RESUMEN
6-benzylaminopurine (6-BA), a multifunctional plant growth regulator, which is frequently used worldwide to improve qualities of various crops, is an important ingredient in production of "toxic bean sprouts." Although there is no direct evidence of adverse effects, its hazardous effects, as well as joint toxicity with other chemicals, have received particular attention and aroused furious debate between proponents and environmental regulators. By use of human umbilical vein endothelial cells (HUVECs), adverse effects of 6-BA to human-derived cells were first demonstrated in this study. A total of 25-50 mg 6-BA/L inhibited proliferation, migration, and formation of tubular-like structures by 50% in vitro. Results of Western blot analyses revealed that exposure to 6-BA differentially modulated the MAPK signal transduction pathway in HUVECs. Specifically, 6-BA decreased phosphorylation of MEK and ERK, but increased phosphorylation of JNK and P38. In addition, 6-BA exacerbated atorvastatin-induced cerebral hemorrhage via increasing hemorrhagic occurrence by 60% and areas by 4 times in zebrafish larvae. In summary, 6-BA elicited toxicity to the endothelial system of HUVECs and zebrafish. This was due, at least in part, to discoordination of MAPK signaling pathway, which should pose potential risks to the cerebral vascular system.
Asunto(s)
Compuestos de Bencilo , Hemorragia Cerebral , Purinas , Pez Cebra , Animales , Humanos , Atorvastatina/metabolismo , Atorvastatina/farmacología , Pez Cebra/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Hemorragia Cerebral/metabolismoRESUMEN
Phosphatidylinositol 4 kinase-ß (PI4KB) plays critical roles in human genetic diseases. In zebrafish, Pi4kb is strongly expressed in hair cells (HCs), which are necessary for detecting sound vibrations, head movements, and water motion. However, the role of PI4KB in HC or semicircular canal development is unclear. Herein, we report that pi4kb morphants exhibit insensitivity to sound stimulation and abnormal morphological vestibular organs, including cilium loss in HCs of the cristae and semicircular canal malformation. As bone morphogenetic protein (BMP) signaling is associated with HC and semicircular canal development, we analyzed the expression of BMP-related genes; the phosphorylated Smad1/5/9 (p-Smad1/5/9) expression was markedly reduced in otic HCs. RNA-sequencing data indicated that the transcriptional levels of BMP membrane receptor 2 (bmpr2a and bmpr2b) and hes-related family of bHLH transcription factors with YRPW motif 1 (hey1), a direct downstream target gene of p-Smad, were significantly reduced in the pi4kb morphants, as verified using quantitative reverse transcription-polymerase chain reaction and in situ hybridization. Co-injection of hey1 mRNA and pi4kb morpholino notably recovered vestibular apparatus development, including the number and length of cilia in HCs of the cristae and semicircular canal formation. Collectively, these results suggest that Pi4kb is involved in vestibular apparatus development in zebrafish by regulating BMP membrane receptor 2 and p-Smad1/5/9 levels, thereby affecting the transcriptional activation of the target gene hey1. This study sheds light on the interaction between Pi4kb and the BMP-Hey1 signaling axis, which is critical for HC and semicircular canal formation.
Asunto(s)
Vestíbulo del Laberinto , Pez Cebra , Animales , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Transducción de Señal , Vestíbulo del Laberinto/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The type VI secretion system (T6SS) is a large secretory device, widely found in Gram-negative bacteria, which plays important roles in virulence, bacterial competition, and environmental adaptation. Vibrio alginolyticus (V. alginolyticus) is an opportunistic pathogen that causes vibriosis in aquaculture animals. V. alginolyticus possesses two type VI secretion systems (named the T6SS1 and T6SS2), but their functions remain largely unclear. In this paper, the roles of the core component of the T6SS2 cluster of V. alginolyticus HY9901, hemolysin-coregulated protein2 coding gene hcp2, are reported. Deletion of hcp2 clearly impaired the swarming motility, adhesive capacity, and pathogenicity of V. alginolyticus against zebrafish. Furthermore, transmission electron microscopy (TEM) found that the abnormal morphology of flagellum filament in the hcp2 mutant strain could be partially restored by hcp2 complementarity. By proteomic and RT-qPCR analysis, we confirmed that the expression levels of flagellar flagellin and assembly-associated proteins were remarkably decreased in an hcp2 mutant strain, compared with the wild-type strain, and could be partially restored with a supply of hcp2. Accordingly, hcp2 had a positive influence on the transcription of flagellar regulons rpoN, rpoS, and fliA; this was verified by RT-qPCR. Taken together, these results suggested that hcp2 was involved in mediating the motility, adhesion, and pathogenicity of Vibrio alginolyticus through positively impacting its flagellar system.
