Language：English   Publishing type：Research paper (scientific journal)   Publisher：mSystems  

Keng Po Lai, Peng Zhu, Delbert Almerick T. Boncan, Lu Yang, Cherry Chi Tim Leung, Jeff Cheuk Hin Ho, Xiao Lin, Ting Fung Chan, Richard Yuen Chong Kong, William Ka Fai Tse

DOI： 10.1128/msystems.00047-22

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Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE:
Cleft lip (CL) is a common congenital anomaly that can be syndromic or non-syndromic. It can be triggered by the mutation of gene or environmental factors. The incidence of CL is about 1 out of 700 live births. Facial development is a complex process, and there is no existing therapy to prevent the disease development. One of the characteristics in this facial malformation is the increased presence of reactive oxygen species (ROS). In this study, we hypothesize that the antioxidant glutathione (GSH) could help to attenuate the oxidative stress in this disease.
METHODS:
Bioinformatics network pharmacology was applied to determine pharmacological targets and molecular mechanisms of GSH treatment for CL. Moreover, RNA-sequencing of the POLR1C knockdown osteoblast CL model was applied to validate the in silico data of using GSH in CL.
RESULTS:
Twenty-two core targets of GSH and CL were identified via various bioinformatics tools. The GO and KEGG analysis indicated that GSH could modulate two major families (matrix metalloproteinase and integrins), which are related to extracellular matrix modification and composition for facial development in CL. The findings from POLR1C knockdown model further supported the rescue response of GSH in CL.
CONCLUSIONS:
The study uncovered the possible pharmacological mechanism of GSH for treating CL. The data helps research group to focus on the specific pathways for understanding the biological action of GSH for treating the CL in the future.

DOI： 10.1016/j.freeradbiomed.2020.12.455

Language：English   Publishing type：Research paper (scientific journal)  

Alteration of the gut microbiota plays an important role in animal health and metabolic diseases. However, little is known with respect to the influence of environmental osmolality on the gut microbial community. The aim of the current study was to determine whether the reduction in salinity affects the gut microbiota and identify its potential role in salinity acclimation. Using Oryzias melastigma as a model organism to perform progressive hypotonic transfer experiments, we evaluated three conditions: seawater control (SW), SW to 50% sea water transfer (SFW) and SW to SFW to freshwater transfer (FW). Our results showed that the SFW and FW transfer groups contained higher operational taxonomic unit microbiota diversities. The dominant bacteria in all conditions constituted the phylum Proteobacteria, with the majority in the SW and SFW transfer gut comprising Vibrio at the genus level, whereas this population was replaced by Pseudomonas in the FW transfer gut. Furthermore, our data revealed that the FW transfer gut microbiota exhibited a reduced renin–angiotensin system, which is important in SW acclimation. In addition, induced detoxification and immune mechanisms were found in the FW transfer gut microbiota. The shift of the bacteria community in different osmolality environments indicated possible roles of bacteria in facilitating host acclimation.

DOI： 10.1111/1462-2920.15150

Language：English   Publishing type：Research paper (scientific journal)  

Treacher Collins Syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects, including the downward slanting palpebral fissures, hypoplasia of the facial bones, and cleft palate (CP). Over 90% of patients with TCS have a mutation in the TCOF1 gene. However, some patients exhibit mutations in two new causative genes, POLR1C and POLR1D, which encode subunits of RNA polymerases I and III, that affect ribosome biogenesis. In this study, we examine the role of POLR1C in TCS using zebrafish as a model system. Our data confirmed that polr1c is highly expressed in the facial region, and dysfunction of this gene by knockdown or knock-out resulted in mis-expression of neural crest cells during early development that leads to TCS phenotype. Next generation sequencing and bioinformatics analysis of the polr1c mutants further demonstrated the up-regulated p53 pathway and predicted skeletal disorders. Lastly, we partially rescued the TCS facial phenotype in the background of p53 mutants, which supported the hypothesis that POLR1C-dependent type 3 TCS is associated with the p53 pathway.

