Production of CaCO3-single-coated probiotics and evaluation of their spectroscopic properties, morphological characteristics, viability, and intestinal delivery efficiency.

The intake of probiotics offers various health benefits; however, their efficacy depends on the maintenance of viability during industrial processing and digestion. Probiotic viability can be compromised during encapsulation, freeze-drying, storage, and digestion, necessitating multiple coatings. This complicates production and raises costs. In this study, CaCO3-single-coated probiotics (CSCPs) were prepared, an approach rarely reported before. Through instrumental analyses, the encapsulation of probiotics within CaCO3 was confirmed, ensuring their high viability. This proposed technology effectively preserves the viability of probiotics during the encapsulation and freeze-drying processes, resulting in minimal cell loss. Moreover, CSCPs demonstrated exceptional viability performance under simulated gastric and intestinal conditions. Notably, 100% of these microorganisms reached the intestines, delivering over 10 billion CFUs of probiotics in a viable state. This study highlights the potential of CSCPs as a feasible solution for overcoming probiotic encapsulation challenges and optimizing therapeutic benefits.

Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model.

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.

CRISPR/Cas9-mediated knockout of tnf-α1 in zebrafish reduces disease resistance after Edwardsiella piscicida bacterial infection.

Tumor necrosis factor (TNF) is an important cytokine involved in immune responses to bacterial infections in vertebrates, including fish. Although Tnf-α is a well-studied cytokine, there are contradictory findings about Tnf-α function following bacterial infection. In this study, we analyzed the expression and function of the Tnf-α-type I isoform (Tnf-α1) in zebrafish by knockout experiments using the CRISPR/Cas9 gene-editing tool. The open reading frame of tnf-α1 encodes a 25.82 kDa protein with 234 amino acids (aa). The expression of tnf-α1 in the early stages of zebrafish was observed from the 2-cell stage. Adult zebrafish spleens showed the highest expression of tnf-α1. To evaluate the function of Tnf-α1, an 8 bp deletion in the target region, resulting in a short truncated protein of 55 aa, was used to create the tnf-α1 knockout mutant. The pattern of downstream gene expression in 7-day larvae in wild-type (WT) and tnf-α1 knockout fish was examined. We also verified the fish mortality rate after Edwardsiella piscicida challenge and found that it was much higher in tnf-α1 knockout fish than in WT fish. Additionally, downstream gene expression analyses after E. piscicida exposure revealed a distinct expression pattern in tnf-α1 knockout fish compared to that in WT fish. Overall, our study using tnf-α1 deletion in zebrafish confirmed that Tnf-α1 is critical for immune regulation during bacterial infection.

Development of a Gene-Based Soybean-Origin Discrimination Method Using Allele-Specific Polymerase Chain Reaction.

A low soybean self-sufficiency rate in South Korea has caused a high import dependence and considerable price variation between domestic and foreign soybeans, causing the false labeling of foreign soybeans as domestic. Conventional soybean origin discrimination methods prevent a single-grain analysis and rely on the presence or absence of several compounds or concentration differences. This limits the origin discrimination of mixed samples, demonstrating the need for a method that analyzes individual grains. Therefore, we developed a method for origin discrimination using genetic analysis. The whole-genome sequencing data of the Williams 82 reference cultivar and 15 soybean varieties cultivated in South Korea were analyzed to identify the dense variation blocks (dVBs) with a high single-nucleotide polymorphism density. The PCR primers were prepared and validated for the insertion-deletion (InDel) sequences of the dVBs to discriminate each soybean variety. Our method effectively discriminated domestic and foreign soybean varieties, eliminating their false labeling.

Ablation of myd88 alters the immune gene expression and immune cell recruitment during VHSV infection in zebrafish.

Myeloid differentiation primary response protein-88 (MYD88) is an essential adaptor molecule in pathogen-related pattern recognition signaling pathways. Toll-like and interleukin receptors recognize numerous signals and are funneled through MyD88 to express genes responsible for the innate and adaptive immune systems. In the present study, the relevance of MyD88 in viral hemorrhagic septicemia virus (VHSV) was investigated by generating myd88-/- zebrafish. The model was challenged with VHSV, and viral propagation was quantified by evaluating clinical symptoms, mortality, and VHSV copy number. The infected fish showed abnormal morphologies, such as subcutaneous hemorrhages, abdominal swelling, and bulging eyes, which were comparatively more intense in myd88-/- fish than in the wild-type. An injury infection experiment conducted in zebrafish larvae indicated a substantial spread of VHSV in the wound site. The number of neutrophils and macrophages recruited to the wounded area were markedly reduced in myd88-/- fish. According to gene expression analysis, VHSV NP gene expression was considerably upregulated in myd88-/- fish. Substantial gene expression and immune cell marker modulation were observed in the mutant model compared to that in the wild-type. These results suggest that the lack of a significant adaptor protein for immune signal transduction results in enhanced VHSV replication.

