Assessment of the inbred C57BL/6 and outbred CD1 mouse strains using a progressive ratio schedule during development.

Inbred strains have a genetic similarity of at least 98.6% compared to their outbred counterparts. Several studies have shown that inbred C57BL/6 mice and outbred ICR (CD1) mice differ in locomotion, cognitive flexibility, and aggression. However, their performance in operant paradigms is not well understood. A progressive ratio (PR) schedule of reinforcement is a method of quantitative estimation of the incentive state of an animal for a reward by increasing response requirements for reinforcer delivery, which is relevant to assess the breakpoint (amount of response effort an animal is willing to invest for a single unit of reward). This study tested male and female C57BL/6 and CD1 mice with an open field to analyze locomotion. Then, we used conditioning chambers with a PR3 schedule for ten consecutive days (P30-P40). PR performance was measured with the breakpoint, and the mathematical principles of reinforcement (MPR) were used to estimate motivation, impulsivity, and motor skills to manipulate the operandum. We found that CD1 mice showed higher locomotor activity than C57BL/6 independently of sex. CD1 mice had a higher breakpoint. However, male CD1 mice gradually increased breakpoint until the last session. In the MPR model, CD1 mice showed decreased fixed paused parameter (impulsivity) than C57BL/6, independent of sex. Our data suggest that the higher breakpoint in CD1 strain may partially be related to impulsivity. Therefore, the MPR model can help identify factors that affect performances, such as motivation, impulsivity, and motor skills during a PR in adolescent CD1 and C57BL/6 mice. These findings are essential to characterize the differences in the behavioral performance between C57BL/6 and CD1 strains and their potential as animal models.

A comparative study on chromium-induced micronuclei assessment in the peripheral blood of Hsd:ICR mice.

This study investigated the genotoxic effects of chromium (Cr) in Hsd:ICR mice, considering factors such as oxidative state, apoptosis, exposure pathway, duration, pregnancy, and transplacental exposure. Genotoxicity was assessed using the erythrocytes' micronucleus (MN) assay, while apoptosis was evaluated in nucleated blood cells. The results showed that Cr(III) (CrK(SO4 )2 and CrCl3 ) did not induce any marked genotoxic damage. However, Cr(VI) (CrO3 , K2 Cr2 O7 , Na2 Cr2 O7 , and K2 CrO4 ) produced varying degrees of genotoxicity, with CrO3 being the most potent. MN frequencies increased following 24-h exposure, with a greater effect in male mice. Administering 20 mg/kg of CrO3 via gavage did not lead to significant effects compared to intraperitoneal administration. Short-term oral treatment with a daily dose of 8.5 mg/kg for 49 days elevated MN levels only on day 14 after treatment. Pregnant female mice exposed to CrO3 on day 15 of pregnancy exhibited reduced genotoxic effects compared to nonpregnant animals. However, significant increases in MN levels were found in their fetuses starting 48 h after exposure. In summary, data indicate the potential genotoxic effects of Cr, with Cr(VI) forms inducing higher genotoxicity than Cr(III). These findings indicate that gender, exposure route, and pregnancy status might influence genotoxic responses to Cr.

Carcinogenicity assessment of tegoprazan in Sprague-Dawley (Crl:CD) rats and ICR (Crl:CD1) mice.

Tegoprazan is a novel potassium-competitive acid blocker (P-CAB) that reversibly inhibits the proton pump in gastric parietal cells and has been approved for the treatment of acid-related diseases in Korea. This study aimed to evaluate the carcinogenic potential of tegoprazan in Sprague-Dawley rats and CD-1 mice. Tegoprazan was administered daily by oral gavage to rats for up to 94 weeks and mice for up to 104 weeks. Evidence of carcinogenic potential of tegoprazan was identified in rats only and was limited to benign and/or malignant neuroendocrine cell tumors at exposures >7-fold of the recommended human dose. Glandular stomach findings were considered secondary to the expected pharmacology of tegoprazan, characterized by their location in the fundic and body regions of the stomach. Overall, tegoprazan induced gastric enterochromaffin-like (ECL) cell tumors in SD rats, but did not produce any treatment-related statistically significant increase in the incidence of neoplasms relevant to humans when administered to SD rats and CD-1 mice by gavage at doses up to 300 and 150 mg/kg/day, respectively. Gastric ECL cell tumors are thought to be induced by the exaggerated indirect pharmacological effect of tegoprazan, similar to that reported for proton pump inhibitors (PPIs) and other P-CABs.

Genotoxicity assessment of root extracts of Paeonia lactiflora Pall.