RESUMEN
The spread of coronavirus infectious disease (COVID-19) is associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has risked public health more than any other infectious disease. Researchers around the globe use multiple approaches to identify an effective approved drug (drug repurposing) that treats viral infections. Most of the drug repurposing approaches target spike protein or main protease. Here we use transmembrane serine protease 2 (TMPRSS2) as a target that can prevent the virus entry into the cell by interacting with the surface receptors. By hypothesizing that the TMPRSS2 binders may help prevent the virus entry into the cell, we performed a systematic drug screening over the current approved drug database. Furthermore, we screened the Enamine REAL fragments dataset against the TMPRSS2 and presented nine potential drug-like compounds that give us clues about which kinds of groups the pocket prefers to bind, aiding future structure-based drug design for COVID-19. Also, we employ molecular dynamics simulations, binding free energy calculations, and well-tempered metadynamics to validate the obtained candidate drug and fragment list. Our results suggested three potential FDA-approved drugs against human TMPRSS2 as a target. These findings may pave the way for more drugs to be exposed to TMPRSS2, and testing the efficacy of these drugs with biochemical experiments will help improve COVID-19 treatment. Supplementary information: The online version contains supplementary material available at 10.1007/s11224-022-01960-w.
RESUMEN
Steinberg's differential adhesion hypothesis suggests that adhesive mechanisms are important for sorting of cells and tissues during morphogenesis (Steinberg, 2007). During zebrafish vasculogenesis, endothelial cells sort into arterial and venous vessel beds but it is unknown whether this involves adhesive mechanisms. Claudins are tight junction proteins regulating the permeability of epithelial and endothelial tissue barriers. Previously, the roles of claudins during organ development have exclusively been related to their canonical functions in determining paracellular permeability. Here, we use atomic force microscopy to quantify claudin-5-dependent adhesion and find that this strongly contributes to the adhesive forces between arterial endothelial cells. Based on genetic manipulations, we reveal a non-canonical role of Claudin-5a during zebrafish vasculogenesis, which involves the regulation of adhesive forces between adjacent dorsal aortic endothelial cells. In vitro and in vivo studies demonstrate that loss of claudin-5 results in increased motility of dorsal aorta endothelial cells and in a failure of the dorsal aorta to lumenize. Our findings uncover a novel role of claudin-5 in limiting arterial endothelial cell motility, which goes beyond its traditional sealing function during embryonic development.
Asunto(s)
Proteínas de Uniones Estrechas , Uniones Estrechas , Animales , Claudina-4 , Claudina-5/genética , Claudinas , Células Endoteliales , Pez Cebra , Proteínas de Pez CebraRESUMEN
Blood-brain barrier (BBB) disruption is a key event in triggering secondary damage to the central nervous system (CNS) under stroke, and is frequently associated with abnormal macroautophagy/autophagy in brain microvascular endothelial cells (BMECs). However, the underlying mechanism of autophagy in maintaining BBB integrity remains unclear. Here we report that in BMECs of patients suffering stroke, CLDN5 (claudin 5) abnormally aggregates in the cytosol accompanied by autophagy activation. In vivo zebrafish and in vitro cell studies reveal that BBB breakdown is partially caused by CAV1 (caveolin 1)-mediated redistribution of membranous CLDN5 into the cytosol under hypoxia. Meanwhile, autophagy is activated and contributes mainly to the degradation of CAV1 and aggregated CLDN5 in the cytosol of BMECs, therefore alleviating BBB breakdown. Blockage of autophagy by genetic methods or chemicals aggravates cytosolic aggregation of CLDN5, resulting in severer BBB impairment. These data demonstrate that autophagy functions in the protection of BBB integrity by regulating CLDN5 redistribution and provide a potential therapeutic strategy for BBB disorder-related cerebrovascular disease.Abbreviations: BBB: blood-brain barrier; BECN1: beclin 1; BMEC: brain microvascular endothelial cell; CAV1: caveolin 1; CCA: common carotid artery; CLDN5: claudin 5; CNS: central nervous system; CQ: chloroquine; HIF1A: hypoxia inducible factor 1 subunit alpha; MCAO: middle cerebral artery occlusion-reperfusion; OCLN: occludin; ROS: reactive oxygen species; STED: stimulated emission depletion; TEER: trans-endothelial electrical resistance; TEM: transmission electron microscopy; TJ: tight junction; TJP1: tight junction protein 1; UPS: ubiquitin-proteasome system.