Nanosized vaterite production through organic-solvent-free indirect carbonation.

Nanosized vaterite, which exhibits characteristics such as high specific surface area, porosity, and biocompatibility, has attracted research attention for use as a drug delivery material. However, fatal drawbacks such as high costs, difficulty in mass production, and toxicity exist in conventional nanosized vaterite production owing to the use of a large amount of organic solvents to forcibly suppress the vaterite recrystallization and particle growth. Therefore, nanosized 100 % vaterite was produced in this study via indirect carbonation without using any organic solvent, which has rarely been achieved previously. Seawater, sucrose, ultrasonication, and aging-which facilitate vaterite production and particle size reduction-exhibited a synergistic effect in producing vaterite. To realize nanosized vaterite production via indirect carbonation, seawater was used as a solvent, sucrose was added when Ca was eluted, and CO2 bubbling was performed under ultrasonication. Furthermore, the CaCO3-containing suspension obtained after the carbonation was aged. Ultrasonic waves were required to generate nanosized vaterite and reducing size at the carbonation stage. This nanosized-vaterite-production strategy involving organic-solvent-free indirect carbonation is meaningful, in that it highlights the potential of synthesizing vaterite in an economically sound, environmentally friendly manner for use as a pharmaceutical raw material.

CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection.

Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(-/-) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(-/-) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(-/-) than in healthy WT fish. The late response was observed in sting(-/-) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS-STING pathway. Furthermore, adult sting(-/-) fish had a high mortality rate and significantly downregulated cGAS-STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

Molecular insights into two ferritin subunits from red-lip mullet (Liza haematocheila): Detectable antibacterial activity with its expressional response against immune stimulants.

Iron (Fe) is considered as an essential micronutrient due to its diverse functions in living systems. However, regulation of free iron levels is essential because free Fe ions, in excess, induce biological toxicity through different routes, including production of reactive oxygen species. Ferritin proteins play a vital role in controlling free Fe ion homeostasis by sequestering excess iron in the body. Ferritins comprise an H subunit with a ferroxidase center and an L subunit with a Fe nucleation site. However, lower vertebrates such as fish harbor an additional subunit termed ferritin M, which shows the characteristic features of both H and L. In this study, two ferritin subunits (H and M) with ferroxidase centers were identified and characterized from red-lip mullet (Liza haematocheila). The open reading frames of red-lip mullet ferritin H (LhFerH) and ferritin M-like (LhFerM) subunits comprise 534 and 531 bps, which encode for putative polypeptides of 177 and 176 amino acids, respectively. LhFerH and LhFerM were found to retain well-conserved residues, including seven ferroxidase di-iron centers, characteristic domains, and signatures of their known homologs. We cloned the open reading frames of the two ferritin subunits to overexpress the corresponding proteins in Escherichia coli and subsequently demonstrated their iron sequestration activity along with antibacterial activity against E. coli using respective purified recombinant proteins in vitro. A basal expression analysis of two LhFer genes in selected tissues using qPCR assays showed pronounced expression in blood cells with respect to both genes. A relatively high expression level of LhFerH was also detected in muscle tissues. The expression level of LhFer in the head kidney was significantly up-regulated following lipopolysaccharides (LPS) and Lactococcus garvieae injection. The resulting gene expression pattern upon immune stimulation suggests that ferritin may contribute to the defense against harmful pathogen infection. Collectively, our results indicate that both LhFerH and LhFerM potentially participate in the homeostasis of free Fe ions and in the host immune defense of red-lip mullet.

Viperin mutation is linked to immunity, immune cell dynamics, and metabolic alteration during VHSV infection in zebrafish.

Viperin is a prominent antiviral protein found in animals. The primary function of Viperin is the production of 3'-deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP), an inhibitory nucleotide involved in viral RNA synthesis. Studies in mammalian models have suggested that ddhCTP interferes with metabolic proteins. However, this hypothesis has yet to be tested in teleost. In this study, the role of Viperin in regulating metabolic alterations during viral hemorrhagic septicemia virus (VHSV) infection was tested. When infected with VHSV, viperin -/- fish showed considerably higher mortality rates. VHSV copy number and the expression of the NP gene were significantly increased in viperin -/- fish. Metabolic gene analysis revealed significant differences in soda, hif1a, fasn, and acc expression, indicating their impact on metabolism. Cholesterol analysis in zebrafish larvae during VHSV infection showed significant upregulation of cholesterol production without Viperin. In vitro analysis of ZF4 cells suggested a considerable reduction in lipid production and a significant upregulation of reactive oxygen species (ROS) generation with the overexpression of viperin. Neutrophil and macrophage recruitment were significantly modulated in viperin -/- fish compared to the wild-type (WT) fish. Thus, we have demonstrated that Viperin plays a role in interfering with metabolic alterations during VHSV infection.

