Allelic Mutations in the Ripening -Inhibitor Locus Generate Extensive Variation in Tomato Ripening.

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that (1) RIN is indispensable for full ripening but not for the induction of ripening; and (2) the rin mutation, which produces nonripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that, contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 µM, and 20.2 µM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.

Preventive Effects of Green Tea Extract against Obesity Development in Zebrafish.

Various natural products (NPs) have been used to treat obesity and related diseases. However, the best way to fight obesity is preventive, with accurate body weight management through exercise, diet, or bioactive NPs to avoid obesity development. We demonstrated that green tea extract (GTE) is an anti-obesity NP using a zebrafish obesity model. Based on a hypothesis that GTE can prevent obesity, the objective of this study was to assess GTE's ability to attenuate obesity development. Juvenile zebrafish were pretreated with GTE for seven days before obesity induction via a high-fat diet; adult zebrafish were pretreated with GTE for two weeks before obesity induction by overfeeding. As a preventive intervention, GTE significantly decreased visceral adipose tissue accumulation in juveniles and ameliorated visceral adiposity and plasma triglyceride levels in adult zebrafish obesity models. RNA sequencing analysis was performed using liver tissues from adult obese zebrafish, with or without GTE administration, to investigate the underlying molecular mechanism. Transcriptome analysis revealed that preventive GTE treatment affects several pathways associated with anti-obesity regulation, including activation of STAT and downregulation of CEBP signaling pathways. In conclusion, GTE could be used as a preventive agent against obesity.

Lecithin-Based Dermal Drug Delivery for Anti-Pigmentation Maize Ceramide.

Ceramides have several well-known biological properties, including anti-pigmentation and anti-melanogenesis, which make them applicable for use in skincare products in cosmetics. However, the efficacy of ceramides is still limited. Dermal or transdermal drug delivery systems can enhance the anti-pigmentation properties of ceramides, although there is currently no systemic evaluation method for the efficacy of these systems. Here we prepared several types of lecithin-based emulsion of maize-derived glucosylceramide, determining PC70-ceramide (phosphatidylcholine-base) to be the safest and most effective anti-pigmentation agent using zebrafish larvae. We also demonstrated the efficacy of PC70 as a drug delivery system by showing that PC70-Nile Red (red fluorescence) promoted Nile Red accumulation in the larval bodies. In addition, PC70-ceramide suppressed melanin in mouse B16 melanoma cells compared to ceramide alone. In conclusion, we developed a lecithin-based dermal delivery method for ceramide using zebrafish larvae with implications for human clinical use.

Anti-Obesity Natural Products Tested in Juvenile Zebrafish Obesogenic Tests and Mouse 3T3-L1 Adipogenesis Assays.

(1) Background: The obesity epidemic has been drastically progressing in both children and adults worldwide. Pharmacotherapy is considered necessary for its treatment. However, many anti-obesity drugs have been withdrawn from the market due to their adverse effects. Instead, natural products (NPs) have been studied as a source for drug discovery for obesity, with the goal of limiting the adverse effects. Zebrafish are ideal model animals for in vivo testing of anti-obesity NPs, and disease models of several types of obesity have been developed. However, the evidence for zebrafish as an anti-obesity drug screening model are still limited. (2) Methods: We performed anti-adipogenic testing using the juvenile zebrafish obesogenic test (ZOT) and mouse 3T3-L1 preadipocytes using the focused NP library containing 38 NPs and compared their results. (3) Results: Seven and eleven NPs reduced lipid accumulation in zebrafish visceral fat tissues and mouse adipocytes, respectively. Of these, five NPs suppressed lipid accumulation in both zebrafish and 3T3-L1 adipocytes. We confirmed that these five NPs (globin-digested peptides, green tea extract, red pepper extract, nobiletin, and Moringa leaf powder) exerted anti-obesity effects in diet-induced obese adult zebrafish. (4) Conclusions: ZOT using juvenile fish can be a high-throughput alternative to ZOT using adult zebrafish and can be applied for in vivo screening to discover novel therapeutics for visceral obesity and potentially also other disorders.

