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1.
Front Cell Infect Microbiol ; 12: 790851, 2022.
Article in English | MEDLINE | ID: covidwho-1775643

ABSTRACT

Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.


Subject(s)
COVID-19 , Zebrafish , Animals , Larva , Mammals , RNA, Viral , SARS-CoV-2 , Urinary Bladder
2.
Sci Total Environ ; 824: 153967, 2022 Jun 10.
Article in English | MEDLINE | ID: covidwho-1757819

ABSTRACT

In coronavirus disease 2019 (COVID-19), among many protocols, lopinavir and ritonavir in individual or combined forms with other drugs have been used, causing an increase in the concentration of antiviral drugs in the wastewater and hospital effluents. In conventional wastewater treatment plants, the removal efficiency of various antiviral drugs is estimated to be low (<20%). The high values of predicted no-effect concentration (PNEC) for lopinavir and ritonavir (in ng∙L-1) reveal their high chronic toxicity to aquatic organisms. This indicates that lopinavir and ritonavir are current priority antiviral drugs that need to be thoroughly monitored and effectively removed from any water and wastewater samples. In this study, we attempt to explore the impacts of two photo-induced processes (photolysis and photocatalysis) on the toxicity of treated water and wastewater samples containing lopinavir and ritonavir to zebrafish (Danio rerio) and marine bacteria (Allivibrio fischeri). The obtained results reveal that traces of lopinavir in water under photo-induced processes may cause severe problems for Danio rerio, including pericardial edema and shortening of the tail, affecting its behavior, and for Allivibrio fischeri as a result of the oxygen-depleted environment, inflammation, and oxidative stress. Hence, lopinavir must be removed from water and wastewater before being in contact with light. In contrast, the photo-induced processes of ritonavir-containing water and wastewater reduce the toxicity significantly. This shows that even if the physicochemical parameters of water and wastewater are within the standard requirements/limits, the presence of traces of antiviral drugs and their intermediates can affect the survival and behavior of Danio rerio and Allivibrio fischeri. Therefore, the photo-induced processes and additional treatment of water and wastewater containing ritonavir can minimize its toxic effect.


Subject(s)
COVID-19 , Ritonavir , Animals , Antiviral Agents , COVID-19/drug therapy , Drug Combinations , Lopinavir/therapeutic use , Lopinavir/toxicity , Ritonavir/therapeutic use , Ritonavir/toxicity , Waste Water , Water , Zebrafish
3.
Brain Behav Immun ; 102: 341-359, 2022 May.
Article in English | MEDLINE | ID: covidwho-1748207

ABSTRACT

Anosmia, loss of smell, is a prevalent symptom of SARS-CoV-2 infection. Anosmia may be explained by several mechanisms driven by infection of non-neuronal cells and damage in the nasal epithelium rather than direct infection of olfactory sensory neurons (OSNs). Previously, we showed that viral proteins are sufficient to cause neuroimmune responses in the teleost olfactory organ (OO). We hypothesize that SARS-CoV-2 spike (S) protein is sufficient to cause olfactory damage and olfactory dysfunction. Using an adult zebrafish model, we report that intranasally delivered SARS-CoV-2 S RBD mostly binds to the non-sensory epithelium of the olfactory organ and causes severe olfactory histopathology characterized by loss of cilia, hemorrhages and edema. Electrophysiological recordings reveal impaired olfactory function to both food and bile odorants in animals treated intranasally with SARS-CoV-2 S RBD. However, no loss of behavioral preference for food was detected in SARS-CoV-2 S RBD treated fish. Single cell RNA-Seq of the adult zebrafish olfactory organ indicated widespread loss of olfactory receptor expression and inflammatory responses in sustentacular, endothelial, and myeloid cell clusters along with reduced numbers of Tregs. Combined, our results demonstrate that intranasal SARS-CoV-2 S RBD is sufficient to cause structural and functional damage to the zebrafish olfactory system. These findings may have implications for intranasally delivered vaccines against SARS-CoV-2.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Anosmia , COVID-19 Vaccines , Humans , Inflammation/metabolism , Olfactory Mucosa/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Zebrafish
4.
Toxicol Appl Pharmacol ; 440: 115913, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1671180

ABSTRACT

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.


