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Calcium imaging has emerged as a powerful tool for studying cellular dynamics, with applications spanning neuroscience, cell biology, and beyond. In this chapter, we present a comprehensive guide to the computational analysis of calcium flux data using the R programming language. Using an example of in vivo live imaging of GCaMP signal in zebrafish hepatocytes, we demonstrate techniques for segmentation, normalization, and quantification of calcium transients. We provide a step-by-step code example showcasing extraction of meaningful information from calcium imaging datasets. The code allows insights into the number of oscillating cells, number of oscillations per cell within a time frame, and generation of publication-ready plots for showcasing calcium dynamics. This chapter serves as a valuable resource for researchers seeking to leverage freely available computational tools for analyzing calcium flux data at cellular resolution and uncovering novel insights into cellular physiology.
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Señalización del Calcio , Calcio , Biología Computacional , Programas Informáticos , Pez Cebra , Animales , Calcio/metabolismo , Pez Cebra/metabolismo , Biología Computacional/métodos , Hepatocitos/metabolismo , Lenguajes de Programación , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Wound closure after brain injury is crucial for tissue restoration but remains poorly understood at the tissue level. We investigated this process using in vivo observations of larval zebrafish brain injury. Our findings show that wound closure occurs within the first 24 h through global tissue contraction, as evidenced by live-imaging and drug inhibition studies. Microglia accumulate at the wound site before closure, and computational models suggest that their physical traction could drive this process. Depleting microglia genetically or pharmacologically impairs tissue repair. At the cellular level, live imaging reveals centripetal deformation of astrocytic processes contacted by migrating microglia. Laser severing of these contacts causes rapid retraction of microglial processes and slower retraction of astrocytic processes, indicating tension. Disrupting the lcp1 gene, which encodes the F-actin-stabilising protein L-plastin, in microglia results in failed wound closure. These findings support a mechanical role of microglia in wound contraction and suggest that targeting microglial mechanics could offer new strategies for treating traumatic brain injury.
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Lesiones Encefálicas , Larva , Microglía , Cicatrización de Heridas , Pez Cebra , Animales , Microglía/metabolismo , Cicatrización de Heridas/fisiología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/etiología , Lesiones Encefálicas/patología , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Movimiento Celular , Encéfalo/metabolismo , Glicoproteínas de MembranaRESUMEN
Photocatalysts are one of the effective methods to degrade antibiotic contamination, but the efficiency is low and the toxicity is not well recognized. Deep lattice doping to induce defect generation is an effective way to improve the performance of photocatalysts. Here, defect-rich bromine-doped BiOCl-XBr photocatalysts were constructed with the help of small molecules inserted into the interlayer. The photocatalytic degradation performance of BiOCl-XBr was significantly enhanced, and its degradation rate was up to about 12 times that of BiOCl monomer. The main reasons for the stronger photocatalytic performance of BiOCl-XBr include Br doping to enhance visible light absorption, surface defects, and Bi valence changes to improve charge transport. The degradation of tetracycline (TC) produced more toxic intermediates, and the biotoxicity experiments also confirmed that the toxicity showed a trend of increasing and then decreasing, indicating that the more toxic intermediates were also mineralized during the degradation process. However, the mortality and hatching rate of zebrafish in the exposed group after degradation recovered but changed their activity pattern under light and dark conditions. This further warns us to focus on the toxicity changes after antibiotic degradation. Finally, based on the free radical analysis, the mechanism of photocatalytic degradation and detoxification of TC by BiOCl-XBr was proposed.
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Antibacterianos , Bismuto , Bromo , Tetraciclina , Pez Cebra , Tetraciclina/química , Tetraciclina/farmacología , Bismuto/química , Animales , Catálisis , Antibacterianos/química , Antibacterianos/farmacología , Bromo/química , Procesos Fotoquímicos , Luz , Fotólisis , Propiedades de SuperficieRESUMEN
Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.