Determination of Anthocyanins and Antioxidants in 'Titanbicus' Edible Flowers In Vitro and In Vivo.

Titanbicus (TB), a hybrid of Hibiscus moscheutos × H. coccineus (Medic.) Walt., has potential to be used as an edible flower. In this study, proximate nutritional content, anthocyanin content, total polyphenol content (TPC), and antioxidant activities in vitro and in vivo were investigated. Three cultivars of TB, namely Artemis (AR), Rhea (R), and Adonis (AD), were used as materials. Protein and carbohydrates were the primary macronutrients, while crude fat and ash were detected in trace amounts. Cyanidin 3-glucoside (Cy3-G) and cyanidin 3-sambubioside (Cy3-Sam), were identified in all TBs. The highest anthocyanin content was observed in AD (47.09 ± 1.45 mg/g extract), followed by R and AR (6.04 ± 0.20 and 2.72 ± 0.11 mg/g extract, respectively). The TPC of AD (225.01 ± 1.97 mg/g extract) was greater than that of AR and R (185.41 ± 3.24 and 144.10 ± 1.71 mg/g extract, respectively). AD exhibited the strongest in vitro antioxidant activity in hydrophilic oxygen radical absorbance capacity, compared to the other two TBs. In addition, AD extract suppressed the generation of reactive oxygen species in caudal fin of wounded zebrafish. Antioxidant activities of AD appeared to be related to its total anthocyanin content, Cy3-G, Cy3-Sam, and TPC. Our findings indicate that TB, particularly the AD cultivar, would be an attractive source of bioactive compounds with antioxidant activities, and can improve both nutritional value and appearance of food.

An Integrative Evaluation Method for the Biological Safety of Down and Feather Materials.

BACKGROUND: Down and feather materials have been commonly used and promoted as natural stuffing for warm clothing and bedding. These materials tend to become more allergenic as they become contaminated with microorganisms, in addition to being subjected to several kinds of chemical treatments. The biological or chemical contaminants in these materials pose a major risk to human health, to consumers and manufacturers alike. Here, we report the development of an integrative evaluation method for down and feather materials to assess bacterial contamination and in vivo toxicity. METHODS: To assess bacterial contamination, we quantified 16S ribosomal RNA, performed culture tests, and established a conversion formula. To determine in vivo toxicity, we performed a zebrafish embryo toxicity testing (ZFET). RESULTS: Washing the material appropriately decreases the actual number of bacteria in the down and feather samples; in addition, after washing, 16S rRNA sequencing revealed that the bacterial compositions were similar to those in rinse water. The ZFET results showed that even materials with low bacterial contamination showed high toxicity or high teratogenicity, probably because of the presence of unknown chemical additives. CONCLUSIONS: We established an integrative evaluation method for down and feather safety, based on bacterial contamination with in vivo toxicity testing.

RNA-seq Based Transcriptome Analysis of the Anti-Obesity Effect of Green Tea Extract Using Zebrafish Obesity Models.

Green tea is a popular beverage that is rich in polyphenolic compounds such as catechins. Its major content, (-)-epigallocatechin-3-gallate, has been shown to have beneficial effects on several diseases including cancer, metabolic syndrome, cardiovascular diseases, and neurodegenerative diseases. The aim of this study was to assess the anti-obesity effects and the underlying molecular mechanisms of green tea extract (GTE) using zebrafish larva and adult obesity models. We administered 100 µg/mL GTE to zebrafish larvae and performed a short-term obesogenic test. GTE significantly decreased the visceral adipose tissue volume induced by a high-fat diet. Oral administration (250 µg/g body weight/day) of GTE to adult diet-induced obese zebrafish also significantly reduced their visceral adipose tissue volume, with a reduction of plasma triglyceride and total cholesterol levels. To investigate the molecular mechanism underlying the GTE effects, we conducted RNA sequencing using liver tissues of adult zebrafish and found that GTE may ameliorate the obese phenotypes via the activation of Wnt/ß-catenin and adenosine monophosphate-activated protein kinase (AMPK) pathway signaling. In addition, the comparative transcriptome analysis revealed that zebrafish and mammals may share a common molecular response to GTE. Our findings suggest that daily consumption of green tea may be beneficial for the prevention and treatment of obesity.