Subject(s)
COVID-19 , Influenza, Human , Animals , Cell Communication , Cetylpyridinium/chemistry , Cetylpyridinium/pharmacology , Dinucleoside Phosphates , Humans , Immunity , Mammals , Mice , Microscopy, Fluorescence , Pandemics , Phosphatidylinositols , Rats , SARS-CoV-2 , Zebrafish
5.
Adv Sci (Weinh) ; 9(4): e2102072, 2022 02.
Article in English | MEDLINE | ID: covidwho-1669350

ABSTRACT

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.


Subject(s)
Liposomes/metabolism , Protein Processing, Post-Translational , RNA, Messenger/metabolism , Animals , Mice , Models, Animal , Zebrafish/metabolism
6.
J Hazard Mater ; 429: 128300, 2022 05 05.
Article in English | MEDLINE | ID: covidwho-1633288

ABSTRACT

The use of antiviral drugs has surged as a result of the COVID-19 pandemic, resulting in higher concentrations of these pharmaceuticals in wastewater. The degradation efficiency of antiviral drugs in wastewater treatment plants has been reported to be too low due to their hydrophilic nature, and an additional procedure is usually necessary to degrade them completely. Photocatalysis is regarded as one of the most effective processes to degrade antiviral drugs. The present study aims at synthesizing multiphase photocatalysts by a simple calcination of industrial waste from ammonium molybdate production (WU photocatalysts) and its combination with WO3 (WW photocatalysts). The X-ray diffraction (XRD) results confirm that the presence of multiple crystalline phases in the synthesized photocatalysts. UV-Vis diffuse reflectance spectra reveal that the synthesized multiphase photocatalysts absorb visible light up to 620 nm. Effects of calcination temperature of industrial waste (550-950 °C) and WO3 content (0-100%) on photocatalytic activity of multiphase photocatalysts (WU and WW) for efficient removal of SARS-CoV-2 antiviral drugs (lopinavir and ritonavir) in model and real wastewaters are studied. The highest k1 value is observed for the photocatalytic removal of ritonavir from model wastewater using WW4 (35.64 ×10-2 min-1). The multiphase photocatalysts exhibit 95% efficiency in the photocatalytic removal of ritonavir within 15 of visible light irradiation. In contrast, 60 min of visible light irradiation is necessary to achieve 95% efficiency in the photocatalytic removal of lopinavir. The ecotoxicity test using zebrafish (Danio rerio) embryos shows no toxicity for photocatalytically treated ritonavir-containing wastewater, and the contrary trend is observed for photocatalytically treated lopinavir-containing wastewater. The synthesized multiphase photocatalysts can be tested and applied for efficient degradation of other SARS-CoV-2 antiviral drugs in wastewater in the future.


Subject(s)
COVID-19 , Waste Water , Animals , Antiviral Agents , Catalysis , Humans , Industrial Waste , Pandemics , SARS-CoV-2 , Zebrafish
7.
J Hazard Mater ; 428: 128186, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1587272

ABSTRACT

Surgical face masks are the most popularised and effective personal equipment for protecting public health during the COVID-19 pandemic. They are composed of plastic polymer fibres with a large amount of inorganic and organic compounds that can be released into aquatic environments through degradation processes. This source of microplastics and inorganic and organic substances could potentially impact aquatic organisms. In this study, the toxicogenomic effects of face masks at different stages of degradation in water were analysed in zebrafish larvae (Danio rerio) through RNA-Seq. Larvae were exposed for 10 days to three treatments: 1) face mask fragments in an initial stage of degradation (poorly degraded masks -PDM- products) with the corresponding water; 2) face mask fragments in an advanced stage of degradation (highly degraded masks -HDM- products) with the corresponding water; and 3) water derived from HDM (W-HDM). Transcriptome analyses revealed that the three treatments provoked the down-regulation of genes related to reproduction, especially the HDM products, suggesting that degradation products derived from face masks could act as endocrine disruptors. The affected genes are involved in different steps of reproduction, including gametogenesis, sperm-egg recognition and binding or fertilisation. Immune-related genes and metabolic processes were also differentially affected by the treatments.


