Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.


Assuntos
Cálcio/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/química , Ferro/metabolismo , Proteínas de Peixe-Zebra/química , Peixe-Zebra/metabolismo , Animais , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Transdução de Sinal Luminoso , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteínas de Peixe-Zebra/metabolismo
18.
Talanta ; 234: 122621, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364430

RESUMO

Altered H2S levels and intracellular viscosity have both been seen in Parkinson's disease (PD). However, how H2S and intracellular viscosity are involved in PD pathogenesis remains unknown. Herein, a dual-function fluorescent probe DF was designed and synthesized to analyze intracellular viscosity and hydrogen sulfide. It is a near-infrared fluorescence probe with improved photostability and large Stokes shift (110 nm). The probe reveals increased viscosity and hydrogen sulfide in zebrafish model of PD for the first time.


Assuntos
Sulfeto de Hidrogênio , Doença de Parkinson , Animais , Corantes Fluorescentes , Células HeLa , Humanos , Viscosidade , Peixe-Zebra
19.
Talanta ; 234: 122688, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364485

RESUMO

Untargeted metabolomics has been widely used for studies with zebrafish embryos. Until now, the number of analytical approaches to determine metabolites in zebrafish is limited, and there is a lack of consensus on the best platforms for comprehensive metabolomics analysis of zebrafish embryos. In addition, the capacity of these methods to detect metabolites is unsatisfactory and the confidence level for identifying compounds is relatively low. To improve the metabolome coverage, we mainly focused on the optimization of separation mechanisms, mobile phase additives, and resuspension solvents based on liquid chromatography (LC) coupling to high-resolution mass spectrometry (HRMS) techniques. Moreover, the procedures for optimizing methods were assessed when taking metabolite profiles in both positive and negative ionization modes into account. Four LC columns were studied: C18, T3, PFP, and HILIC. In positive ionization mode, it was strongly recommended to employ the HILIC approach operated at the neutral condition, which led to the presence of more than 4700 features and the annotation of 151 metabolites, mainly zwitterionic and basic compounds, in comparison to reverse phase (RP)-based methods with less than 1000 features. In negative ionization mode, the PFP column operated at 0.02% acetic acid showed the best performance in terms of metabolite coverage: 3100 metabolic features were detected and 218 metabolites were annotated in zebrafish embryos. Metabolite profiles mainly contained acidic and zwitterionic compounds. HILIC-based platforms were complementary to RP columns when analyzing highly polar metabolites. Additionally, it was preferable to reconstitute zebrafish extracts in 100% water for analysis of metabolites on RP columns, with a 20-30% increase in the number of identified metabolites compared to a 50% water in methanol solution. However, water/methanol (1:9, v/v), as resuspension solution, was advantageous over water/methanol (1:1, v/v) for HILIC analysis showing an 8-15% increase in detected metabolites. In total 336 polar metabolites were annotated by the combination of the optimized HILIC (positive) and PFP (negative) approaches. The largest metabolome coverage of polar metabolites in zebrafish embryos was obtained when three approaches were combined (negative PFP and HILIC, and HILIC positive) resulting in more than 420 annotated compounds.


Assuntos
Metaboloma , Peixe-Zebra , Animais , Cromatografia Líquida , Metabolômica , Solventes
20.
ACS Appl Mater Interfaces ; 13(33): 39088-39099, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433242

RESUMO

In this work, we demonstrated that building different linking groups between nanodiamond (ND) and TiO2 (P25) could provide more effective protection under oxidative stress and ultraviolet (UV) light irradiation compared with the use of TiO2 alone. The establishment of ester (-C-O-O-R), amide (-CONH-), and epoxide-amine adduct (-NHCCO-) groups between ND-TiO2 composites was found to be critical in the generation of reactive oxygen species (ROS) by controlling their charge transfer behaviors. We hypothesized that linking groups between the composites dictate the performance of ROS generation from nano-TiO2 under UV-light irradiation due to the differences in linking groups. The results showed that hydroxyl radicals were attenuated by the incorporation of ND. An MTT cell proliferation assay was performed in human cells under the treatment of ND-TiO2 composites to investigate the impacts of composites on cell viability. The results from the luciferase reporter assay suggested they have anti-inflammatory activity and can reduce cellular DNA damage under ROS stimulation. A zebrafish model was also applied with the ND-TiO2 composite treatment to demonstrate the safety aspects of the composites in vivo and their biomedical application potential. Studies exploring ROS generation behaviors in different linking groups suggested that interactive functionalization between nanoparticles might be an ideal antioxidant and anti-inflammatory strategy.


Assuntos
Anti-Inflamatórios/química , Sequestradores de Radicais Livres/química , Nanocompostos/química , Nanodiamantes/química , Titânio/química , Amidas/química , Animais , Anti-Inflamatórios/farmacologia , Carbodi-Imidas/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Esterificação , Sequestradores de Radicais Livres/farmacologia , Células HEK293 , Humanos , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Propriedades de Superfície , Nanomedicina Teranóstica , Raios Ultravioleta , Peixe-Zebra
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