DOI： 10.1016/j.bbadis.2016.03.005

Publisher：Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics  

Gills and gut are the two primary osmoregulatory organs in fish. Recently, studies have expanded beyond the osmoregulatory mechanisms of these organs to explore the microbiota communities inhabiting them. It is now known that microbial communities in both organs shift in response to osmotic stress. However, there are limited studies identifying the major contributors and co-occurrence among these microbiota in both organs under seawater and freshwater transfer conditions. The current data mining report performed a bioinformatics analysis on two previous published datasets from our group, aiming to provide insights into host-bacteria relationships under osmotic stress. We divided the samples into four groups: control seawater gills (LSW); control seawater gut (TSW); freshwater transfer gills (LFW); and freshwater transfer gut (TFW). Our results showed that LSW had higher diversities, richness, and evenness compared to TSW. However, both the LFW and LSW did not show any significant differences after the freshwater transfer experiment. We further applied co-occurrence network analysis and, for the first time, reported on the interactions of taxa shaping the community structure in these two organs. Moreover, we identified enriched ectoine biosynthesis in seawater samples, suggesting its potential role in seawater environments. Increased mRNA expression levels of Na+/K+-atpase, and cftr, were observed in gills after 6 h of ectoine treatment. These findings provide a foundation for future studies on host-bacteria interactions under osmotic stress.

DOI： 10.1016/j.cbd.2024.101285

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Publisher：International Journal of Biological Macromolecules  

Nager syndrome (NS, OMIM 154400) is a rare disease characterized by craniofacial and limb malformations due to variants in the gene encoding splicing factor 3B subunit 4 (SF3B4). Although various noncanonical functions of SF3B4 unrelated to splicing have been previously described, limited studies elucidate molecular mechanisms underlying NS pathogenesis. Here we showed that sf3b4-deficient fish displayed craniofacial and segmentation defects associated with suppression of fgf8 levels, which perturbed FGF signaling and neural crest cell (NCC) expression. Our finding also pointed out that oxidative stress-induced apoptosis was prominently detected in sf3b4-deficient fish and may further exaggerate the bone remodeling process. Notably, injection of exogenous FGF8 significantly rescued the demonstrated defects in sf3b4-deficient fish, which further supported the participation of Fgf8 in NS pathogenesis. Overall, our study provides valuable insights into the molecular mechanism underlying developmental abnormalities observed in NS and suggests future therapeutic strategies to protect against the pathogenesis of NS and possibilities for preventing severe outcomes.

DOI： 10.1016/j.ijbiomac.2024.135692

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Publisher：Toxicology  

Introduction: Growing concerns regarding the reproductive toxicity associated with daily life exposure to micro-/nano-plastics (abbreviated as MNPs) have become increasingly prevalent. In reality, MNPs exposure involves a heterogeneous mixture of MNPs of different sizes rather than a single size. Methods: In this study, an oral exposure mouse model was used to evaluate the effects of MNPs of four size ranges: 25–30 nm, 1–5 µm, 20–27 µm, and 125–150 µm. Adult male C57BL/6 J mice were administered environmentally relevant concentrations of 0.1 mg MNPs/day for 21 days. After that, open field test and computer assisted sperm assessment (CASA) were conducted. Immunohistochemical analyses of organ and cell type localization of MNPs were evaluated. Testicular transcriptome analysis was carried out to understand the molecular mechanisms. Results: Our result showed that MNPs of different size ranges all impaired sperm motility, with a decrease in progressive sperm motility, linearity and straight-line velocity of sperm movement. Alterations did not manifest in animal locomotion, body weight, or sperm count. Noteworthy effects were most pronounced in the smaller MNPs size ranges (25–30 nm and 1–5 µm). Linear regression analysis substantiated a negative correlation between the size of MNPs and sperm curvilinear activity. Immunohistochemical analysis unveiled the intrusions of 1–5 µm MNPs, but not 20–27 µm and 125–150 µm MNPs, into Leydig cells and testicular macrophages. Further testicular transcriptomic analysis revealed perturbations in pathways related to spermatogenesis, oxidative stress, and inflammation. Particularly within the 1–5 µm MNPs group, a heightened perturbation in pathways linked to spermatogenesis and oxidative stress was observed. Conclusions: Our data support the size-dependent impairment of MNPs on sperm functionality, underscoring the pressing need for apprehensions about and interventions against the escalation of environmental micro-/nano-plastics contamination. This urgency is especially pertinent to small-sized MNPs.