Generation of viperin-knockout zebrafish by CRISPR/Cas9-mediated genome engineering and the effect of this mutation under VHSV infection.

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin-/- compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin-/- fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin-/- fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.

Developmental toxicity of 3-phenoxybenzoic acid (3-PBA) and endosulfan sulfate derived from insecticidal active ingredients: Abnormal heart formation by 3-PBA in zebrafish embryos.

Pyrethroid and organochlorine insecticides are enormously used to control agricultural and indoor insect pests. The metabolites of pyrethroid and endosulfan were used to evaluate environmental toxicities using a representative animal model, zebrafish (Danio rerio) embryos in this study. The LC50 values in 3-phenoxy benzoic acid (3-PBA) and endosulfan sulfate (ES) were 1461 µg/L and 1459 µg/L, respectively. At the concentration of 2000 µg/L, spine curvature was observed in the ES-treated embryos. ES showed seizure-like events with an EC50 value of 354 µg/L. At the concentration of 1000 µg/L, the pericardial edema was observed in 3-PBA-treated embryos. The inhibition of heart development and the reduction of beating rates were observed in Tg(cmlc2:EGFP) embryos after the exposure to 3-PBA. Down-regulation of the vmhc gene coding ventricular myosin during heart development was significantly found in 3-PBA-treated embryos at 48 hpf, but recovered afterward. It indicates that ventricular malformation occurred at the initial stage of 3-PBA exposure. Considered together, both 3-PBA and ES need public concerns with periodic monitoring of these metabolites in households and agricultural areas to prevent humans and environmental organisms from their unexpected attacks.

Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame).

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.

Acute toxicity of the insecticide EPN upon zebrafish (Danio rerio) embryos and its related adverse effects: Verification of abnormal cardiac development and seizure-like events.

Toxicological studies of O-ethyl-O-(4-nitrophenyl) phenylphosphonothioate (EPN) to aquatic vertebrates have been reported, but no reports on toxic mechanism was reported. As zebrafish (Danio rerio) embryos were exposed to EPN, no changes in their survival and hatching rates were observed until 96 h post fertilization (hpf), even at the highest treated concentration of 500 µg/L. In both 250 µg/L and 500 µg/L, edemas were observed in the heart and yolk sac, and a blood pool was also found. Acridine orange staining confirmed apoptotic phynotype, which was the strongest in embryos at 48 hpf. No noticeable difference in the formation and the shape of blood vessels of Tg(fli1a:EGFP) was observed. However, the total body length and number of somite were decreased. Heart formation in Tg(cmlc2:EGFP) were not properly proceeded, and the ventricle did not beat normally at 500 µg/L level. Cardiac development-related genes, myl7 and nppa, were significantly down- and up-regulated in a concentration-dependent manner. The slowed heartbeat was confirmed using Tg(gata1:EGFP), showing stagnant blood flow and seizure-like events were observed. Altogether, EPN can be the cause for the abnormal heart development accompanied by blood stagnation in embryos, interfering normal development with their inner circulatory system.

Molecular Mechanisms of Anti-Melanogenic Gedunin Derived from Neem Tree (Azadirachta indica) Using B16F10 Mouse Melanoma Cells and Early-Stage Zebrafish.

Melanogenesis represents a series of processes that produce melanin, a protective skin pigment (against ultraviolet rays), and determines human skin color. Chemicals reducing melanin production have always been in demand in the cosmetic market because of skincare interests, such as whitening. The main mechanism for inhibiting melanin production is the inhibition of tyrosinase (TYR), a key enzyme for melanogenesis. Here, we evaluated gedunin (Ged), a representative limonoid, for its anti-melanogenesis action. Melanin production in vitro was stimulated by alpha-melanocyte stimulating hormone (α-MSH) in B16F10 mouse melanoma cells. Ged reduced α-MSH-stimulated melanin production, inhibiting TYR activity and protein amount. We confirmed this result in vivo in a zebrafish model for melanogenesis. There was no sign of toxicity and malformation of zebrafish embryos during development in all treated concentrations. Ged reduced the number of produced zebrafish embryo pigment dots and melanin contents of embryos. The highly active concentration of Ged (100 µM) was much lower than the positive control, kojic acid (8 mM). Hence, Ged could be a fascinating candidate for anti-melanogenesis reagents.

Generation of cd63-deficient zebrafish to analyze the role of cd63 in viral infection.