Indices of Visual Improvement after Conservative Treatment for Branch Retinal Vein Occlusion with Macular Edema.

Purpose: To identify indices of visual improvement after conservative treatment for branch retinal vein occlusion (BRVO) with macular edema. Methods: We retrospectively reviewed the charts of 33 eyes of 33 patients with BRVO with macular edema. Inclusion criteria were 1) onset within 4 months, 2) decimal visual acuity of 0.05 to 0.5, and 3) minimum central subfield thickness (CST) of 250 µm. After 3 months of treatment with oral aspirin and kallidinogenase, the patients were divided into two groups: those with logarithmic minimum angle of resolution (logMAR) visual improvement of 0.3 or more (14 eyes) and less than 0.3 (19 eyes). We then compared systemic and ocular findings in the groups. Results: The groups differed significantly in logMAR improvement after 1 month (p<0.01) and in CST change after 1 month (p<0.05). Multiple logistic regression analysis showed that CST change after 1 month was a significant index of visual improvement (p<0.05). The cutoff value for visual improvement was -30 µm (sensitivity: 78.6, specificity: 68.4, positive predictive value: 64.7, negative predictive value: 81.3). Conclusion: A decrease in CST of more than 30 µm 1 month after conservative treatment indicates that visual acuity is likely to be improved after 3 months.

Lipid- and glucose-lowering effects of Rhamnan sulphate from Monostroma nitidum with altered gut microbiota in mice.

Rhamnan sulphate (RS) is a sulphated polysaccharide found in green algae such as Monostroma nitidum that exhibits various biological functions, including anticoagulant, antitumour, antiviral, and anti-obesity properties. In our previous clinical trial, we demonstrated that RS intake improves constipation. However, no specific bacteria showed a significant (p < .05) change. Notably, these results were obtained after a short RS inoculation period of only 2 weeks. In the present study, to evaluate the long-term effects of RS on the gut microbiota, we orally administered RS to BALB/c mice for 11 weeks, analyzed their blood biochemical data, and performed 16s rRNA-sequencing. Oral administration of RS increased body weight with increased food intake, whereas plasma total cholesterol and fasting plasma glucose levels decreased. RS-fed mice showed lower fasting insulin levels (p < .1) and decreased homeostatic model assessment for insulin resistance (HOMA-IR, p < .0001), suggesting that RS improved insulin resistance. In the feces of mice, the amounts of acetic and propionic acids increased. In the gut microbiota, predictive metagenomic profiling using the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) revealed functional alterations in Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways in RS-fed mice. Corresponding to the blood glucose-lowering effect, the glycolysis and tricarboxylic acid (TCA) cycle pathways were activated. In addition, the Firmicutes/Bacteroides (F/B) ratio, which may be associated with various health outcomes, was also reduced. These results suggest that the blood glucose-lowering effect, improvement in insulin resistance, and lipid-lowering effect of RS may be due to changes in the intestinal microbiota.

Paraburkholderia sabiae administration alters zebrafish anxiety-like behavior via gut microbial taurine metabolism.

