Performance Comparison of Five Methods for Tetrahymena Number Counting on the ImageJ Platform: Assessing the Built-in Tool and Machine-Learning-Based Extension.

Previous methods to measure protozoan numbers mostly rely on manual counting, which suffers from high variation and poor efficiency. Although advanced counting devices are available, the specialized and usually expensive machinery precludes their prevalent utilization in the regular laboratory routine. In this study, we established the ImageJ-based workflow to quantify ciliate numbers in a high-throughput manner. We conducted Tetrahymena number measurement using five different methods: particle analyzer method (PAM), find maxima method (FMM), trainable WEKA segmentation method (TWS), watershed segmentation method (WSM) and StarDist method (SDM), and compared their results with the data obtained from the manual counting. Among the five methods tested, all of them could yield decent results, but the deep-learning-based SDM displayed the best performance for Tetrahymena cell counting. The optimized methods reported in this paper provide scientists with a convenient tool to perform cell counting for Tetrahymena ecotoxicity assessment.

L-Carnitine ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish.

BACKGROUND: Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. METHODS: Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. RESULTS: While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment. CONCLUSIONS: These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Evaluation of the Effects of Carbon 60 Nanoparticle Exposure to Adult Zebrafish: A Behavioral and Biochemical Approach to Elucidate the Mechanism of Toxicity.

There is a growing concern for the potential toxicity of engineered nanomaterials that have made their way into virtually all novel applications in the electronics, healthcare, cosmetics, technology, and engineering industries, and in particular, biomedical products. However, the potential toxicity of carbon 60 (C60) at the behavioral level has not been properly evaluated. In this study, we used idTracker, a multitracking algorithm to quantitatively assess behavioral toxicity induced by C60 nanoparticles (C60 NPs) in adult zebrafish. We demonstrated that locomotion, novel tank exploration, aggression, shoaling, and color preference activities of the C60 NPs-treated fish was significantly reduced. In addition, the C60 NPs-treated fish also displayed dysregulation of the circadian rhythm by showing lower locomotion activities in both day and night cycles. The biochemical results showed that C60 NPs exposure at low concentration induced oxidative stress and DNA damage, reduced anti-oxidative capacity and ATP (adenosine triphosphate) levels, and induced stress-associated hormones, hypoxia, as well as inflammation marker upregulation in muscle and gill tissues. Together, this work, for the first time, provide direct evidence showing that the chronic exposure of C60 NPs induced multiple behavioral abnormalities in adult zebrafish. Our findings suggest that the ecotoxicity of C60 NPs towards aquatic vertebrates should be carefully evaluated.

Zinc Chloride Exposure Inhibits Brain Acetylcholine Levels, Produces Neurotoxic Signatures, and Diminishes Memory and Motor Activities in Adult Zebrafish.

In this study, we evaluated the acute (24, 48, 72, and 96 h) and chronic (21 days) adverse effects induced by low doses (0.1, 0.5, 1, and 1.5 mg/L) of zinc chloride (ZnCl2) exposure in adult zebrafish by using behavioral endpoints like three-dimensional (3D) locomotion, passive avoidance, aggression, circadian rhythm, and predator avoidance tests. Also, brain tissues were dissected and subjected to analysis of multiple parameters related to oxidative stress, antioxidant responses, superoxide dismutase (SOD), neurotoxicity, and neurotransmitters. The results showed that ZnCl2-exposed fishes displayed decreased locomotor behavior and impaired short-term memory, which caused an Alzheimer's Disease (AD)-like syndrome. In addition, low concentrations of ZnCl2 induced amyloid beta (amyloid ß) and phosphorylated Tau (p-Tau) protein levels in brains. In addition, significant induction in oxidative stress indices (reactive oxygen species (ROS) and malondialdehyde (MDA)), reduction in antioxidant defense system (glutathione (GSH), GSH peroxidase (GSH-Px) and SOD) and changes in neurotransmitters were observed at low concentrations of ZnCl2. Neurotoxic effects of ZnCl2 were observed with significant inhibition of acetylcholine (ACh) activity when the exposure dose was higher than 1 ppm. Furthermore, we found that zinc, metallothionein (MT), and cortisol levels in brain were elevated compared to the control group. A significantly negative correlation was observed between memory and acetylcholinesterase (AChE) activity. In summary, these findings revealed that exposure to ZnCl2 affected the behavior profile of zebrafish, and induced neurotoxicity which may be associated with damaged brain areas related to memory. Moreover, our ZnCl2-induced zebrafish model may have potential for AD-associated research in the future.

