The Cyanotoxin 2,4-DAB Reduces Viability and Causes Behavioral and Molecular Dysfunctions Associated with Neurodegeneration in Larval Zebrafish

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s disease. While the cyanotoxin β-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA, including 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether 2,4-DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB; however, only modulated startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB’s effects, we analyzed the protein profile of larval zebrafish exposed to 500 µM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

Gut-derived Metabolites Influence Neurodevelopmental Gene Expression and Wnt Signalling Events in a Germ-free Zebrafish Model

Background : Small molecule metabolites produced by the microbiome are known to be neuroactive and are capable of directly impacting the brain and central nervous system, yet there is little data on the contribution of these metabolites to the earliest stages of neural development and neural gene expression. Here, we explore the impact of deriving zebrafish embryos in the absence of microbes on early neural development as well as investigate whether any potential changes can be rescued with treatment of metabolites derived from the zebrafish gut microbiota. Results : Overall, we did not observe any gross morphological changes between treatments but did observe a significant decrease in neural gene expression in embryos raised germ-free, which was rescued with the addition of zebrafish metabolites. Specifically, we identified 354 genes significantly down regulated in germ-free embryos compared to conventionally raised embryos via RNA-Seq analysis. Of these, 42 were rescued with a single treatment of zebrafish gut-derived metabolites to germ-free embryos. Gene ontology analysis revealed that these genes are involved in prominent neurodevelopmental pathways including transcriptional regulation and Wnt signalling. Consistent with the ontology analysis, we found alterations in the development of Wnt dependent events which was rescued in the germ-free embryos treated with metabolites. Conclusions : These findings demonstrate that gut-derived metabolites are in part responsible for regulating critical signalling pathways in the brain, especially during neural development.

Myocardial Afterload Is a Key Biomechanical Regulator of Atrioventricular Myocyte Differentiation in Zebrafish

Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development.

The Role of Microtubule Associated Serine/Threonine Kinase 3 Variants in Neurodevelopmental Diseases: Genotype-Phenotype Association

Objective: To prove microtubule associated serine/threonine kinase 3 (MAST3) gene is associated with neurodevelopmental diseases (NDD) and the genotype-phenotype correlation.Methods: Trio exome sequencing (trio ES) was performed on four NDD trios. Bioinformatic analysis was conducted based on large-scale genome sequencing data and human brain transcriptomic data. Further in vivo zebrafish studies were performed.Results: In our study, we identified four de novo MAST3 variants (NM_015016.1: c.302C > T:p.Ser101Phe; c.311C > T:p.Ser104Leu; c.1543G > A:p.Gly515Ser; and c.1547T > C:p.Leu516Pro) in four patients with developmental and epileptic encephalopathy (DEE) separately. Clinical heterogeneities were observed in patients carrying variants in domain of unknown function (DUF) and serine-threonine kinase (STK) domain separately. Using the published large-scale exome sequencing data, higher CADD scores of missense variants in DUF domain were found in NDD cohort compared with gnomAD database. In addition, we obtained an excess of missense variants in DUF domain when compared autistic spectrum disorder (ASD) cohort with gnomAD database, similarly an excess of missense variants in STK domain when compared DEE cohort with gnomAD database. Based on Brainspan datasets, we showed that MAST3 expression was significantly upregulated in ASD and DEE-related brain regions and was functionally linked with DEE genes. In zebrafish model, abnormal morphology of central nervous system was observed in mast3a/b crispants.Conclusion: Our results support the possibility that MAST3 is a novel gene associated with NDD which could expand the genetic spectrum for NDD. The genotype-phenotype correlation may contribute to future genetic counseling.

Management of Hypercholesterolemia Through Dietary ß-glucans–Insights From a Zebrafish Model

Consumption of lipid-rich foods can increase the blood cholesterol content. β-glucans have hypocholesterolemic effect. However, subtle changes in their molecular branching can influence bioactivity. Therefore, a comparative investigation of the cholesterol-lowering potential of two β-glucans with different branching patterns and a cholesterol-lowering drug, namely simvastatin was undertaken employing the zebrafish (Danio rerio) model of diet-induced hypercholesterolemia. Fish were allocated to 5 dietary treatments; a control group, a high cholesterol group, two β-glucan groups, and a simvastatin group. We investigated plasma total cholesterol, LDL and HDL cholesterol levels, histological changes in the tissues, and explored intestinal transcriptomic changes induced by the experimental diets. Dietary cholesterol likely caused the suppression of endogenous cholesterol biosynthesis, induced dysfunction of endoplasmic reticulum and mitochondria, and altered the histomorphology of the intestine. The two β-glucans and simvastatin significantly abated the rise in plasma cholesterol levels and restored the expression of specific genes to alleviate the endoplasmic reticulum-related effects induced by the dietary cholesterol. Furthermore, the distinct patterns of transcriptomic changes in the intestine elicited by the oat and microalga β-glucans impacted processes such as fatty acid metabolism, protein catabolic processes, and nuclear division. Oat and microalgal β-glucans also altered the pattern of lipid deposition in the liver. Our study provides insights into the effectiveness of different β-glucans to alleviate dysfunctions in lipid metabolism caused by dietary cholesterol.