The roots of Paeonia lactiflora Pall., (Paeoniae Radix, PL) are a well-known herbal remedy used to treat fever, rheumatoid arthritis, systemic lupus erythematosus, hepatitis, and gynecological disorders in East Asia. Here we evaluated the genetic toxicity of PL extracts (as a powder [PL-P] and hot-water extract [PL-W]) in accordance with the Organization for Economic Co-operation and Development guidelines. The Ames test revealed that PL-W was not toxic to S. typhimurium strains and E. coli in absence and presence of the S9 metabolic activation system at concentrations up to 5000 µg/plate, but PL-P produced a mutagenic response to TA100 in the absence of S9 mix. PL-P was cytotoxic in in vitro chromosomal aberrations (more than a 50 % decrease in cell population doubling time), and it increased the frequency of structural and numerical aberrations in absence and presence of S9 mix in a concentration-dependent manner. PL-W was cytotoxic in the in vitro chromosomal aberration tests (more than a 50 % decrease in cell population doubling time) only in the absence of S9 mix, and it induced structural aberrations only in the presence of S9 mix. PL-P and PL-W did not produce toxic response during the in vivo micronucleus test after oral administration to ICR mice and did not induce positive results in the in vivo Pig-a gene mutation and comet assays after oral administration to SD rats. Although PL-P showed genotoxic in two in vitro tests, the results from physiologically relevant in vivo Pig-a gene mutation and comet assays illustrated that PL-P and PL-W does not cause genotoxic effects in rodents.

Safety assessment of marigold flavonoids from marigold inflorescence residue.

ETHNOPHARMACOLOGICAL RELEVANCE: Marigold flavonoids, extracted from marigold (Tagetes erecta L.) inflorescence residues, have attracted significant attention with respect to antioxidant, anti-inflammatory and chelating properties. However, the toxicity of marigold flavonoids have not yet been fully investigated. AIM OF THE STUDY: The main purpose of this study was to assess the safety of marigold flavonoids extracted from Marigold (Tagetes erecta L.) in order to provide information on its nonclinical safety. Thus, the acute oral toxicity, in vitro Ames test, sperm aberration study, bone marrow micronucleus test, subchronic oral toxicity test, and teratogenic potential were carried out in rats or mice. MATERIALS AND METHODS: For an acute oral toxicity test, SD rats and ICR mice (male and female, n = 5) orally received a single dose of 5000 mg/kg marigold flavonoids. Evaluation of marigold flavonoids genotoxic potential with a battery of tests, including an in vitro bacterial reverse mutation test using four mutant strains of Salmonella typhimurium (TA97、TA98、TA100、TA102), an sperm aberration test and an in vivo micronucleus test using bone marrow cells ICR mice that were orally administered marigold flavonoids, an subchronic oral toxicity study and teratogenic test employing male and female SD rats that were orally administered marigold flavonoids. All animals tests were completed in accordance with GB 15193 for toxicity tests. RESULTS: In the acute oral toxicity test, marigold flavonoids given at the dose of 5000 mg/kg body weight for 14 days didn't produce any abnormal clinical symptoms or mortality in SD rats and ICR mice (both sex, n = 5). There was no evidence of genotoxicity of marigold flavonoids based on the results of the in vitro bacterial reverse mutation test (up to 1250 µg/plate), the sperm aberration test (up to 5000 mg/kg body weight), the in vivo micronucleus test (up to 5000 mg/kg body weight), the subchronic oral toxicity study (up to 10 g/kg feed dose) and the teratogenic test (up to 1250 mg/kg body weight). CONCLUSIONS: We found that marigold flavonoids are safe with regard to acute toxicity in rats or mice as well as genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of marigold flavonoids as a potential therapeutic material for the traditional use of herbal medicines and for the further development of novel antioxidant.

Safety assessment of functional oligooctasaccharide riclinoctaose: A pilot study of genotoxicity, acute toxicity, and subchronic toxicity.