DOI： 10.1016/j.tox.2024.153834

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Publisher：Marine Pollution Bulletin  

The COVID-19 pandemic has resulted in unprecedented plastic pollution from single-used personal protective equipment (PPE), especially face masks, in coastal and marine environments. The secondary pollutants, microplastics from face masks (mask MP), rise concern about their detrimental effects on marine organisms, terrestrial organisms and even human. Using a mouse model, oral exposure to mask MP at two doses, 0.1 and 1 mg MP/day for 21 days, caused no change in animal locomotion, total weight, or sperm counts, but caused damage to sperm motility with increased curvilinear velocity (VCL). The high-dose mask MP exposure caused a significant decrease in linearity (LIN) of sperm motility. Further testicular transcriptomic analysis revealed perturbed pathways related to spermatogenesis, oxidative stress, inflammation, metabolism and energy production. Collectively, our findings substantiate that microplastics from face masks yield adverse effects on mammalian reproductive capacity, highlighting the need for improved plastic waste management and development of environmentally friendly materials.

DOI： 10.1016/j.marpolbul.2024.116422

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.1016/j.bbadis.2024.167128

Language：English   Publisher：Biochimica et Biophysica Acta - Molecular Basis of Disease  

Nager syndrome (NS) is a rare acrofacial dysostosis caused by heterozygous loss-of-function variants in the splicing factor 3B subunit 4 (SF3B4). The main clinical features of patients with NS are characterized by facial-mandibular and preaxial limb malformations. The migration and specification of neural crest cells are crucial for craniofacial development, and mitochondrial fitness appears to play a role in such processes. Here, by analyzing our previously published transcriptome dataset, we aim to investigate the potential involvement of mitochondrial components in the pathogenesis of craniofacial malformations, especially in sf3b4 mutant zebrafish. We identified that oxidative phosphorylation (OXPHOS) defects and overproduction of reactive oxygen species (ROS) due to decreased antioxidants defense activity, which leads to oxidative damage and mitochondrial dysfunction. Furthermore, our results highlight that fish lacking sf3b4 gene, primarily display defects in mitochondrial complex I. Altogether, our findings suggest that mitochondrial dysfunction may contribute to the development of the craniofacial anomalies observed in sf3b4-depleted zebrafish.

DOI： 10.1016/j.bbadis.2024.167128

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.1016/j.exer.2024.109819

Language：English   Publisher：Experimental Eye Research  

Posterior subcapsular cataract (PSC) frequently develops as a complication in patients with retinitis pigmentosa (RP). Despite numerous scientific investigations, the intricate pathomechanisms underlying cataract formation in individuals affected by RP remain elusive. Therefore, our study aims to elucidate the potential pathogenesis of cataracts in an RP model using splicing factor subunit 3b (sf3b4) mutant zebrafish. By analyzing our previously published transcriptome dataset, we identified that, in addition to RP, cataract was listed as the second condition in our transcriptomic analysis. Furthermore, we confirmed the presence of nucleus retention in the lens fiber cells, along with abnormal cytoskeleton expression in both the lens fiber cells and lens epithelial cells in sf3b4-depleted fish. Upon closer examination, we identified 20 differentially expressed genes (DEGs) that played a role in cataract formation, with 95 % of them related to the downregulation of structural lens proteins. Additionally, we also identified that among all the DEGs, 13 % were associated with fibrotic processes. It seems that the significant upregulation of inflammatory mediators, in conjunction with TGF-β signaling, plays a central role in the cellular biology of PSC and posterior capsular opacification (PCO) in sf3b4 mutant fish. In summary, our study provides valuable insights into cataract formation in the RP model of sf3b4 mutants, highlighting its complexity driven by changes in structural lens proteins and increased cytokines/growth factors.