Elaborate the Mechanism of Ancient Classic Prescriptions (Erzhi Formula) in Reversing GIOP by Network Pharmacology Coupled with Zebrafish Verification

Osteoporosis is a degenerative disease that endangers human health. At present, chemical drugs used for osteoporosis have serious side effects. Therefore, it is valuable to search herbs with high safety and good curative effect in antiosteoporosis. Erzhi formula (EZF), an ancient classic compound, has been reported to have a beneficial effect in antiosteoporosis, but its mechanism is unclear. In this paper, the active compounds of EZF were found in Systems Pharmacology Database, and gene targets related to osteoporosis were obtained in GeneCards. The GO functional and KEGG pathway enrichment analysis were performed by Metascape. The network of “components-targets-signal pathway” was constructed by Cytoscape. Next, molecular docking between the active components and hub genes related to the PI3K-Akt signaling pathway was conducted by Autodock. In the verification experiment, the zebrafish induced by prednisolone (PNSL) was used to reproduce glucocorticoid-induced osteoporosis (GIOP) model, and then the reversal effects of EZF were systematically evaluated according to the behavior, skull staining area, bone mineralization area (BMA), average optical density (AOD), and cumulative optical density (COD). Finally, it was shown that 24 components in EZF could regulate 39 common gene targets to exert antiosteoporosis effect. Besides, the main regulatory mechanisms of EZF were 4 signaling pathways: PI3K-Akt, JAK-STAT, AGE-RAGE, and cancer pathway. In PI3K-Akt signaling pathway, wedelolactone, dimethyl wedelolactone, specnuezhenide, ursolic acid, acacetin, beta-sitosterol, apigenin, and kaempferol can bind tightly with EGF, IL-2, and IL-4 genes. Compared with the model group, the moving distance, swimming speed, and cumulative swimming time of zebrafish in EZF group were significantly increased ( P < 0.05 ). Meanwhile, the BMA and COD of zebrafish were significantly improved after the intervention of EZF ( P < 0.05 ). In summary, the 24 components of EZF exert their antiosteoporosis effects by regulating 39 related gene targets, among which the PI3K signaling pathway is crucial. EZF can promote bone formation and reversed GIOP through “multicomponent/multitarget/multipathway” and the medium dose of EZF may be the most suitable concentration for the treatment of GIOP in zebrafish model.

Discovery and identification of potential anti-melanogenic active constituents of Bletilla striata by zebrafish model and molecular docking

Background Bletilla striata is the main medicine of many skin whitening classic formulas in traditional Chinese medicine (TCM) and is widely used in cosmetic industry recently. However, its active ingredients are still unclear and its fibrous roots are not used effectively. The aim of the present study is to discover and identify its potential anti-melanogenic active constituents by zebrafish model and molecular docking. Methods The antioxidant activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′-azino-bis-(3-ethylbenthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity and ferric reducing antioxidant power (FRAP) assay. The anti-melanogenic activity was assessed by tyrosinase inhibitory activity in vitro and melanin inhibitory in zebrafish. The chemical profiles were performed by ultra-high-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Meanwhile, the potential anti-melanogenic active constituents were temporary identified by molecular docking. Results The 95% ethanol extract of B. striata fibrous roots (EFB) possessed the strongest DPPH, ABTS, FRAP and tyrosinase inhibitory activities, with IC50 5.94 mg/L, 11.69 mg/L, 6.92 mmol FeSO4/g, and 58.92 mg/L, respectively. In addition, EFB and 95% ethanol extract of B. striata tuber (ETB) significantly reduced the melanin synthesis of zebrafish embryos in a dose-dependent manner. 39 chemical compositions, including 24 stilbenoids were tentatively identified from EFB and ETB. Molecular docking indicated that there were 83 (including 60 stilbenoids) and 85 (including 70 stilbenoids) compounds exhibited stronger binding affinities toward tyrosinase and adenylate cyclase. Conclusion The present findings supported the rationale for the use of EFB and ETB as natural skin-whitening agents in pharmaceutical and cosmetic industries.

Lipid Profiles of the Heads of Four Shrimp Species by UPLC–Q–Exactive Orbitrap/MS and Their Cardiovascular Activities

Lipids are key factors in nutrition, structural function, metabolic features, and other biological functions. In this study, the lipids from the heads of four species of shrimp (Fenneropenaeus chinensis (FC), Penaeus japonicus (PJ), Penaeus vannamei (PV), and Procambarus clarkia (PCC)) were compared and characterized based on UPLC–Q–Exactive Orbitrap/MS. We compared the differences in lipid composition of four kinds of shrimp head using multivariate analysis. In addition, a zebrafish model was used to evaluate pro-angiogenic, anti-inflammatory, anti-thrombotic, and cardioprotective activities of the shrimp head lipids. The lipids from the four kinds of shrimp head had different degrees of pro-angiogenic activities, and the activities of PCC and PJ shrimp lipids were more significant than those of the other two species. Four lipid groups displayed strong anti-inflammatory activities. For antithrombotic activity, only PCC (25 μg/mL) and PV (100 μg/mL) groups showed obvious activity. In terms of cardioprotective activity, the four kinds of lipid groups significantly increased the zebrafish heart rhythms. The heart distances were shortened, except for those of the FC (100 μg/mL) and PJ (25 μg/mL) groups. Our comprehensive lipidomics analysis and bioactivity study of lipids from different sources could provide a basis for the better utilization of shrimp.