Riclinoctaose was produced by enzymatic hydrolysis of a succinoglycan-type exopolysaccharide riclin. It can be used as a prebiotic to regulate the composition of gut microbiota. Therefore, a safety evaluation is needed. Here, we reported the safety data generated on riclinoctaose. Standard in vitro genotoxicity tests such as the bacterial reverse mutation assay and in vivo micronucleus assay were performed and no mutagenic or clastogenic potential was found. In the acute toxicity study, ICR mice were administered with riclinoctaose via gavage in 14-day studies at the level corresponding to 3000 mg/kg BW/day. In the subchronic study, the diets containing 0%, 1.0%, 2.5%, and 5.0% of riclinoctaose (weight/weight) were prepared for ICR mice for 13 weeks. No test item-related adverse effects were observed in the acute and subchronic studies. No riclinoctaose-induced differences in the overall health, body weight gain, food and water consumption, hematology, blood chemistry, gross pathology, histopathology, or animal death were observed. A no-observed-adverse-effect level of 8842 mg/kg BW/day for male and 9230 mg/kg BW/day for female mice was identified for riclinoctaose when administered for 13 weeks. In conclusion, these findings demonstrated the safety of riclinoctaose and indicated the possibility that riclinoctaose may be used as a functional food. PRACTICAL APPLICATION: Functional oligosaccharide is a low-calorie sweetener, which is beneficial to human health. Dietary riclinoctaose can improve intestinal health and understanding the safety of riclinoctaose is the first step to evaluate its potential use in functional food. Therefore, genotoxicity, acute toxicity, and subchronic toxicity of riclinoctaose were studied.

Female mouse model of diabetes mellitus induced by streptozotocin and high-carbohydrate high-fat diet.

With the improvement of people's living standards and rapid economic development, the incidence of diabetes mellitus (DM) is increasing in most parts of the world. DM presents an important potential threat to human health. In the present study, a model of diabetes in female mice was established, and fasting blood glucose was detected at week 4, after which the biochemical profiles were evaluated by histopathological analysis. The success rate of modeling in the normal control (NC) group and the low/ middle/high-dose streptozotocin (STZ) group were 0, 0, 25% and 60%, respectively. In the middle-dose and high-dose STZ groups, the liver index was increased significantly compared with the NC group (p⟨0.05). The blood biochemical indicators of total cholesterol and low density lipoprotein cholesterol in three STZ injection groups were as follows: alanine aminotransferase and aspartate transaminase in middle- and high-dose STZ groups, high-density lipoprotein cholesterol and serum creatinine in the high-dose STZ group, and blood urea nitrogen in the middle-dose STZ group were significantly increased (p⟨0.05). The level of total triglycerides was lower, obviously, in the high-dose STZ group than in the NC group (p⟨0.05). The mice showed marked steatosis, green-dyed fiber tissue coloring in varying degrees, and the contour of the hepatic lobules basically disappeared in STZ injection groups. The results suggest that to establish a diabetes model for female ICR mice, the optimum dose of STZ is 100 mg/kg.

In vivo impact assessment of orally administered polystyrene nanoplastics: biodistribution, toxicity, and inflammatory response in mice.

To assess the in vivo impact of nanoplastics (NP) and coagulation-based purified NP (PurNP), this study analyzed for alterations in the biodistribution, toxicity and inflammatory response in ICR mice exposed to three different doses of NP (5, 25, and 50 mg/kg) and PurNP for 2 weeks. Except water consumption, which was dose-dependently and significantly increased in all NP-treated groups, most factors assessed for feeding behaviors and excretions remained constant, without any significant change. Orally administered NP was detected in the intestine, kidneys, and liver at all concentrations, although the accumulation was higher in the intestine than in the kidneys and liver. No significant alterations were detected in the levels of serum biochemical markers and histopathological structures. However, compared to the vehicle group, expressions of the inflammatory response proteins (iNOS and COX-2) and mRNA levels of the inflammatory cytokines were remarkably increased in the liver, kidneys, and intestine of NP-treated mice. A similar increase was detected in the oxidative stress responses, including ROS concentration, SOD activity, and Nrf2 expression. Furthermore, similar inflammatory responses were observed in the PurNP-treated group, as compared to the vehicle-treated group. The results presented in this study provide the first strong evidence that oral administration of NP for 2 weeks results in high accumulation in the liver, kidneys, and intestine of ICR mice, and induces severe inflammatory and oxidative stress responses. These results additionally confirm the efficacy of water purification using the tannic acid-mediated coagulation removal technique.

[Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15].

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"¼ and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

FEMA GRAS assessment of natural flavor complexes: Origanum oil, thyme oil and related phenol derivative-containing flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients, mostly consisting of a variety of essential oils and botanical extracts. This publication, seventh in the series, re-evaluates NFCs with constituent profiles dominated by phenolic derivatives including carvacrol, thymol and related compounds using a constituent-based procedure first published in 2005 and updated in 2018. The procedure is based on the chemical characterization of each NFC as intended for commerce and the estimated intake of the constituent congeneric groups. The procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology of the constituent congeneric groups and the NFC under evaluation. Herein, the FEMA Expert Panel affirmed the generally recognized as safe (GRAS) status of seven phenolic derivative-based NFCs, Origanum Oil (Extractive) (FEMA 2828), Savory Summer Oil (FEMA 3013), Savory Summer Oleoresin (FEMA 3014), Savory Winter Oil (FEMA 3016), Savory Winter Oleoresin (FEMA 3017), Thyme Oil (FEMA 3064) and Thyme White Oil (FEMA 3065) under their conditions of intended use as flavor ingredients.