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Interacciones Huésped-Patógeno , Infecciones por Klebsiella , Klebsiella pneumoniae , Larva , Microinyecciones , Microscopía Fluorescente , Pez Cebra , Animales , Pez Cebra/microbiología , Klebsiella pneumoniae/inmunología , Microinyecciones/métodos , Larva/microbiología , Larva/inmunología , Microscopía Fluorescente/métodos , Interacciones Huésped-Patógeno/inmunología , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/inmunología , Modelos Animales de EnfermedadRESUMEN
Recent technological advances in single-cell RNA sequencing (scRNA-Seq) have enabled scientists to answer novel questions in biology with unparalleled precision. Indeed, in the field of ocular development and regeneration, scRNA-Seq studies have resulted in a number of exciting discoveries that have begun to revolutionize the way we think about these processes. Despite the widespread success of scRNA-Seq, many scientists are wary to perform scRNA-Seq experiments due to the uncertainty of obtaining high-quality viable cell populations that are necessary for the generation of usable data that enable rigorous computational analyses. Here, we describe methodology to reproducibility generate high-quality single-cell suspensions from embryonic zebrafish eyes. These single-cell suspensions served as inputs to the 10× Genomics v3.1 system and yielded high-quality scRNA-Seq data in proof-of-principle studies. In describing methodology to quantitatively assess cell yields, cell viability, and other critical quality control parameters, this protocol can serve as a useful starting point for others in designing their scRNA-Seq experiments in the zebrafish eye and in other developing or regenerating tissues in zebrafish or other model systems.
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Retina , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/embriología , Análisis de la Célula Individual/métodos , Retina/citología , Retina/embriología , Retina/metabolismo , Análisis de Secuencia de ARN/métodos , Separación Celular/métodosRESUMEN
Zebrafish maintain a remarkable ability to regenerate their neural retina following rapid and extensive loss of retinal neurons. This is mediated by Müller glial cells (MG), which re-enter the cell cycle to produce amplifying progenitor cells that eventually differentiate into the lost retinal neurons. For example, exposing adult albino zebrafish to intense light destroys large numbers of rod and cone photoreceptors, which are then restored by MG-mediated regeneration. Here, we describe an updated method for performing these acute phototoxic lesions to adult zebrafish retinas. Next, we contrast this method to a chronic, low light lesion model that results in a more muted and sustained damage to photoreceptors and does not trigger a MG-mediated regeneration response. Thus, these two methods can be used to compare and contrast the genetic and morphological changes associated with acute and chronic methods of photoreceptor degeneration.
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Modelos Animales de Enfermedad , Degeneración Retiniana , Pez Cebra , Animales , Degeneración Retiniana/patología , Degeneración Retiniana/genética , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Luz , Células Fotorreceptoras de Vertebrados/patología , Retina/patología , Retina/metabolismoRESUMEN
Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.
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Pez Cebra , Animales , Retina/citología , Retina/metabolismo , Fenotipo , Proliferación Celular , Regeneración , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Células Madre/citología , Células Madre/metabolismo , Cinética , Regeneración Nerviosa/fisiologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of Qi and blood in Traditional Chinese Medicine (TCM), the combination of Qi-reinforcing herbs and blood-activating herbs has a synergistic effect in improving blood stasis syndrome, especially in tumor treatment. The classic "Radix Astragali - Salvia miltiorrhiza" duo exemplifies this principle, renowned for invigorating Qi and activating blood flow, employed widely in tumor therapies. Our prior research underscores the potent inhibition of pancreatic tumor xenografts by the combination of Formononetin (from Radix Astragali) and Salvianolic acid B (from Salvia miltiorrhiza) in vitro. However, it remains unclear whether this combination can inhibit the abnormal vascularization of pancreatic tumors to achieve its anti-cancer effect. AIM OF THE STUDY: Abnormal vasculature, known to facilitate tumor growth and metastasis. Strategies to normalize tumor-associated blood vessels provide a promising avenue for anti-tumor therapy. This study aimed to unravel the therapeutic potential of Formononetin combined with Salvianolic acid B (FcS) in modulating pancreatic cancer's impact on endothelial cells, illuminate the underlying mechanisms that govern this therapeutic interaction, thereby advancing