Interventions to the gut microbiome manipulate the gut-brain axis and could be useful in the treatment of anxiety and depression. In this study, we demonstrated that administration of the bacterium Paraburkholderia sabiae reduces anxiety-like behavior in adult zebrafish. P. sabiae administration increased the diversity of the zebrafish gut microbiome. Linear discriminant analysis Effect Size (LEfSe) analysis revealed that the populations of Actinomycetales including Nocardiaceae, Nocardia, Gordoniaceae, Gordonia, Nakamurellaceae, and Aeromonadaceae were reduced, whereas those of Rhizobiales including Xanthobacteraceae, Bradyrhizobiaceae, Rhodospirillaceae, and Pirellulaceae were increased in the gut microbiome. Functional analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) predicted that P. sabiae administration altered taurine metabolism in the zebrafish gut, and we demonstrated that P. sabiae administration increased the taurine concentration in the brain. Since taurine functions as an antidepressant neurotransmitter in vertebrates, our results suggest that P. sabiae could improve anxiety-like behavior in zebrafish via the gut-brain axis.

Zebrafish obesogenic test identifies anti-adipogenic fraction in Moringa oreifera leaf extracts.

The zebrafish obesogenic test (ZOT) is a powerful tool for identifying anti-adipogenic compounds for in vivo screening. In our previous study, we found that Moringa oleifera (MO) leaf powder suppressed the accumulation of visceral adipose tissue (VAT) in ZOT. MO demonstrates a wide range of pharmacological effects; however, little is known about its functional constituents. To identify the anti-adipogenic components of MO leaves, we prepared extracts using different extraction methods and tested the obtained extracts and fractions using ZOT. We found that the dichloromethane extract and its hexane:EtOAc = 8:2 fraction reduced VAT accumulation in young zebrafish fed a high-fat diet. We also performed gene expression analysis in the zebrafish VAT and found that CCAAT/enhancer-binding protein beta and CCAAT/enhancer-binding protein delta (associated with early stages of adipogenesis) gene expression was downregulated after fraction 2 administration. We identified a new MO fraction that suppressed VAT accumulation by inhibiting early adipogenesis using the ZOT. Phenotype-driven zebrafish screening is a reasonable strategy for identifying bioactive components in natural products.

Effects of nanoplastic on toxicity of azole fungicides (ketoconazole and fluconazole) in zebrafish embryos.

The ubiquity of nanoplastics (NPs) raises concerns about their interactions and combined toxicity with other common contaminants. Although azoles are present throughout the natural environment, their interactions with NP are not well known. We investigated the effects of polystyrene (PS) NP on the toxicity of ketoconazole (KCZ) and fluconazole (FCZ) in zebrafish embryos using the developmental toxicity, oxidative-stress-related biochemical parameters, and expression of genes related to neurotoxicity (ache), cardiotoxicity (gata4, bmp4), inflammation (il1b), oxidative stress (sod1, sod2, cyp1a), and apoptosis (bax, bcl2). Co-exposure to NP (1 mg/L) and KCZ/FCZ (1 mg/L) for 96 h reduced the hatching rate, survival rate, and heart rate and increased the malformation rate and catalase activity. The bax/bcl2 ratio, an apoptosis indicator, was higher after NP, KCZ, or FCZ treatment. However, the bax/bcl2 ratio after exposure to NP + KCZ or NP + FCZ was much higher than that after single exposure. Overall, the results indicated that NP aggravated the toxicity of azole by significantly increasing the reactive oxygen species, lipid peroxidation and altering the expression of oxidative-stress- and apoptosis-related genes. The interactive toxicity of PS NP with KCZ/FCZ reported in this study emphasises the need for caution in the release of azole fungicides in the environment.

10-Gingerol Suppresses Osteoclastogenesis in RAW264.7 Cells and Zebrafish Osteoporotic Scales.