DOI： 10.1016/j.exer.2024.109819

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.3390/genes15010093

Language：English   Publisher：Genes  

Perfluorohexanesulfonic acid (PFHxS) is a highly prevalent environmental pollutant, often considered to be less toxic than other poly- and perfluoroalkyl substances (PFASs). Despite its relatively lower environmental impact compared to other PFASs, several studies have suggested that exposure to PFHxS may be associated with disruptions of liver function in humans. Nevertheless, the precise pathomechanisms underlying PFHxS-induced non-alcoholic fatty liver disease (NAFLD) remain relatively unclear. Therefore, this study applied our previously published transcriptome dataset to explore the effects of PFHxS exposure on the susceptibility to NAFLD and to identify potential mechanisms responsible for PFHxS-induced NAFLD through transcriptomic analysis conducted on zebrafish embryos. Results showed that exposure to PFHxS markedly aggravated hepatic symptoms resembling NAFLD and other metabolic syndromes (MetS) in fish. Transcriptomic analysis unveiled 17 genes consistently observed in both NAFLD and insulin resistance (IR), along with an additional 28 genes identified in both the adipocytokine signaling pathway and IR. These shared genes were also found within the NAFLD dataset, suggesting that hepatic IR may play a prominent role in the development of PFHxS-induced NAFLD. In conclusion, our study suggests that environmental exposure to PFHxS could be a potential risk factor for the development of NAFLD, challenging the earlier notion of PFHxS being safer as previously claimed.

DOI： 10.3390/genes15010093

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Publisher：Zoological Research  

The organ-specific toxicity resulting from microplastic (MP) exposure has been extensively explored, particularly concerning the gut, liver, testis, and lung. However, under natural conditions, these effects are not restricted to specific organs or tissues. Investigating whether MP exposure presents a systemic threat to an entire organism, impacting factors such as lifespan, sleep, and fecundity, is essential. In this study, we investigated the effects of dietary exposure to two different doses of MPs (1–5 μm) using the terrestrial model organism Drosophila melanogaster. Results indicated that the particles caused gut damage and remained within the digestive system. Continuous MP exposure significantly shortened the lifespan of adult flies. Even short-term exposure disrupted sleep patterns, increasing the length of daytime sleep episodes. Additionally, one week of MP exposure reduced ovary size, with a trend towards decreased egg-laying in mated females. Although MPs did not penetrate the brain or ovaries, transcriptome analysis revealed altered gene expression in these tissues. In the ovary, Gene Ontology (GO) analysis indicated genotoxic effects impacting inflammation, circadian regulation, and metabolic processes, with significant impacts on extracellular structure-related pathways. In the brain, GO analysis identified changes in pathways associated with proteolysis and carbohydrate metabolism. Overall, this study provides compelling evidence of the systemic negative effects of MP exposure, highlighting the urgent need to address and mitigate environmental MP pollution.

DOI： 10.24272/j.issn.2095-8137.2024.038

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.1016/j.scitotenv.2023.166833

Language：English   Publisher：Science of the Total Environment  

Perfluorohexanesulfonic acid (PFHxS) is a short-chain perfluoroalkyl substance widely used to replace the banned perfluorooctanesulfonic acid (PFOS) in various industrial and household products. It can be found in the environment and human bodies; however, its potential toxicities are not well studied. Zebrafish have been extensively used as a model for studying toxicants, and currently, two studies have reported on the toxicity of PFHxS in zebrafish from different approaches. Ulhaq and Tse (J Hazard Mater. 2023; 457: 131722) conducted general biological experiments and applied transcriptomics to demonstrate that PFHxS at a concentration of 5 μM could affect glucose and fatty acid metabolism, leading to oxidative stress, developmental defects, and cell cycle arrest. Xu et al. (Sci Total Environ. 2023; 887: 163770) employed metabolomics and showed that concentrations of various metabolites changed after exposure to 3 and 10 μM PFHxS. As we observed a match between the metabolomics data and our biochemistry experimental findings, we integrated the two studies, which enabled us to unfold the possible mechanism of the deregulated metabolites. We identified 22 differential expressed genes (DEGs) in the tricarboxylic acid (TCA) cycle, 17 DEGs in glcyolytic process, including the critical glucokinase under the carbon metabolism. Besides, genes likes aldehyde dehydrogenases, and histone-lysine N-methyltransferases that participate in lipid peroxidation and amino metabolism respectively were spotted. Lastly, we further strengthen our discoveries by undergoing the gene set enrichment analysis. This article could provide insights into the toxicity of PFHxS, as well as prospects for environmental studies.