strategies to normalize tumor vasculature and combat cancer progression. MATERIALS AND METHODS: A co-culture system involving Human Umbilical Vein Endothelial Cells (HUVECs) and PANC-1 cells was established to investigate the potential of targeting abnormal vasculature as a novel anti-tumor therapeutic strategy. We systematically compared HUVEC proliferation, migration, invasion, and lumenogenesis in both mono- and co-culture conditions with PANC-1 (H-P). Subsequently, FcS treatment of the H-P system was evaluated for its anti-angiogenic properties. Molecular docking was utilized to predict the interactions between Formononetin and Salvianolic acid B with RhoA, and the post-treatment expression of RhoA in HUVECs was assessed. Furthermore, we utilized shRhoA lentivirus to elucidate the role of RhoA in FcS-mediated effects on HUVECs. In vivo, a zebrafish xenograft tumor model was employed to assess FcS's anti-tumor potential, focusing on cancer cell proliferation, migration, apoptosis, and vascular development. RESULTS: FcS treatment demonstrated a significant, dose-dependent inhibition of PANC-1-induced alterations in HUVECs, including proliferation, migration, invasion, and tube formation capabilities. Molecular docking analyses indicated potential interactions between FcS and RhoA. Further, FcS treatment was found to downregulate RhoA expression and modulated the PI3K/AKT signaling pathway in PANC-1-induced HUVECs. Notably, the phenotypic inhibitory effects of FcS on HUVECs were attenuated by RhoA knockdown. In vivo zebrafish studies validated FcS's anti-tumor activity, inhibiting cancer cell proliferation, metastasis, and vascular sprouting, while promoting tumor cell apoptosis. CONCLUSIONS: This study underscores the promising potential of FcS in countering pancreatic cancer-induced endothelial alterations. FcS exhibits pronounced anti-abnormal vasculature effects, potentially achieved through downregulation of RhoA and inhibition of the PI3K/Akt signaling pathway, thereby presenting a novel therapeutic avenue for pancreatic cancer management.
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Benzofuranos , Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana , Isoflavonas , Neoplasias Pancreáticas , Proteína de Unión al GTP rhoA , Isoflavonas/farmacología , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Animales , Benzofuranos/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Pez Cebra , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Antineoplásicos Fitogénicos/farmacología , DepsidosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver-related morbidity and mortality, with hepatic steatosis being the hallmark symptom. Salvia miltiorrhiza Bunge (Smil, Dan-Shen) and Ligusticum striatum DC (Lstr, Chuan-Xiong) are commonly used to treat cardiovascular diseases and have the potential to regulate lipid metabolism. However, whether Smil/Lstr combo can be used to treat NAFLD and the mechanisms underlying its lipid-regulating properties remain unclear. PURPOSE: To assess the feasibility and reliability of a short-term high-fat diet (HFD) induced zebrafish model for evaluating hepatic steatosis phenotype and to investigate the liver lipid-lowering effects of Smil/Lstr, as well as its active components. METHODS: The phenotypic alterations of liver and multiple other organ systems were examined in the HFD zebrafish model using fluorescence imaging and histochemistry. The liver-specific lipid-lowering effects of Smil/Lstr combo were evaluated endogenously. The active molecules and functional mechanisms were further explored in zebrafish, human hepatocytes, and hamster models. RESULTS: In 5-day HFD zebrafish, significant lipid accumulation was detected in the blood vessels and the liver, as evidenced by increased staining with Oil Red O and fluorescent lipid probes. Hepatic hypertrophy was observed in the model, along with macrovesicular steatosis. Smil/Lstr combo administration effectively restored the lipid profile and alleviated hepatic hypertrophy in the HFD zebrafish. In oleic-acid stimulated hepatocytes, Smil/Lstr combo markedly reduced lipid accumulation and cell damage. Subsequently, based on zebrafish phenotypic screening, the natural phthalide senkyunolide I (SEI) was identified as a major molecule mediating the lipid-lowering activities of Smil/Lstr combo in the liver. Moreover, SEI upregulated the expression of the lipid metabolism regulator PPARα and downregulated fatty acid translocase CD36, while a PPARα antagonist sufficiently blocked the regulatory effect of SEI on hepatic steatosis. Finally, the roles of SEI on hepatic lipid accumulation and PPARα signaling were further verified in the hamster model. CONCLUSIONS: We proposed a zebrafish-based screening strategy for modulators of hepatic steatosis and discovered the regulatory roles of Smil/Lstr combo and its component SEI on liver lipid accumulation and PPARα signaling, suggesting their potential value as novel candidates for NAFLD treatment.