Subject(s)
COVID-19 , Environmental Pollutants , Water Pollutants, Chemical , Animals , Humans , Masks , Pandemics , Plastics , RNA-Seq , SARS-CoV-2 , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/toxicity , Zebrafish/genetics
8.
Sci Total Environ ; 813: 152345, 2022 Mar 20.
Article in English | MEDLINE | ID: covidwho-1586548

ABSTRACT

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Female , Humans , SARS-CoV-2 , Zebrafish
9.
Sci Rep ; 11(1): 23670, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1560986

ABSTRACT

Among cases of SARS-CoV-2 infections that result in serious conditions or death, many have pre-existing conditions such as hypertension and are on renin-angiotensin-aldosterone system (RAAS) inhibitors. The angiotensin-converting-enzyme-2 (ACE2), a key protein of the RAAS pathway, also mediates cellular entry of SARS-CoV-2. RAAS inhibitors might affect the expression levels of ace2, which could impact patient susceptibility to SARS-CoV-2. However, multi-organ-specific information is currently lacking and no species other than rodents have been examined. To address this knowledge gap, we treated adult zebrafish with the RAAS inhibitors aliskiren, olmesartan, and captopril for 7 consecutive days and performed qRT-PCR analysis of major RAAS pathway genes in the brain, gill, heart, intestine, kidney, and liver. Both olmesartan and captopril significantly increased ace2 expression in the heart, gill, and kidney. Olmesartan also increased ace2 expression in the intestine. Conversely, aliskiren significantly decreased ace2 expression in the heart. Discontinuation of compound treatments for 7 days did not return ace2 expression to baseline levels. While potential risks or benefits of antihypertensive RAAS inhibitors to SARS-CoV-2 infections in humans remain uncertain, this study provides new insights regarding the impact of RAAS inhibitors on organ-specific ace2 expression in another vertebrate model, thereby providing comparative data and laying scientific groundwork for future clinical decisions of RAAS inhibitor use in the context of COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Down-Regulation/drug effects , Up-Regulation/drug effects , Zebrafish/metabolism , Amides/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Animals , Brain/drug effects , Brain/metabolism , COVID-19/pathology , COVID-19/virology , Fumarates/pharmacology , Gills/drug effects , Gills/metabolism , Humans , Imidazoles/pharmacology , Liver/drug effects , Liver/metabolism , Models, Animal , SARS-CoV-2/isolation & purification , Tetrazoles/pharmacology
10.
eNeuro ; 8(3)2021.
Article in English | MEDLINE | ID: covidwho-1551335

ABSTRACT

The Coronavirus disease-2019 (COVID-19) presents a variability of clinical symptoms, ranging from asymptomatic to severe respiratory and systemic conditions. In a cohort of patients, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), beyond the classical respiratory manifestations, induces anosmia. Evidence has suggested SARS-CoV-2-induced anosmia can be the result of neurodegeneration of the olfactory pathway. Neurologic symptoms associated with COVID-19 have been reported; however, the precise mechanism and possible long-lasting effects remain poorly investigated. Preclinical models are valuable tools for describing and testing new possible treatments for neurologic disorders. In this way, the zebrafish (Danio rerio) organism model represents an attractive tool in the field of neuroscience, showing economic and logistic advantages besides genetic and physiologic similarities with mammalian, including the brain structure and functions. Besides, its external embryonic development, high availability of eggs, and fast development allows easy genetic manipulation and fast replications. In the present review, we suggest that the zebrafish model can be advantageous to investigate the neurologic features of COVID-19.