DOI： 10.1016/j.scitotenv.2023.166833

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.3389/fphar.2023.1272523

Language：English   Publisher：Frontiers in Pharmacology  

DOI： 10.3389/fphar.2023.1272523

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Fatty acid oxidation disorders (FAODs) are a group of rare genetic metabolic disorders caused by mutations in genes responsible for transporting and metabolizing fatty acids in the mitochondria. One crucial enzyme involved in this process is carnitine palmitoyltransferase I (CPT1), which transports long-chain fatty acids to the mitochondrial matrix for beta-oxidation. Defects in beta-oxidation enzymes often lead to pigmentary retinopathy; however, the underlying mechanisms are not entirely understood. To investigate FAOD and its impact on the retina, we employed zebrafish as a model organism. Specifically, we used antisense-mediated knockdown strategies to target the cpt1a gene and examined the resulting retinal phenotypes. We demonstrated that the cpt1a MO-injected fish significantly reduced the length of connecting cilia and severely affected photoreceptor cell development. Moreover, our findings highlight that the loss of functional cpt1a disrupted energy homeostasis in the retina, leading to lipid droplet deposition and promoting ferroptosis, which is likely attributed to the photoreceptor degeneration and visual impairments observed in the cpt1a morphants.

DOI： 10.1016/j.bbrc.2023.04.096

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.18632/aging.204733

Language：English   Publisher：Aging  

Hepatocellular carcinoma (HCC) is the sixth most common cancer and third leading cause of cancer-related deaths worldwide. HCC is a multistep disease marked by various signaling alterations. A better understanding of the new molecular drivers of HCC could therefore provide an opportunity to develop effective diagnostic and therapeutic targets. Ubiquitin-specific protease 44 (USP44), a member of the cysteine protease family, has been reported to play a role in many cancer types. However, its contribution to HCC development remains unknown. In the present study, we observed suppression of USP44 expression in HCC tissue. Clinicopathologic analysis further showed that low USP44 expression correlated with poorer survival and a later tumor stage in HCC, suggesting that USP44 could be a predictor of poor prognosis in patients with HCC. Gain-of-function analysis in vitro demonstrated the importance of USP44 in HCC cell growth and G0/G1 cell cycle arrest. To investigate the downstream targets of USP44 and the molecular mechanisms underlying its regulation of cell proliferation in HCC, we conducted a comparative transcriptomic analysis and identified a cluster of proliferation-related genes, including CCND2, CCNG2, and SMC3. Ingenuity Pathway Analysis further delineated the gene networks controlled by USP44 through the regulation of membrane proteins and receptors, enzymes, transcriptional factors, and cyclins involved in the control of cell proliferation, metastasis, and apoptosis in HCC. To summarize, our results highlight, for the first time, the tumor-suppression role of USP44 in HCC and suggest a new prognostic biomarker in this disease.

DOI： 10.18632/aging.204733

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Variants in the gene encoding trafficking protein particle complex 11 (TRAPPC11) cause limb-girdle muscular dystrophy R18 (LGMD R18). Although recently several genes related to myopathies have been identified, correlations between genetic causes and signaling events that lead from mutation to the disease phenotype are still mostly unclear. Here, we utilized zebrafish to model LGMD R18 by specifically inactivating trappc11 using antisense-mediated knockdown strategies and evaluated the resulting muscular phenotypes. Targeted ablation of trappc11 showed compromised skeletal muscle function due to muscle disorganization and myofibrosis. Our findings pinpoint that fish lacking functional trappc11 suppressed FGF8, which resulted in the aberrant activation of Notch signaling and eventually stimulated epithelial-mesenchymal transition (EMT) and fibrotic changes in the skeletal muscle. In summary, our study provides the role of FGF8 in the pathogenesis and its therapeutic potential of LGMD R18.

DOI： 10.1016/j.bbrc.2023.02.046

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers in Endocrinology  

DOI： 10.3389/fendo.2023.1151942

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Clinical Genetics  

Treacher Collins syndrome (TCS, OMIM: 154500) is a rare congenital craniofacial disorder that is caused by variants in the genes TCOF1, POLR1D, POLR1C, and POLR1B. Studies on the association between phenotypic variability and their relative variants are very limited. This systematic review summarized the 53 literatures from PubMed and Scopus to explore the potential TCS genotype–phenotype correlations with statistical analysis. Studies reporting both complete molecular genetics and clinical data were included. We identified that the molecular anomaly within TCOF1 (88.71%) accounted for most TCS cases. The only true hot spot for TCOF1 was detected in exon 24, with recurrent c.4369_4373delAAGAA variant is identified. While the hot spot for POLR1D, POLR1C, and POLR1B were identified in exons 3, 8, and 15, respectively. Our result suggested that the higher severity level was likely to be observed in Asian patients harboring TCOF1 variants rather than POLR1. Moreover, common 5-bp deletions tended to have a higher severity degree in comparison to any variants within exon 24 of TCOF1. In summary, this report suggested the relationship between genetic and clinical data in TCS. Our findings could be used as a reference for clinical diagnosis and further biological studies.