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PPAR alfa , Transducción de Señal , Pez Cebra , Animales , Cricetinae , Humanos , Masculino , Benzofuranos/farmacología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hígado Graso/tratamiento farmacológico , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Mesocricetus , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , PPAR alfa/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.
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Cisteína , Colorantes Fluorescentes , Análisis de los Alimentos , Glutatión , Lisosomas , Espectrometría de Fluorescencia , Pez Cebra , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Lisosomas/química , Lisosomas/metabolismo , Células HeLa , Cisteína/análisis , Animales , Análisis de los Alimentos/métodos , Glutatión/análisis , Espectrometría de Fluorescencia/métodos , Homocisteína/análisis , Arabidopsis/química , Límite de Detección , Microscopía ConfocalRESUMEN
Epilepsy is one of the most commonly-seen neurological disorders, and both endoplasmic reticulum stress (ERS) and oxidative stress (OS) have been demonstrated to be associated with epileptic seizures. As one of the three endogenous thiol-containing amino acids, cysteine (Cys) is recognized not only as an important biomarker of various biological processes but also widely used as a significant additive in the food industry. However, the exact role that Cys plays in ERS has not been well answered up to now. In this paper, we reported the first flavone-based fluorescent probe (namely BFC) with nice endoplasmic reticulum (ER)-targeting ability, which was capable of monitoring Cys in a fast response (3.0 min), large stokes shift (130 nm) and low detection limit (10.4 nM). The recognition mechanism of Cys could be attributed to the addition-cyclization reaction involving a Cys residue and an acrylate group, resulting in the release of the strong excited-state intramolecular proton transfer (ESIPT) emission molecule of benzoflavonol (BF). The low cytotoxicity and good biocompatibility of the probe BFC allowed for monitoring the fluctuation of endogenous Cys levels under both ERS and OS processes, as well as in zebrafish models of epilepsy. Quantitative determination of Cys with the probe BFC was also achieved in three different food samples. Additionally, a probe-immersed test strips integrated with a smartphone device was successfully constructed for on-site colorimetric detection of Cys. Undoubtedly, our work provided a valuable tool for tracking Cys levels in both an epilepsy model and real food samples.
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Cisteína , Retículo Endoplásmico , Epilepsia , Flavonas , Colorantes Fluorescentes , Análisis de los Alimentos , Pez Cebra , Colorantes Fluorescentes/química , Cisteína/análisis , Animales , Epilepsia/diagnóstico , Flavonas/análisis , Flavonas/química , Retículo Endoplásmico/metabolismo , Análisis de los Alimentos/métodos , Espectrometría de Fluorescencia/métodos , Humanos , Modelos Animales de Enfermedad , Límite de Detección , Estrés del Retículo EndoplásmicoRESUMEN
The role of ClO- in the physiological functioning of organisms is significant. In this paper, the four fluorescent probes HONx (HON1, HON2, HON3 and HON4) were prepared based on oxyanthracene through the introduction of different substituents, and their photophysical properties were investigated, among which the AIE effect of HON1 was the most significant, and therefore the fluorescent "turn-off" ClO- probe HON1-CN was chosen to be prepared by constructing the ClO- recognition site hydrazone bond at HON1. The ClO- recognises the hydrazone group in the probe HON1-CN, and when the hydrazone bond is broken, the aldehyde group is released, generating HON1 with yellow fluorescence. The probe HON1-CN is highly selective and stable for the detection of ClO- with a detection limit of 0.48 µM and a more than 10-fold increase in fluorescence intensity when the fluorescence is 'switched on', and to a lesser extent, the probe is also very good for the detection of hypochlorite ClO- in the pericarp. Finally, HON1-CN has also been used to detect the presence of ClO- in HeLa cells and zebrafish.