Subject(s)
COVID-19 , Nervous System Diseases , Animals , Anosmia , Humans , SARS-CoV-2 , Zebrafish
11.
Expert Opin Drug Discov ; 17(2): 121-137, 2022 02.
Article in English | MEDLINE | ID: covidwho-1500936

ABSTRACT

INTRODUCTION: The search for an animal model capable of reproducing the physiopathology of the COVID-19, and also suitable for evaluating the efficacy and safety of new drugs has become a challenge for many researchers. AREAS COVERED: This work reviews the current animal models for in vivo tests with SARS-CoV-2 as well as the challenges involved in the safety and efficacy trials. EXPERT OPINION: Studies have reported the use of nonhuman primates, ferrets, mice, Syrian hamsters, lagomorphs, mink, and zebrafish in experiments that aimed to understand the course of COVID-19 or test vaccines and other drugs. In contrast, the assays with animal hyperimmune sera have only been used in in vitro assays. Finding an animal that faithfully reproduces all the characteristics of the disease in humans is difficult. Some models may be more complex to work with, such as monkeys, or require genetic manipulation so that they can express the human ACE2 receptor, as in the case of mice. Although some models are more promising, possibly the use of more than one animal model represents the best scenario. Therefore, further studies are needed to establish an ideal animal model to help in the development of other treatment strategies besides vaccines.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cricetinae , Disease Models, Animal , Ferrets , Humans , Mice , Zebrafish
12.
Genome ; 65(2): 57-74, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1450618

ABSTRACT

Human Angiotensin I Converting Enzyme 2 (ACE2) plays an essential role in blood pressure regulation and SARS-CoV-2 entry. ACE2 has a highly conserved, one-to-one ortholog (ace2) in zebrafish, which is an important model for human diseases. However, the zebrafish ace2 expression profile has not yet been studied during early development, between genders, across different genotypes, or in disease. Moreover, a network-based meta-analysis for the extraction of functionally enriched pathways associated with differential ace2 expression is lacking in the literature. Herein, we first identified significant development-, tissue-, genotype-, and gender-specific modulations in ace2 expression via meta-analysis of zebrafish Affymetrix transcriptomics datasets (ndatasets = 107); and the correlation analysis of ace2 meta-differential expression profile revealed distinct positively and negatively correlated local functionally enriched gene networks. Moreover, we demonstrated that ace2 expression was significantly modulated under different physiological and pathological conditions related to development, tissue, gender, diet, infection, and inflammation using additional RNA-seq datasets. Our findings implicate a novel translational role for zebrafish ace2 in organ differentiation and pathologies observed in the intestines and liver.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Zebrafish Proteins/genetics , Zebrafish , Animals , Female , Gene Expression Regulation, Developmental , Male , RNA-Seq , Zebrafish/genetics
13.
Dis Model Mech ; 14(11)2021 11 01.
Article in English | MEDLINE | ID: covidwho-1430507

ABSTRACT

Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, including cancer, stroke, cardiovascular disease, retinal conditions and COVID-19-associated pulmonary edema, sepsis and acute lung injury. Understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics to inhibit vascular permeability, while preserving tissue-restorative angiogenesis. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. We show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9-generated Stat3 knockout zebrafish. Intercellular adhesion molecule 1 (ICAM-1) expression is transcriptionally regulated by STAT3, and VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse and human endothelium. Collectively, our findings suggest that VEGF/VEGFR-2/JAK2/STAT3 signaling regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability. This article has an associated First Person interview with the first author of the paper.


Subject(s)
Capillary Permeability , Endothelium, Vascular/metabolism , STAT3 Transcription Factor/genetics , Vascular Endothelial Growth Factor A/metabolism , Animals , CRISPR-Cas Systems , Humans , Intercellular Adhesion Molecule-1/metabolism , Janus Kinase 2/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , STAT3 Transcription Factor/metabolism , Signal Transduction , Zebrafish
14.
J Hazard Mater ; 423(Pt B): 127152, 2022 02 05.
Article in English | MEDLINE | ID: covidwho-1401614