Osteoporosis is the most common aging-associated bone disease and is caused by hyperactivation of osteoclastic activity. We previously reported that the hexane extract of ginger rhizome [ginger hexane extract (GHE)] could suppress receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. However, the anti-osteoclastic components in GHE have not yet been identified. In this study, we separated GHE into several fractions using silica gel column chromatography and evaluated their effects on osteoclastogenesis using a RAW264.7 cell osteoclast differentiation assay (in vitro) and the zebrafish scale model of osteoporosis (in vivo). We identified that the fractions containing 10-gingerol suppressed osteoclastogenesis in RAW264.7 cells detected by tartrate-resistant acid phosphatase (TRAP) staining. In zebrafish, GHE and 10-gingerol suppressed osteoclastogenesis in prednisolone-induced osteoporosis regenerated scales to promote normal regeneration. Gene expression analysis revealed that 10-gingerol suppressed osteoclast markers in RAW264.7 cells [osteoclast-associated immunoglobulin-like receptor, dendrocyte-expressed seven transmembrane protein, and matrix metallopeptidase-9 (Mmp9)] and zebrafish scales [osteoclast-specific cathepsin K (CTSK), mmp2, and mmp9]. Interestingly, nuclear factor of activated T-cells cytoplasmic 1, a master transcription regulator of osteoclast differentiation upstream of the osteoclastic activators, was downregulated in zebrafish scales but showed no alteration in RAW264.7 cells. In addition, 10-gingerol inhibited CTSK activity under cell-free conditions. This is the first study, to our knowledge, that has found that 10-gingerol in GHE could suppress osteoclastic activity in both in vitro and in vivo conditions.

Rhamnan sulphate from green algae Monostroma nitidum improves constipation with gut microbiome alteration in double-blind placebo-controlled trial.

Rhamnan sulphate (RS), a sulphated polysaccharide from Monostroma nitidum, possesses several biological properties that help in treating diseases such as viral infection, thrombosis, and obesity. In the present study, we first administered RS (0.25 mg/g food volume) orally to high-fat diet-treated mice for 4 weeks. RS increased the faecal volume and calorie excretion with decreased plasma lipids, which was in accordance with the results of our previous zebrafish study. Notably, as the excretion amount by RS increased in the mice, we hypothesised that RS could decrease the chance of constipation in mice and also in human subjects because RS is considered as a dietary fibre. We administrated RS (100 mg/day) to subjects with low defaecation frequencies (3-5 times/week) for 2 weeks in double-blind placebo-controlled manner. As a result, RS administration significantly increased the frequency of dejection without any side effects, although no effect was observed on the body weight and blood lipids. Moreover, we performed 16s rRNA-seq analysis of the gut microbiota in these subjects. Metagenomics profiling using PICRUSt revealed functional alternation of the KEGG pathways, which could be involved in the therapeutic effect of RS for constipation.

Globin Digest Improves Visceral Adiposity Through UCP1 Upregulation in Diet-Induced Obese Zebrafish and Mice.

Globin digest (GD), a bioactive oligopeptide derived from porcine hemoglobin proteins, has been demonstrated to have beneficial effects on improving postprandial hyperlipidemia, hyperglycemia, and liver injury. We previously reported the lipid-lowering effects of GD using a zebrafish obesogenic test. Here, we sought to evaluate the effect of GD on visceral adiposity and the underlying molecular mechanisms using zebrafish and mouse obesity models. GD ameliorated dyslipidemia and suppressed the accumulation of visceral adipose tissue (VAT) in adult obese zebrafish. Transcriptomic analysis by RNA sequencing of GD-treated adult zebrafish revealed that GD upregulated UCP1-related pathways. Further, we performed mouse experiments and found that GD intake (2 mg/g body weight/day) was associated with lowered plasma triglyceride and total cholesterol levels, decreased VAT accumulation, and improved adipocyte hypertrophy with the upregulation of Ucp1 expression in white adipose tissue at both the mRNA and protein levels. Taken together, these results indicate that GD improves visceral adiposity by upregulating UCP1 expression, providing a novel perspective on combating obesity.

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells.

Sofalcone, 2'-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.

Water Extract of Yamato Tachibana (Citrus tachibana) Induces Food Intake in Adult and Larval Zebrafish.