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Colorantes Fluorescentes , Ácido Hipocloroso , Espectrometría de Fluorescencia , Xantonas , Pez Cebra , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Xantonas/química , Animales , Ácido Hipocloroso/análisis , Humanos , Células HeLa , Frutas/química , Límite de DetecciónRESUMEN
Mercury ion (Hg2+), a heavy metal cation with greater toxicity, is widely present in the ecological environment and has become a serious threat to human health and environmental safety. Currently, developing a solution to simultaneously visualize and monitor Hg2+ in environmental samples, including water, soil, and plants, remains a great challenge. In this work, we created and synthesized a near-infrared fluorescent probe, BBN-Hg, and utilized Hg2+ to trigger the partial cleavage of the carbon sulfate ester in BBN-Hg as a sensing mechanism, and the fluorescence intensity of BBN-Hg was significantly enhanced at 650 nm, thus realizing the visualization of Hg2+ with good selectivity (detection limit, 53 nM). In live cells and zebrafish, the probe BBN-Hg enhances the red fluorescence signal in the presence of Hg2+, and successfully performs 3D imaging on zebrafish, making it a powerful tool for detecting Hg2+ in living systems. More importantly, with BBN-Hg, we are able to detect Hg2+ in actual water samples, soil and plant seedling roots. Furthermore, the probe was prepared as a test strip for on-site determination of Hg2+ with the assistance of a smartphone. Therefore, this study offers an easy-to-use and useful method for tracking Hg2+ levels in living organisms and their surroundings.
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Colorantes Fluorescentes , Mercurio , Pez Cebra , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Mercurio/análisis , Animales , Humanos , Espectrometría de Fluorescencia/métodos , Límite de DetecciónRESUMEN
Fluoride ions (F-) are one of the essential trace elements for the human body and are widely existed in nature. In this study, we present a novel fluorescent probe (YF-SZ-F) designed and synthesized for the specific detection of F-. The probe exhibits high sensitivity, excellent selectivity, and low cytotoxicity, making it a promising tool for biomedical applications. Imaging experiments conducted on zebrafish and Arabidopsis roots demonstrate the probe's remarkable cellular permeability and biocompatibility, laying a solid foundation for its potential biomedical utility. Furthermore, the probe holds potential for practical applications in environmental monitoring and public health through its capability to detect fluoride ions in water samples and via mobile phone software. This multifaceted functionality underscores the broad applicability and significance of the fluorescent probe, not only in scientific research but also in real-world scenarios, contributing to the development of more convenient and precise methods for fluoride detection.
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Benzotiazoles , Colorantes Fluorescentes , Fluoruros , Pez Cebra , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Fluoruros/análisis , Animales , Benzotiazoles/química , Humanos , Arabidopsis/química , Espectrometría de Fluorescencia/métodos , Imagen ÓpticaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Mountain-cultivated Panax ginseng C.A.Mey. (MCG) with high market price and various properties was valuable special local product in Northeast of Asia. MCG has been historically used to mitigate heart failure (HF) for thousand years, HF is a clinical manifestation of deficiency of "heart-qi" in traditional Chinese medicine. However, there was little report focus on the activities of extracted residue of MCG. AIM OF THE STUDY: A novel glycopeptide (APMCG-1) was isolated from step ethanol precipitations of alkaline protease-assisted extract from MCG residue. MATERIALS AND METHODS: The molecular weight and subunit structure of APMCG-1 were determined by FT-IR, HPLC and GPC technologies, as well as the H9c2 cells, Tg (kdrl:EGFP) zebrafish were performed to evaluated the protective effect of APMCG-1. RESULTS: APMCG-1 was identified as a glycopeptide containing seven monosaccharides and seven amino acids via O-lined bonds. Further, in vitro, APMCG-1 significantly decreased reactive oxygen species production and lactate dehydrogenase contents in palmitic acid (PA)-induced H9c2 cells. APMCG-1 also attenuated endoplasmic reticulum stress and mitochondria-mediated apoptosis in H9c2 cells via the PI3K/AKT signaling pathway. More importantly, APMCG-1 reduced the blood glucose, lipid contents, the levels of heart injury, oxidative stress and inflammation of 5 days post fertilization Tg (kdrl:EGFP) zebrafish with type 2 diabetic symptoms in vivo. CONCLUSIONS: APMCG-1 protects PA-induced H9c2 cells while reducing cardiac dysfunction in zebrafish with type 2 diabetic symptoms. The present study provides a new insight into the development of natural glycopeptides as heart-related drug therapies.