ABSTRACT

Chlorine disinfection is a key global public health strategy for the prevention and control of diseases, such as COVID-19. However, little is known about effects of low levels of residual chlorine on freshwater microbial communities and antibiotic resistomes. Here, we treated freshwater microcosms with continuous low concentrations of chlorine and quantified the effects on aquatic and zebrafish intestinal microbial communities and antibiotic resistomes, using shotgun metagenome and 16S rRNA gene sequencing. Although chlorine rapidly degraded, it altered the aquatic microbial community composition over time and disrupted interactions among microbes, leading to decreases in community complexity and stability. However, community diversity was unaffected. The majority of ecological functions, particularly metabolic capacities, recovered after treatment with chlorine for 14 d, due to microbial community redundancy. There were also increased levels of antibiotic-resistance gene dissemination by horizontal and vertical gene transfer under chlorine treatment. Although the zebrafish intestinal microbial community recovered from temporary dysbiosis, growth and behavior of zebrafish adults were negatively affected by chlorine. Overall, our findings demonstrate the negative effects of residual chlorine on freshwater ecosystems and highlight a possible long-term risk to public health.


Subject(s)
COVID-19 , Microbiota , Animals , Chlorine/toxicity , Drug Resistance, Microbial , Fresh Water , RNA, Ribosomal, 16S/genetics , SARS-CoV-2 , Zebrafish
15.
Nat Commun ; 12(1): 3358, 2021 06 07.
Article in English | MEDLINE | ID: covidwho-1397869

ABSTRACT

Early stages of embryogenesis depend on subcellular localization and transport of maternal mRNA. However, systematic analysis of these processes is hindered by a lack of spatio-temporal information in single-cell RNA sequencing. Here, we combine spatially-resolved transcriptomics and single-cell RNA labeling to perform a spatio-temporal analysis of the transcriptome during early zebrafish development. We measure spatial localization of mRNA molecules within the one-cell stage embryo, which allows us to identify a class of mRNAs that are specifically localized at an extraembryonic position, the vegetal pole. Furthermore, we establish a method for high-throughput single-cell RNA labeling in early zebrafish embryos, which enables us to follow the fate of individual maternal transcripts until gastrulation. This approach reveals that many localized transcripts are specifically transported to the primordial germ cells. Finally, we acquire spatial transcriptomes of two xenopus species and compare evolutionary conservation of localized genes as well as enriched sequence motifs.


Subject(s)
Cell Tracking/methods , Embryo, Nonmammalian/metabolism , RNA, Messenger/genetics , Transcriptome/genetics , Zebrafish/genetics , Animals , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Female , Gene Expression Regulation, Developmental , Oocytes/cytology , Oocytes/metabolism , RNA, Messenger/metabolism , Single-Cell Analysis/methods , Spatio-Temporal Analysis , Species Specificity , Xenopus/embryology , Xenopus/genetics , Xenopus laevis/embryology , Xenopus laevis/genetics , Zebrafish/embryology
16.
Molecules ; 25(21)2020 Nov 02.
Article in English | MEDLINE | ID: covidwho-1389462

ABSTRACT

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV-V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1ß induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.


Subject(s)
Antiviral Agents/therapeutic use , Coronavirus Infections/drug therapy , Plant Extracts/therapeutic use , Pneumonia, Viral/drug therapy , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Air Sacs/drug effects , Air Sacs/virology , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , COVID-19 , Chromatography, High Pressure Liquid/methods , Coronavirus Infections/pathology , Coronavirus Infections/physiopathology , Disease Models, Animal , Fever/drug therapy , Fever/etiology , Hemorrhage/prevention & control , Humans , Interleukin-6/metabolism , Kidney/drug effects , Necrosis/pathology , Necrosis/prevention & control , Pandemics , Phytotherapy , Pneumonia, Viral/pathology , Pneumonia, Viral/physiopathology , Respiratory Mucosa/transplantation , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/metabolism , Zebrafish
17.
Mar Drugs ; 19(8)2021 Jul 29.
Article in English | MEDLINE | ID: covidwho-1376892