Yamato Tachibana (Tachibana; Citrus tachibana) is an endemic fruit and represents one of the oldest citrus species in Japan; it is grown in the Mie Prefecture. It has been attracting attention for its cultural heritage and unique scent. To evaluate biological activities of Tachibana, we fed several parts of the Tachibana fruit (whole fruit, pulp [albedo and segment wall], and flavedo) to adult zebrafish and found that Tachibana increased body weight and plasma triglycerides besides increasing overall food intake. We then created a simple fluorescence-based feeding assay using dried rotifer sheets and larval zebrafish (6 days postfertilization) to screen the various extracts of Tachibana parts. We found that water extracts of Tachibana pulp increased feeding volume in zebrafish. Although citrus species are believed to prevent obesity and obesity-associated diseases in general, our findings showed that water extracts of Tachibana increase food intake in zebrafish and lead to an increase in body weight. We suggest that Tachibana might reverse appetite loss in lean populations and may prove beneficial in aiding fish cultivation.

Carotid artery intima-media thickness, HDL cholesterol levels, and gender associated with poor visual acuity in patients with branch retinal artery occlusion.

PURPOSE: To investigate factors associated with poor visual acuity (VA) in branch retinal artery occlusion (BRAO). METHODS: This was a retrospective cross-sectional study of 72 eyes with BRAO of 72 patients. For statistical comparison, we divided the patients into worse-VA (decimal VA < 0.5) and better-VA (decimal VA > = 0.5) groups. We examined the association of clinical findings, including blood biochemical test data and carotid artery ultrasound parameters, with poor VA. RESULTS: Median age, hematocrit, hemoglobin and high-density lipoprotein (HDL) differed significantly between the groups (P = 0.018, P < 0.01, P < 0.01, and P = 0.025). There was a tendency towards higher median IMT-Bmax in the worse-VA group (worse-VA vs. better-VA: 2.70 mm vs. 1.60 mm, P = 0.152). Spearman's rank correlation test revealed that logMAR VA was significantly correlated to IMT-Bmax (rs = 0.31, P < 0.01) and IMT-Cmax (rs = 0.24, P = 0.035). Furthermore, logMAR VA was significantly correlated to HDL level (rs = -0.33, P < 0.01). Multivariate logistic regression analysis revealed that IMT-Bmax (odds ratio [OR] = 2.70, P = 0.049), HDL level (OR = 0.91, P = 0.032), and female gender (OR = 15.63, P = 0.032) were independently associated with worse VA in BRAO. CONCLUSIONS: We found that increased IMT-Bmax, decreased HDL, and female sex were associated with poor VA in BRAO patients. Our findings might suggest novel risk factors for visual dysfunction in BRAO and may provide new insights into the pathomechanisms underlying BRAO.

ZF-Mapper: Simple and Complete Freeware for Fluorescence Quantification in Zebrafish Images.

Zebrafish embryos and larvae have become popular vertebrate models because their body walls are transparent, which enables live imaging of target organs using fluorescent protein transgenes or dye staining. Software packages for the quantification of these fluorescent signals are available from both commercial and noncommercial sources; however, their algorithms are complicated and their resources (code) have mostly not been openly shared. In this study, we developed a simple and robust open-source software tool named "ZF-Mapper" for the quantification of the fluorescence intensity of each pixel in zebrafish images with batch image file processing capability. Using this software, we can evaluate the three-dimensional (3D) distribution of fluorescence intensity among zebrafish cells by analyzing each image pixel. We tested ZF-Mapper for the analysis of zebrafish with macrophage-specific enhanced green fluorescent protein (EGFP) and obtained results that were equivalent to those acquired using the conventional image analysis software ImageJ. We further applied ZF-Mapper to the analysis of zebrafish with cancer cell xenografts and quantified the amount of implanted melanoma cells labeled with a tdTomato red fluorescent protein in the whole body and the tail region. In addition, by combining ZF-Mapper with R freeware, we created an interactive 3D scatter plot of the fluorescence intensities of macrophage-EGFPs in zebrafish. In summary, we developed the Python-based freeware ZF-Mapper for the quantification of fluorescent signals in multiple zebrafish images, which enables fluorescence-based zebrafish screening. We provide the source code and the executable application software for Windows (.exe) and macOS (.app).