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Diabetes Mellitus Tipo 2 , Glicopéptidos , Insuficiencia Cardíaca , Panax , Pez Cebra , Animales , Panax/química , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/prevención & control , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ratas , Línea Celular , Glicopéptidos/farmacología , Glicopéptidos/química , Apoptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/química , Cardiotónicos/farmacología , Cardiotónicos/química , Cardiotónicos/aislamiento & purificación , Cardiotónicos/uso terapéutico , Miocitos Cardíacos/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. AIM OF THE STUDY: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. MATERIALS AND METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 µg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 µg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.
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Boraginaceae , Extractos Vegetales , Cicatrización de Heridas , Pez Cebra , Cicatrización de Heridas/efectos de los fármacos , Animales , Extractos Vegetales/farmacología , Humanos , Boraginaceae/química , Bioensayo , Línea Celular , Queratinocitos/efectos de los fármacos , Sudáfrica , Células HaCaT , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacosRESUMEN
Immunosuppressive tumor microenvironment (ITM) severely limited the efficacy of immunotherapy against triple-negative breast cancer (TNBC). Herein, Apt-LPR, a light-activatable photodynamic therapy (PDT)/RNAi immune synergy-enhancer was constructed by co-loading miR-34a and photosensitizers in cationic liposomes (in phase III clinical trial). Interestingly, the introduction of tumor-specific aptamers creates a special "Liposome-Aptamer-Target" interface, where the aptamers are initially in a "lying down" state but transform to "standing up" after target binding. The interfacing mechanism was elaborately revealed by computational and practical experiments. This unique interface endowed Apt-LPR with neutralized surface potential of cationic liposomes to reduce non-specific cytotoxicity, enhanced DNase resistance to protect aptamers, and preserved target-binding ability for selective drug delivery. Upon near-infrared irradiation, the generated reactive oxygen species would oxidize unsaturated phospholipids to destabilize both liposomes and lysosomes, realizing stepwise lysosomal escape of miR-34a for tumor cell apoptosis and downregulation of PD-L1 to suppress immune escape. Together, tumor-associated antigens released from PDT-damaged mitochondria and endoplasmic reticulum could activate the suppressive immune cells to establish an "immune hot" milieu. The collaborative immune-enhancing strategy effectively aroused systemic antitumor immunity and inhibited primary and distal tumor progression as well as lung metastasis in 4T1 xenografted mouse models. The photo-controlled drug release and specific tumor-targeting capabilities of Apt-LPR were also visualized in MDA-MB-231 xenografted zebrafish models. Therefore, this photoswitchable PDT/RNAi immune stimulator offered a powerful approach to reprogramming ITM and reinforcing cancer immunotherapy efficacy.