DOI： 10.1111/cge.14243

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cleft Palate Craniofacial Journal  

Objective: Genetic variants in EFTUD2 were proven to influence variable phenotypic expressivity in mandibulofacial dysostosis Guion-Almeida type (MFDGA) or mandibulofacial dysostosis with microcephaly (MFDM). Yet, the association between the severity of clinical findings with variants within the EFTUD2 gene has not been established. Thus, we aim to elucidate a possible genotype–phenotype correlation in MFDM. Methods: Forty articles comprising 156 patients were evaluated. The genotype–phenotype correlation was analyzed using a chi-square or Fisher's exact test. Results: The proportion of patients with MFDM was higher in Caucasian relative to Asian populations. Although, in general, there was no apparent genotype–phenotype correlation in patients with MFDM, Asians tended to have more severe clinical manifestations than Caucasians. In addition, cardiac abnormality presented in patients with intronic variants located in canonical splice sites was a predisposing factor in affecting MFDM severity. Conclusion: Altogether, this article provides the pathogenic variants observed in EFTUD2 and possible genotype–phenotype relationships in this disease.

DOI： 10.1177/10556656221136177

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Anatomy  

Bone has multiple functions in animals, such as supporting the body for mobility. The zebrafish skeleton is composed of craniofacial and axial skeletons. It shares a physiological curvature and consists of a similar number of vertebrae as humans. Bone degeneration and malformations have been widely studied in zebrafish as human disease models. High-resolution imaging and different bone properties such as density and volume can be obtained using micro-computed tomography (micro-CT). This study aimed to understand the possible changes in the structure and bone mineral density (BMD) of the vertebrae and craniofacial skeleton with age (4, 12 and 24 months post fertilisation [mpf]) in zebrafish. Our data showed that the BMD in the vertebrae and specific craniofacial skeleton (mandibular arch, ceratohyal and ethmoid plate) of 12 and 24 mpf fish were higher than that of the 4 mpf fish. In addition, we found the age-dependent increase in BMD was not ubiquitously observed in facial bones, and such differences were not correlated with bone type. In summary, such additional information on the craniofacial skeleton could help in understanding bone development throughout the lifespan of zebrafish.

DOI： 10.1111/joa.13780

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biology  

The fish gill is the first tissue that is exposed to the external media and undergoes continuous osmotic challenges. Recently, our group published an article entitled “Integrated Omics Approaches Revealed the Osmotic Stress-Responsive Genes and Microbiota in Gill of Marine Medaka” in the journal mSystems (e0004722, 2022), and suggested the possible host-bacterium interaction in the fish gill during osmotic stress. The previous study was performed by the progressive fresh water transfer (i.e., seawater to fresh water transfer via 50% seawater (FW)). Our group hypothesized that osmotic gradient could be a factor that determines the microbiota communities in the gill. The current 16S rRNA metagenomic sequencing study found that the direct transfer (i.e., seawater to fresh water (FWd)) could result in different gill microbiota communities in the same fresh water endpoints. Pseduomonas was the dominant bacteria (more than 55%) in the FWd gill. The Kyoto Encyclopedia of Genes and Genomes and MetaCyc analysis further suggested that the FWd group had enhanced osmosensing pathways, such as the ATP-binding cassette transporters, taurine degradation, and energy-related tricarboxylic acid metabolism compared to the FW group.

DOI： 10.3390/biology11101528

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

Fish gills are the major osmoregulatory tissue that contact the external water environment and have developed an effective osmoregulatory mechanism to maintain cellular function. Marine medaka (Oryzias melastigma) has the ability to live in both seawater and fresh water environments. The present study performed a seawater (SW) to 50% seawater (SFW) transfer, and the gill samples were used for comparative transcriptomic analysis to study the alteration of hypo-osmotic stress on immune responsive genes in this model organism. The result identified 518 differentiated expressed genes (DEGs) after the SW to SFW transfer. Various pathways such as p53 signaling, forkhead box O signaling, and the cell cycle were enriched. Moreover, the immune system was highlighted as one of the top altered biological processes in the enrichment analysis. Various cytokines, chemokines, and inflammatory genes that participate in the IL-17 signaling pathway were suppressed after the SW to SFW transfer. On the other hand, some immunoglobulin-related genes were up-regulated. The results were further validated by real-time qPCR. Taken together, our study provides additional gill transcriptome information in marine medaka; it also supports the notion that osmotic stress could influence the immune responses in fish gills.