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

Molecular mechanisms of developmental toxicities of azoxystrobin and pyraclostrobin toward zebrafish (Danio rerio) embryos: Visualization of abnormal development using two transgenic lines.

Azoxystrobin (AZ) and pyraclostrobin (PY) are strobilurin fungicides that inhibit fungal mitochondrial respiration. In this study, a representative model, zebrafish (Danio rerio), was used as a test species for acute and developmental toxicity. Survival and malformation rates were observed only PY-treated embryos, with an LC50 value of 77.75 ppb accompanied by a dramatic decrease in hatching rate, while AZ did not show great mortality. Morphological changes were observed in PY-treated embryos with the occurrence of pericadial edema at 25 ppb. A delay in growth was observed after treatment with pyraclostrobin at 50 ppb. Use of genetically engineered Tg(cmlc:EGFP) allowed fluorescence observation during heart development. PY interfered with normal heart development via upregulation of the nppa gene responsible for the expression of natriuretic peptides. Heart function was dramatically reduced as indicated by reduced heart rates. Increased expression of the nppa gene was also seen in AZ-treated embryos. The expression level of cyp24a1 was also up-regulated, while ugt1a1 and sult1st6 were down-regulated after treatment of zebrafish embryos with AZ or PY. Overall, strobilurin fungicides might inhibit normal heart formation and function within the range of concentrations tested.

Influencing nutritional habits of college students using a food pantry.

Objective: Food insecurity in college students is associated with poor eating habits. The purpose of this project was to increase consumption of fruits and vegetables in college students using a campus food pantry. Participants: Twenty-nine college students completed the study. Methods: Participants viewed weekly food preparation videos demonstrating cost-effective recipes containing fruits and vegetables and a key take-away message. Participants were provided a recipe and food to cook at home based on the Cooking Matters Food Pantry Toolkit. A 24-h dietary recall was collected pre- and postintervention using the Automated Self-Administered 24-H Dietary Assessment. This descriptive comparative study used repeated measure ANOVA. Results: Females (n = 19) increased their fruit and vegetable consumption by 1.3 cups daily (p < .05) while males (n = 10) decreased fruit and vegetable consumption at the postintervention measurement. Conclusion: Increased consumption of fruits and vegetables in female college students using the food pantry was a modifiable behavior in this study.

Flipping to Motivate: Perceptions Among Prelicensure Nursing Students.

BACKGROUND: Research suggests flipped learning may improve student motivation to learn. PURPOSE: This study examined motivation and learning strategies among prelicensure BSN students in a course taught using the flipped learning model. The hypothesis was that flipped learning may improve motivation and learning strategies of students. METHODS: A descriptive, comparative design examined motivation and learning strategies used by students in a flipped learning course. The Motivated Strategies for Learning Questionnaire and a demographic survey were administered to 25 BSN students at a Midwestern university. RESULTS: Substantial decreases were found from pretest to posttest mean scores for the task value, control of learning beliefs, and help seeking subscales. CONCLUSION: The results did not support the hypothesis that flipped learning would improve motivation and learning strategies. Further research in larger diverse samples of students in a flipped learning course is recommended.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Acute toxicities of fluorene, fluorene-1-carboxylic acid, and fluorene-9-carboxylic acid on zebrafish embryos (Danio rerio): Molecular mechanisms of developmental toxicities of fluorene-1-carboxylic acid.

In this study, fluorene (FL), FL-1-carboxylic acid (FC-1), and FL-9-carboxylic acid (FC-9) were investigated to understand their acute toxicity by measuring inhibitory effects on hatching rates and developmental processes of zebrafish embryos (Danio rerio). For exposure concentrations up to 3000 µg/L, FC-1 alone showed acute toxicity at 1458 µg/L for LC50 value. FC-1 caused yolk sac and spinal deformities, and pericardial edema. Molecular studies were undertaken to understand FC-1 toxicity examining 61 genes after exposure to 5 µM (equivalent to LC20 value of FC-1) in embryos. In the FC-1-treated embryos, the expression of the cyp7a1 gene, involved in bile acid biosynthesis, was dramatically decreased, while the expression of the Il-1ß gene involved in inflammation was remarkably increased. In addition to these findings, in FC-1-treated embryos, the expression of nppa gene related to the differentiation of the myocardium was 3-fold increased. On the other hand, cyp1a, cyp3a, ugt1a1, abcc4, mdr1, and sult1st1 responsible for detoxification of xenobiotics were upregulated in FC-9-treated embryos. Taken together, carboxylation on carbon 1 of FL increased acute toxicity in zebrafish embryos, and its toxicity might be related to morphological changes with modification of normal biological functions and lowered defense ability.