Zebrafish Mutants Carrying Leptin a (lepa) Gene Deficiency Display Obesity, Anxiety, Less Aggression and Fear, and Circadian Rhythm and Color Preference Dysregulation.

Leptin, a hormone secreted by peripheral adipose tissues, regulates the appetite in animals. Recently, evidence has shown that leptin also plays roles in behavioral response in addition to controlling appetite. In this study, we examined the potential function of leptin on non-appetite behaviors in zebrafish model. By using genome editing tool of Transcription activator-like effector nuclease (TALEN), we successfully knocked out leptin a (lepa) gene by deleting 4 bp within coding region to create a premature-translation stop. Morphological and appetite analysis showed the lepa KO fish display a phenotype with obese, good appetite and elevation of Agouti-related peptide (AgRP) and Ghrelin hormones, consistent with the canonical function of leptin in controlling food intake. By multiple behavior endpoint analyses, including novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm, and color preference assay, we found the lepa KO fish display an anxiogenic phenotype showing hyperactivity with rapid swimming, less freezing time, less fear to predator, loose shoaling area forming, and circadian rhythm and color preference dysregulations. Using biochemical assays, melatonin, norepinephrine, acetylcholine and serotonin levels in the brain were found to be significantly reduced in lepa KO fish, while the levels of dopamine, glycine and cortisol in the brain were significantly elevated. In addition, the brain ROS level was elevated, and the anti-oxidative enzyme catalase level was reduced. Taken together, by performing loss-of-function multiple behavior endpoint testing and biochemical analysis, we provide strong evidence for a critical role of lepa gene in modulating anxiety, aggression, fear, and circadian rhythm behaviors in zebrafish for the first time.

The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence.

Carassius auratus complex formula (CACF) is a traditional Chinese medicine known for its antidiabetic effects. Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide, and there are currently no effective therapies for advanced HCC. This study explores the comprehensive effects and possible mechanisms of CACF on HCC. The results show that CACF reduces the viability of hepatoma cells in vitro, while benefiting normal hepatocytes. In addition, CACF inhibits hepatoma cell growth in a zebrafish xenotransplantation model and decreases lipid accumulation, represses inflammation and cell proliferation markers in fatty acid translocase (CD36) transgenic zebrafish, and inhibits the expression of cell proliferation and ß-catenin downstream targets in telomerase (tert) transgenic zebrafish models. Ingenuity Pathway Analysis reveals that CACF exerts multiple functions, including reduction of inflammation and inhibition of lipid transporter and PPAR signaling pathway. Surprisingly, CACF also regulates the expression of genes and reduces coronavirus infection and pathogenesis in a zebrafish model. CACF treatment is validated to regulate the expression of genes for anti-coronavirus activity. Mechanistically, CACF stabilizes G-quadruplex and reduces cell senescence associated ß-galactosidase activity. In summary, CACF may be a promising therapeutic agent with multiple functions including anticancer, anti-inflammation, and anti-microorganisms in a zebrafish model.

TCMacro: A Simple and Robust ImageJ-Based Method for Automated Measurement of Tail Coiling Activity in Zebrafish.