Asunto(s)
Autofagia , Barrera Hematoencefálica , Claudina-5 , Células Endoteliales , Accidente Cerebrovascular , Animales , Barrera Hematoencefálica/patología , Claudina-5/genética , Claudina-5/metabolismo , Células Endoteliales/metabolismo , Humanos , Hipoxia , Accidente Cerebrovascular/patología , Pez Cebra/metabolismoRESUMEN
Congenital hearing loss is a common disorder worldwide. Heterogeneous gene variation accounts for approximately 20-25% of such patients. We investigated a five-generation Chinese family with autosomal-dominant nonsyndromic sensorineural hearing loss (SNHL). No wave was detected in the pure-tone audiometry, and the auditory brainstem response was absent in all patients. Computed tomography of the patients, as well as of two sporadic SNHL cases, showed bilateral inner ear anomaly, cochlear maldevelopment, absence of the osseous spiral lamina, and an enlarged vestibular aqueduct. Such findings were absent in nonaffected persons. We used linkage analysis and exome sequencing and uncovered a heterozygous missense mutation in the PI4KB gene (p.Gln121Arg) encoding phosphatidylinositol 4-kinase ß (PI4KB) from the patients in this family. In addition, 3 missense PI4KB (p.Val434Gly, p.Glu667Lys, and p.Met739Arg) mutations were identified in five patients with nonsyndromic SNHL from 57 sporadic cases. No such mutations were present within 600 Chinese controls, the 1000 genome project, gnomAD, or similar databases. Depleting pi4kb mRNA expression in zebrafish caused inner ear abnormalities and audiosensory impairment, mimicking the patient phenotypes. Moreover, overexpression of 4 human missense PI4KB mutant mRNAs in zebrafish embryos resulted in impaired hearing function, suggesting dominant-negative effects. Taken together, our results reveal that PI4KB mutations can cause SNHL and inner ear malformation. PI4KB should be included in neonatal deafness screening.
Asunto(s)
Predisposición Genética a la Enfermedad , Pérdida Auditiva Sensorineural/genética , Enfermedades del Laberinto/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Cóclea/patología , Oído Interno/patología , Femenino , Ligamiento Genético/genética , Pérdida Auditiva Sensorineural/patología , Humanos , Lactante , Recién Nacido , Enfermedades del Laberinto/patología , Masculino , Mutación Missense/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Secuenciación del Exoma , Pez Cebra/genéticaRESUMEN
Traumatic brain injury (TBI) is a major public and socioeconomic problem throughout the world. The establishment of an effective and cost-effective TBI model for developing new therapeutic agents is challenging. Microglia are considered the resident macrophages of the central nervous system (CNS) that normally do not enter the brain. As the primary mediators of the innate immune response in the CNS, microglia play a critical role in neuroinflammation and secondary injury after TBI. In this study, we established an in vivo TBI zebrafish model using Tg(coro1a:EGFP) line where the green fluorescent protein-labeled microglia were present. We demonstrated that microglia accumulated rapidly in response to neuronal injuries. To clear away injured neurons and restore the CNS homeostasis, activated microglia secreted two types of functional cytokines, including pro-inflammatory interleukins (IL) of IL-1ß and IL-6 and anti-inflammatory factors of IL-4 and IL-10 in the lesioned larvae. Cytidine 5'-Diphosphocholine (CDP-choline), as an effective and clinical neuroprotective drug, could further activate microglia, expressing high levels of il-1ß, il-6, il-4, and il-10 in the TBI model. Moreover, CDP-choline reduced neuronal apoptosis and promoted neuronal proliferation around the lesioned site. Based on these results, the TBI model established in this study represents a suitable model for developing new therapeutic agents for CNS-associated diseases.
Asunto(s)
Lesiones Traumáticas del Encéfalo/fisiopatología , Cerebro/fisiopatología , Modelos Animales de Enfermedad , Inflamación/fisiopatología , Microglía/metabolismo , Pez Cebra , Animales , Animales Modificados Genéticamente , Lesiones Traumáticas del Encéfalo/patología , Cerebro/patología , Inflamación/patologíaRESUMEN
The monolayer of endothelial cells (ECs) lining the intima of all blood vessel wall forms a semipermeable barrier that regulates tissue-fluid homeostasis, transport of nutrients, and migration of blood cells across the barrier. A number of signaling pathways and molecules mediate endothelial permeability, which plays important roles in a variety of the physiological and pathological conditions. Fatty acid binding proteins (FABPs) are able to bind various hydrophobic molecules, such as long-chain fatty acids, prostaglandins and eicosanoids. FABP4, a member of the family of FABPs, plays an important role in maintenance of glucose and lipid homeostasis as well as angiogenesis. In the present study, we found that fabp11a, the ortholog of mammalian FABP4, was highly expressed in developing brain vessels of zebrafish. Knockout of fabp11a gene caused hemorrhage in zebrafish brain. Morpholino mediated fabp11a gene knockdown phenocopied the hemorrhage in mutants. Furthermore, we demonstrated permeability of brain vessels in fabp11a mutant is significantly higher than that of control. In addition, COX and LOX inhibition partially rescued the brain vessel integrity defects caused by fabp11a loss-of-function, suggesting the integrity defect was relevant to the Fatty Acid function.