ABSTRACT

Seaweed of Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO3 residues and (1→6)-ß-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKß) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Aquatic Organisms/chemistry , Fucose/pharmacology , Galactose/pharmacology , Seaweed/chemistry , Animals , Inhibitory Concentration 50 , Mice , RAW 264.7 Cells/drug effects , Zebrafish
18.
Sci Total Environ ; 790: 148129, 2021 Oct 10.
Article in English | MEDLINE | ID: covidwho-1253606

ABSTRACT

One of the most impact issues in recent years refers to the COVID-19 pandemic, the consequences of which thousands of deaths recorded worldwide, are still inferior understood. Its impacts on the environment and aquatic biota constitute a fertile field of investigation. Thus, to predict the impact of the indiscriminate use of azithromycin (AZT) and hydroxychloroquine (HCQ) in this pandemic context, we aim to assess their toxicological risks when isolated or in combination, using zebrafish (Danio rerio) as a model system. In summary, we observed that 72 h of exposure to AZT and HCQ (alone or in binary combination, both at 2.5 µg/L) induced the reduction of total protein levels, accompanied by increased levels of thiobarbituric acid reactive substances, hydrogen peroxide, reactive oxygen species and nitrite, suggesting a REDOX imbalance and possible oxidative stress. Molecular docking analysis further supported this data by demonstrating a strong affinity of AZT and HCQ with their potential antioxidant targets (catalase and superoxide dismutase). In the protein-protein interaction network analysis, AZT showed a putative interaction with different cytochrome P450 molecules, while HCQ demonstrated interaction with caspase-3. The functional enrichment analysis also demonstrated diverse biological processes and molecular mechanisms related to the maintenance of REDOX homeostasis. Moreover, we also demonstrated an increase in the AChE activity followed by a reduction in the neuromasts of the head when zebrafish were exposed to the mixture AZT + HCQ. These data suggest a neurotoxic effect of the drugs. Altogether, our study demonstrated that short exposure to AZT, HCQ or their mixture induced physiological alterations in adult zebrafish. These effects can compromise the health of these animals, suggesting that the increase of AZT and HCQ due to COVID-19 pandemic can negatively impact freshwater ecosystems.


Subject(s)
COVID-19 , Hydroxychloroquine , Animals , Azithromycin , COVID-19/drug therapy , Ecosystem , Humans , Hydroxychloroquine/toxicity , Molecular Docking Simulation , Pandemics , SARS-CoV-2 , Zebrafish
19.
Mol Pharmacol ; 100(2): 155-169, 2021 08.
Article in English | MEDLINE | ID: covidwho-1242189

ABSTRACT

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.


Subject(s)
14-3-3 Proteins/chemistry , 14-3-3 Proteins/metabolism , Cysteine/genetics , Isoindoles/pharmacology , Organoselenium Compounds/pharmacology , 14-3-3 Proteins/genetics , Animals , Binding Sites/drug effects , Brain/metabolism , Cell Line , Circular Dichroism , Down-Regulation , Female , Humans , Male , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding/drug effects , Protein Conformation , Protein Stability/drug effects , Tyrosine 3-Monooxygenase/metabolism , Zebrafish , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolism
20.
Eur J Med Chem ; 221: 113514, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1228023

ABSTRACT

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 = 0.43 ± 0.11 and 0.54 ± 0.19 µM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/drug therapy , Isocoumarins/therapeutic use , Phosphodiesterase 4 Inhibitors/therapeutic use , Sulfonamides/therapeutic use , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/toxicity , Arthritis, Experimental/pathology , Catalysis , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Embryo, Nonmammalian/drug effects , Female , Isocoumarins/chemical synthesis , Isocoumarins/metabolism , Isocoumarins/toxicity , Knee Joint/drug effects , Knee Joint/pathology , Male , Mice , Molecular Docking Simulation , Molecular Structure , Palladium/chemistry , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/metabolism , Phosphodiesterase 4 Inhibitors/toxicity , Protein Binding , RAW 264.7 Cells , Rats, Wistar , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/metabolism , Sulfonamides/toxicity , Zebrafish
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