Tail coiling is a reflection response in fish embryos that can be used as a model for neurotoxic analysis. The previous method to analyze fish tail coiling is largely based on third-party software. In this study, we aim to develop a simple and cost-effective method called TCMacro by using ImageJ macro to reduce the operational complexity. The basic principle of the current method is based on the dynamic change of pixel intensity in the region of interest (ROI). When the fish tail is moving, the average intensity is increasing. In time when the fish freeze, the peak of mean intensity is maintaining at a relatively low level. By using the optimized macro settings and excel VBA scripts, all the tail coiling measurement processes can be archived with few operation steps with high precision. Three major endpoints of tail coiling counts, tail coiling duration and tail coiling intervals can be obtained in batch. To validate this established method, we tested the potential neurotoxic activity of Tricaine (methanesulfonate, MS-222) and psychoactive compound of caffeine. Zebrafish embryos after Tricaine exposure displayed significantly less tail coiling activity in a dose-dependent manner, and were comparable to manual counting through the Wilcoxon test and Pearson correlation double validation. Zebrafish embryos after caffeine exposure displayed significantly high tail coiling activity. In conclusion, the TCMacro method presented in this study provides a simple and robust method that is able to measure the relative tail coiling activities in zebrafish embryos in a high-throughput manner.

Low molecular weight fucoidan inhibits hepatocarcinogenesis and nonalcoholic fatty liver disease in zebrafish via ASGR/STAT3/HNF4A signaling.

BACKGROUND: Hepatocellular carcinoma ranks fourth in cancer-related mortality currently lacks effective therapeutics. Fucoidan is sulfated polysaccharide that is mainly found in brown seaweeds. In this study, we investigated the effects and mechanisms of low molecular weight fucoidan (i.e. oligo-fucoidan [OF]) preventing hepatocarcinogenesis. METHODS: We used [HBx,src], [HBx,src,p53-/+ ], and [CD36] transgenic zebrafish liver cancer model treated with OF, and performed molecular and histopathological analysis. Transcriptomic and pathways analysis was performed. RESULTS: Decreased expression of lipogenic enzymes, fibrosis markers, and cell cycle/proliferation markers by OF in [HBx,src] and [HBx,src,p53-/+ ] transgenic fish. Liver fibrosis was decreased as revealed by Sirius Red staining, and the liver cancer formation was eventually reduced by feeding OF. OF was also found to be capable of reducing lipid accumulation and cancer formation in non-B non-C Hepatocellular carcinoma (HCC) model in CD36 transgenic zebrafish. Whole-genome expression analysis showed that 661 genes were up-regulated, and 451 genes were downregulated by feeding OF. Upregulated genes were mostly found in protein transporter activity, and downregulated genes were enriched with response to extracellular stimulus and metal binding in gene ontology analysis. The driver gene was HNF4A revealed by NetworkAnalyst from OF differential regulated genes at various insults. OF is able to bind the asialoglycoprotein receptor (ASGR) in hepatoma cells, and increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in both hepatoma cells and [HBx,src,p53-/+ ] transgenic fish liver cancer model. Using chromatin-immunoprecipitation, we found pSTAT3 could associate with the P1 promoter of HNF4A. Knockdown of either ASGR or HNF4A reversed OF mediated anti-cancer cell proliferation. CONCLUSIONS: Taken together, we provide evidence that OF exhibits the anti-HCC, anti-steatosis, and anti-fibrosis effect for liver in zebrafish models, and the anti-cancer potential of OF attributed to the binding to ASGR and activation of STAT3/HNF4A signaling. OF might be potentially valuable for the management of HCC.

WNK1 Kinase Stimulates Angiogenesis to Promote Tumor Growth and Metastasis.