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Liposomas , MicroARNs , Fotoquimioterapia , Fármacos Fotosensibilizantes , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Animales , Humanos , Liposomas/química , MicroARNs/genética , MicroARNs/metabolismo , Fotoquimioterapia/métodos , Microambiente Tumoral/efectos de los fármacos , Línea Celular Tumoral , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Femenino , Neoplasias de la Mama Triple Negativas/terapia , Neoplasias de la Mama Triple Negativas/patología , Ratones , Aptámeros de Nucleótidos/química , Preparaciones de Acción Retardada/química , Interferencia de ARN , Pez CebraRESUMEN
Long interspersed nuclear element1 (L1) is highly expressed in the early embryos of humans, rodents and fish. To investigate the molecular mechanisms underlying high expression of L1 during early embryonic development, a C1open reading frame (ORF)2 vector was constructed in which ORF2 of human L1 (L1ORF2) was inserted into a pEGFPC1 plasmid. C1ORF2 vector was injected into early zebrafish embryos (EZEs) to observe expression of EGFP reporter protein by fluorescence microscopy. RNAseq and RTqPCR were used to detect the effects of lipovitellin (LV) on gene expression in EZEs. The binding ability of LV to L1ORF2 DNA was detected by electrophoretic mobilityshift assay (EMSA). The chromatin recombinant DNase I digestion and ATACseq assay were used to evaluate the accessibility of plasmid DNA. C1ORF2 vector induced high expression of enhanced green fluorescent protein (EGFP) reporter gene after it had been injected into 0 h postfertilization (hpf) zebrafish embryos, although histone octamer inhibited expression of EGFP in C1ORF2. SDSPAGE was used to show that LV was the predominant protein binding ORF2 DNA in 0 hpf zebrafish embryo lysate (ZEL). Both ZEL and purified LV from ZEL attenuated the inhibitory effects induced by histone. LV bound histone to interfere with the binding of histone to ORF2 DNA. Both in vitro chromatin reconstitution experiments and assay for transposaseaccessible chromatin with sequencing with HeLa cells were utilized to demonstrate that the interference induced by LV resulted in increased accessibility of C1ORF2. Transcription experiments in vitro verified that LV could enhance the mRNA levels of zebrafish early embryo expression genes grainyheadlike transcription factor 3 (GRHL3), SRYbox transcription factor 19a (SOX19A) and nanor (NNR) and also of the EGFP gene. LV was found to increase the expression levels of the zebrafish early embryo expression genes in liver tissue after LV had been injected into the abdominal cavity of adult male zebrafish. Taken together, the findings of the present study demonstrated that LV activates the expression of EGFP induced by ORF2 in EZEs by enhancing the accessibility of ORF2 DNA.
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Histonas , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/embriología , Histonas/metabolismo , Histonas/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Humanos , Sistemas de Lectura Abierta/genética , Elementos de Nucleótido Esparcido Largo/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Unión ProteicaRESUMEN
Phenanthrene (Phe) is one of the common polycyclic aromatic hydrocarbons in the environment, and recent studies show that it can cause cardiac developmental toxicity and immunotoxicity. However, it is still unknown whether it can affect the hematopoietic development in aquatic organisms. To address this question, zebrafish (Danio rerio) were chronically exposed to Phe at different concentrations. We found that Phe caused structural damage to the renal tubules in the kidney, induced malformed erythrocytes in peripheral blood, and decreased the proportion of myeloid cells in adult zebrafish, suggesting possible negative impacts that Phe posed to hematopoietic development. Then, using in situ hybridization technology, we found that Phe decreased the expression of primitive hematopoietic marker genes, specifically gata1 and pu.1, accompanied by an obstruction of primitive erythrocyte circulation. Furthermore, Phe impaired definitive hematopoiesis, increased aberrations of the transient hematopoietic site (PBI), and reduced the generation of hematopoietic stem cells, ultimately influencing the number of erythrocytes and myeloid cells. The findings suggested that Phe could induce hematopoietic toxicity in zebrafish embryos and pose unknown ecological risks.
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Hematopoyesis , Fenantrenos , Pez Cebra , Animales , Pez Cebra/embriología , Fenantrenos/toxicidad , Hematopoyesis/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Embrión no Mamífero/efectos de los fármacosRESUMEN
With the global population continuously rising, efficient bioconversion of inedible agricultural by-products is crucial for human food and energy sustainability. We here propose solid-state fermentation approaches to efficiently convert biopolymers into oligomers/monomers by accelerating the natural degradation process of the versatile Streptomyces sp. strain SCUT-3. Using fish skin as a representative by-product, 54.3 g amino acids and 14.7 g peptides (91 % < 2500 Da) were recovered from 89.0 g protein in 100 g tilapia skin sample by collagenase-overexpressed SCUT-3 for seven days at a 1:4 substrate:liquid ratio. Fish skin collagen hydrolysates exhibited excellent anti-oxidation, anti-hypertension, scratch-repairing, anti-aging, anti-ultraviolet radiation, and anti-inflammation effects on human skin fibroblasts In vitro and zebrafish larvae in vivo, indicating their potential applications in healthcare/skincare and anti-atopic dermatitis. As Laozi said, the divine law follows nature. This study underscores the efficacy of genetically engineered SCUT-3 according to its natural biomass utilization laws in large-scale biopolymer conversion.