Effect of Echinostoma caproni on Presumptive Lactic Acid Bacteria Abundance and Salmonella enterica Serovar Typhimurium Colonization in the Mouse Gut.

Co-infections of mammalian hosts with intestinal helminths and bacterial pathogens are common, especially in areas with inadequate sanitation. Interactions between co-infecting species and host microbiota can cause significant changes in host immunity, disease severity, and pathogen transmission, requiring unique treatment for each case. A greater understanding of the influences of parasite-bacteria co-infections will improve diagnosis and therapeutic approaches to control infectious diseases. To study the influence of the trematode parasite Echinostoma caproni on commensal and pathogenic bacteria in the mouse gut, we examined the abundance of intestinal lactic acid bacteria and Salmonella enterica serovar Typhimurium in control mice not exposed to E. caproni (P-) or S. Typhimurium (S-), E. caproni-infected (P+S-), S. Typhimurium-infected (P-S+), and E. caproni-S. Typhimurium co-infected (P+S+) mice, and determined bacterial burdens in the livers and spleens of the P-S+ and P+S+ mice. We also examined a subset of P+S- and P+S+ mice for survival and the relative location of E. caproni in the small intestine. The numbers of presumptive lactic acid bacteria were significantly higher in the P+S+ and P-S+ mice compared to the uninfected mice, and S. Typhimurium colonization in the liver and spleen was significantly reduced in the P+S+ mice compared to the P-S+ mice. Echinostoma caproni were located anteriorly in the intestine of P+S- mice, while in the P+S+ mice, the parasites were distributed more posteriorly. Survival of E. caproni was unaffected in either group. The results of our study suggest that E. caproni facilitates a higher abundance of presumptive lactic acid bacteria in the mouse intestine and reduces colonization of S. Typhimurium in the liver and spleen of the co-infected host.

Toxicological assessment and food allergy of silk fibroin derived from Bombyx mori cocoons.

Recent studies have demonstrated silk fibroin protein's (SF) ability to extend the shelf life of foods by mitigating the hallmarks of spoilage, namely oxidation and dehydration. Due to the potential for this protein to become more widespread, its safety was evaluated comprehensively. First, a bacterial reverse mutation test (Ames test) was conducted in five bacterial strains. Second, an in vivo erythrocyte test was conducted with Sprague Dawley rats at doses up to 1,000mg/kg-bw/day. Third, a range-finder study was conducted with Sprague Dawley rats at the highest consumption amount given solubility and oral gavage volume constrains (500mg/kg-bw/day). Fourth, a 28-day sub-chronic study in Sprague Dawley rats was conducted with the high dose set at 500mg/kg-bw/day, as limited by solubility of the protein in a single-gavage per-day study. Fifth, an in vitro pepsin digestion assay was performed to assess the potential for protein allergenicity. Sixth, allergenic potential was further assessed using liquid chromatography-mass spectroscopy for detection of allergenic insect proteins. Seventh, the SF protein sequences were subjected to bioinformatic analyses. Together, these studies raise no mutagenic, genotoxic, toxicological, or allergenic concerns with the oral consumption of silk fibroin.

Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone.

In this study, we examined the immunolocalization of podoplanin/E11, CD44, actin filaments, and phosphorylated ezrin in the osteoblasts on the verge of differentiating into osteocytes in murine femora and tibiae. When observing under stimulated emission depletion microscopy, unlike podoplanin-negative osteoblasts, podoplanin-positive osteoblasts showed a rearranged assembly of actin filaments along the cell membranes which resembled that of embedded osteocytes. In the metaphysis, i.e., the bone remodeling site, CD44-bearing osteoclasts were either proximal to or in contact with podoplanin-positive osteoblasts, but the podoplanin-positive osteoblasts also localized CD44 on their own cell surface. These podoplanin-positive osteoblasts, which either possessed CD44 on their cell surface or were close to CD44-bearing osteoclasts, showed phosphorylated ezrin-positivity on the cell membranes. Therefore, the CD44/podoplanin interaction on the cell surface may be involved in the osteoblastic differentiation into osteocytes in the metaphyses, via the mediation of podoplanin-driven ezrin phosphorylation and the subsequent reorganized assembly of actin filaments. Consistently, the protein expression of phosphorylated ezrin was increased after CD44 administration in calvarial culture. Conversely, in modeling sites such as the cortical bones, podoplanin-positive osteoblasts were uniformly localized at certain intervals even without contact with CD44-positive bone marrow cells; furthermore, they also exhibited phosphorylated ezrin immunoreactivity along their cell membranes. Taken together, it seems likely that the CD44/podoplanin interaction is involved in osteoblastic differentiation into osteocytes in the bone remodeling area but not in modeling sites.