With-no-lysine (K)-1 (WNK1) is the founding member of family of four protein kinases with atypical placement of catalytic lysine that play important roles in regulating epithelial ion transport. Gain-of-function mutations of WNK1 and WNK4 cause a mendelian hypertension and hyperkalemic disease. WNK1 is ubiquitously expressed and essential for embryonic angiogenesis in mice. Increasing evidence indicates the role of WNK kinases in tumorigenesis at least partly by stimulating tumor cell proliferation. Here, we show that human hepatoma cells xenotransplanted into zebrafish produced high levels of vascular endothelial growth factor (VEGF) and WNK1, and induced expression of zebrafish wnk1. Knockdown of wnk1 in zebrafish decreased tumor-induced ectopic vessel formation and inhibited tumor proliferation. Inhibition of WNK1 or its downstream kinases OSR1 (oxidative stress responsive kinase 1)/SPAK (Ste20-related proline alanine rich kinase) using chemical inhibitors decreased ectopic vessel formation as well as proliferation of xenotransplanted hepatoma cells. The effect of WNK and OSR1 inhibitors is greater than that achieved by inhibitor of VEGF signaling cascade. These inhibitors also effectively inhibited tumorigenesis in two separate transgenic zebrafish models of intestinal and hepatocellular carcinomas. Endothelial-specific overexpression of wnk1 enhanced tumorigenesis in transgenic carcinogenic fish, supporting endothelial cell-autonomous effect of WNK1 in tumor promotion. Thus, WNK1 can promote tumorigenesis by multiple effects that include stimulating tumor angiogenesis. Inhibition of WNK1 may be a potent anti-cancer therapy.

Establishing simple image-based methods and a cost-effective instrument for toxicity assessment on circadian rhythm dysregulation in fish.

Analysis of circadian rhythm behavior alteration in fish for toxicity assessment usually requires expensive commercial equipment and laborious and complicated tweaking. Here, we report a simple setup that consists of a custom-made light box equipped with white and 940 nm light-emitting diode (LED) light strips as light sources, where the locomotion activities of zebrafish or catfish are captured using an infrared-sensitive coupled charged device (CCD). The whole setup was housed in a temperature-controlled incubator to isolate external noise and to maintain consistent experimental conditions. The video recording and light triggering were synchronized using Total Recorder, a recording scheduling software. By using the setup mentioned above and open source software such as ImageJ or idTracker, the locomotion activities of diurnal (e.g. zebrafish) and nocturnal (e.g. catfish) fish during day and night cycles can be quantitatively analyzed. We used simple image-based methods and a cost-effective instrument to assess the circadian rhythm of multiple fish species, as well as other parameters such as age, ambient temperature and chemical toxicology with high precision and reproducibility. In conclusion, the instrument setting and analysis methods established in this study provide a reliable and easy entry point for toxicity assessment on circadian rhythm dysregulation in fish.

Cardiac Rhythm and Molecular Docking Studies of Ion Channel Ligands with Cardiotoxicity in Zebrafish.

Safety is one of the most important and critical issues in drug development. Many drugs were abandoned in clinical trials and retracted from the market because of unknown side effects. Cardiotoxicity is one of the most common reasons for drug retraction due to its potential side effects, i.e., inducing either tachycardia, bradycardia or arrhythmia. The zebrafish model could be used to screen drug libraries with potential cardiotoxicity in a high-throughput manner. In addition, the fundamental principles of replacement, reduction, and refinement of laboratory animal usage, 3R, could be achieved by using zebrafish as an alternative to animal models. In this study, we used a simple ImageJ-based method to evaluate and screen 70 ion channel ligands and successfully identify six compounds with strong cardiotoxicity in vivo. Next, we conducted an in silico-based molecular docking simulation to elucidate five identified compounds that might interact with domain III or domain IV of the Danio rerio L-type calcium channel (LTCC), a known pharmaceutically important target for arrhythmia. In conclusion, in this study, we provide a web lab and dry lab combinatorial approach to perform in vivo cardiotoxicity drug screening and in silico mechanistic studies.

A Novel AURKA Mutant-Induced Early-Onset Severe Hepatocarcinogenesis Greater than Wild-Type via Activating Different Pathways in Zebrafish.