In Vitro and In Vivo Antimalarial Activities and Toxicological Assessment of Pogostemon Cablin (Blanco) Benth.

The aim of this study was to investigate the antimalarial activities and toxicity of Pogostemon cablin extracts. In vitro activities against the chloroquine-resistant Plasmodium falciparum K1 strain were assessed by using the Plasmodium lactate dehydrogenase enzyme (pLDH) assay, while in vivo activity against the Plasmodium berghei ANKA strain in mice was investigated using a 4-day suppressive test. The in vitro and in vivo toxicity were determined in Vero cells and mice, respectively. The ethanolic extract possessed antimalarial activity with an IC50 of 24.49 ± 0.01 µg/ml, whereas the aqueous extract showed an IC50 of 549.30 ± 0.07 µg/ml. Cytotoxic analyses of the ethanolic and aqueous extracts revealed a nontoxic effect on Vero cells at a concentration of 80 µg/ml. Based on a preliminary study of in vitro antimalarial activity, the ethanolic extract was chosen as a potential agent for further in vivo antimalarial activity analysis in mice. The ethanolic extract, which showed no toxic effect on mice at a dose of 2000 mg/kg body weight, significantly suppressed parasitemia in mice by 38.41%, 45.12% and 89.00% at doses of 200, 400 and 600 mg/kg body weight, respectively. In conclusion, this study shows that the ethanolic P. cablin extract possesses in vitro and in vivo antimalarial activity without toxic effects.

Assessment of nest building and social interaction behavior in mice exposed to acute social defeat stress using a three-dimensional depth camera.

Nest building is an instinctive behavior toward protection from predators, body temperature regulation, and courtship. Previously, we discovered that acute and chronic social defeat stress suppresses the onset of nest-building behavior in male mice (C57BL/6J). Here, we analyzed nest building and other behavioral deficits induced by acute social defeat stress (ASDS). We utilized a customized cage and specifically developed observational programs for nest building, social avoidance, and other behaviors using an infrared depth camera to acquire three-dimensional (3D) data of animal behavior (Negura system). We determined the volume of nesting materials from these 3D depth images. Mice exposed to ASDS showed increased spontaneous activities, decreased rearing, and delayed nest building; however, nest-building activity was gradually recovered during the dark period of the 24 hr observation interval. At the endpoint following 24 hr, the ASDS and control groups showed no differences in nest volumes. Furthermore, we observed the time courses of both nest building and social avoidance behaviors and their relationship using the Negura system. Our data demonstrated a weak positive correlation between nest-building delay and social avoidance in ASDS mice. The Negura system can observe various behaviors that reflect the effects of social defeat stress.

Toxicity Assessment of SiO2 and TiO2 in Normal Colon Cells, In Vivo and in Human Colon Organoids.

We conducted systemic assessments on the toxicity of silicon dioxide (SiO2) and titanium dioxide (TiO2) nanoparticles using different forms of normal colon cells (CCD-18Co), in vivo and in human colon organoids. The in vivo acute oral toxicity data showed that the LD50 values are greater than 2000 mg/kg for both the SiO2 and TiO2 nanoparticles; however, the SiO2 and TiO2 nanoparticles induced cytotoxicity in two-dimensional CCD-18Co cells and three-dimensional CCD-18Co spheroids and human colon organoids, with IC50 values of 0.6, 0.8 and 0.3 mM for SiO2 and 2.5, 1.1 and 12.5 mM for TiO2 nanoparticles, respectively. The data suggest that, when SiO2 and TiO2 are in nanoparticle form, cytotoxicity is induced; thus, care should be taken with these materials.

Simultaneous quantitative assessment of two distinct cell lineages with a nuclear-localized dual genetic reporter.

Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo.

Metabolite Profiling of Javanese Ginger Zingiber purpureum and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

The rhizomes of Zingiber purpureum, "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were isolated, and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of Z. purpureum active compounds, its chemical profile was compared to that of Z. officinale. Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of Z. purpureum composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.