Aurora A kinase (AURKA) is an important regulator in mitotic progression and is overexpressed frequently in human cancers, including hepatocellular carcinoma (HCC). Many AURKA mutations were identified in cancer patients. Overexpressing wild-type Aurka developed a low incidence of hepatic tumors after long latency in mice. However, none of the AURKA mutant animal models have ever been described. The mechanism of mutant AURKA-mediated hepatocarcinogenesis is still unclear. A novel AURKA mutation with a.a.352 Valine to Isoleucine (V352I) was identified from clinical specimens. By using liver-specific transgenic fish overexpressing both the mutant and wild-type AURKA, the AURKA(V352I)-induced hepatocarcinogenesis was earlier and much more severe than wild-type AURKA. Although an increase of the expression of lipogenic enzyme and lipogenic factor was observed in both AURKA(V352I) and AURKA(WT) transgenic fish, AURKA(V352I) has a greater probability to promote fibrosis at 3 months compared to AURKA(WT). Furthermore, the expression levels of cell cycle/proliferation markers were higher in the AURKA(V352I) mutant than AURKA(WT) in transgenic fish, implying that the AURKA(V352I) mutant may accelerate HCC progression. Moreover, we found that the AURKA(V352I) mutant activates AKT signaling and increases nuclear ß-catenin, but AURKA(WT) only activates membrane form ß-catenin, which may account for the differences. In this study, we provide a new insight, that the AURKA(V352I) mutation contributes to early onset hepatocarcinogenesis, possibly through activation of different pathways than AURKA(WT). This transgenic fish may serve as a drug-screening platform for potential precision medicine therapeutics.

Identification of Novel Anti-Liver Cancer Small Molecules with Better Therapeutic Index than Sorafenib via Zebrafish Drug Screening Platform.

Hepatocellular carcinoma (HCC) ranks as the fourth leading cause of cancer-related deaths worldwide. Sorafenib was the only U.S. Food and Drug Administration (FDA) approved drug for treating advanced HCC until recently, so development of new target therapy is urgently needed. In this study, we established a zebrafish drug screening platform and compared the therapeutic effects of two multiple tyrosine kinase inhibitors, 419S1 and 420S1, with Sorafenib. All three compounds exhibited anti-angiogenesis abilities in immersed fli1:EGFP transgenic embryos and the half inhibition concentration (IC50) was determined. 419S1 exhibited lower hepatoxicity and embryonic toxicity than 420S1 and Sorafenib, and the half lethal concentration (LC50) was determined. The therapeutic index (LC50/IC50) for 419S1 was much higher than for Sorafenib and 420S1. The compounds were either injected retro-orbitally or by oral gavage to adult transgenic zebrafish with HCC. The compounds not only rescued the pathological feature, but also reversed the expression levels of cell-cycle-related genes and protein levels of a proliferation marker. Using a patient-derived-xenograft assay, we found that the effectiveness of 419S1 and 420S1 in preventing liver cancer proliferation is better than that of Sorafenib. With integrated efforts and the advantage of the zebrafish platform, we can find more effective and safe drugs for HCC treatment and screen for personalized medicine.

Genome skimming yields the complete mitogenome of Chromodoris annae (Mollusca: Chromodorididae).

In this study, the complete mitogenome sequence of sea slug, Chromodoris annae (Mollusca: Chromodorididae), has been decoded for the first time by genome skimming method. The overall base composition of C. annae mitogenome is 30.6% for A, 14.5% for C, 17.8% for G, and 37.2% for T, and has GC content of 32.2%. The assembled mitogenome, consisting of 14,260 bp, has unique 13 protein-coding genes (PCGs), 22 transfer RNAs, and two ribosomal RNAs genes. The C. annae has the common mitogenome gene organization and feature of Chromodorididae. The complete mitogenome of C. annae provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for sea slugs.