DOI： 10.3390/ijms232012417

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers in Physiology  

DOI： 10.3389/fphys.2022.977185

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cancers  

Deubiquitinases (DUBs) deconjugate ubiquitin (UBQ) from ubiquitylated substrates to regulate its activity and stability. They are involved in several cellular functions. In addition to the general biological regulation of normal cells, studies have demonstrated their critical roles in various cancers. In this review, we evaluated and grouped the biological roles of DUBs, including proliferation, metastasis, and apoptosis, in the most common cancers in the world (liver, breast, prostate, colorectal, pancreatic, and lung cancers). The current findings in these cancers are summarized, and the relevant mechanisms and relationship between DUBs and cancers are discussed. In addition to highlighting the importance of DUBs in cancer biology, this study also provides updated information on the roles of DUBs in different types of cancers.

DOI： 10.3390/cancers14143547

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cleft Palate Craniofacial Journal  

Nager syndrome (NS) is a rare disease marked with craniofacial and preaxial limb anomalies. In this report, we summarized the current evidence to determine a possible genotype–phenotype association among NS individuals. Twenty-four articles comprising of 84 NS (including 9 patients with a severe form of NS [Rodriguez syndrome]) patients were examined, of which 76% were caused by variants in SF3B4 (OMIM *605593, Splicing Factor 3B, Subunit 4). Within the SF3B4 gene, variants located in exon 3 commonly occurred (20%) from a total identified variant, while hotspot location was identified in exon 1 (12%), and primarily occurred as frameshift variants (64%). Thirty-five distinct pathogenic variants within SF3B4 gene were identified with two common sites, c.1A > G and c.1060dupC in exons 1 and 5, respectively. Although no significant genotype–phenotype association was found, it is notable that patients with frameshift SF3B4 variants and predicted to lead to nonsense-mediated RNA decay (NMD) of the transcripts tended to have a more severe clinical manifestation. Additionally, patients harboring variants in exons 2 and 3 displayed a higher proportion of cardiac malformations. Taken together, this article summarizes the pathogenic variants observed in SF3B4 and provides a possible genotype–phenotype relationship in this disease.

DOI： 10.1177/10556656221089156

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers in Marine Science  

Recent studies have shown hypoxia to be an endocrine disruptor that impairs sex differentiation and reproductive function, leading to male-biased F1 populations in fish. However, the molecular mechanisms through which hypoxia alters fish sex differentiation and therefore sex ratios remain poorly understood. In order to understand the potential role of miRNAs in mediating hypoxia-altered sex determination and differentiation in fish, we conducted small RNA sequencing and transcriptome sequencing on marine medaka (Oryzias melastigma) embryos that were exposed to hypoxia (2.0 ± 0.2 mg O2 L–1) for 40 h (encompassing a critical window of sex determination). We identified dysregulated miRNAs and mRNAs in the hypoxia-exposed embryo, and bioinformatic analysis of the integrative small RNA sequencing and transcriptome sequencing results revealed hypoxia to cause alterations of genes related to embryonic development through miRNA regulation. Importantly, we have identified miRNA-mRNA pairs that were reported to play roles in gonad development (novel miR-145-col9a3 and novel miRNA-94- arid5b), in sex hormone response (novel miRNA-210-ca2, novel miRNA-106-nr2f2, nbr-miR-29c-nr4a1, and ola-miR-92b-akr1d1), and in sex characteristic development (novel miRNA-145-mns1, nle-miR-20-sord, and ipu-miR-219b-abcc8). Our findings highlighted the possible roles of miRNA–mRNA in regulation of embryonic development and sex determination in response to hypoxic stress.

DOI： 10.3389/fmars.2021.736362

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Ecotoxicology and Environmental Safety  

2,4-dichlorophenol (2,4-DCP) is commonly found in the aquatic environment that can be formed by the conversion of triclosan, which is a high production volume endocrine disturbing chemical. The study aims to understand the potential developmental toxicity of 2,4-DCP by using the in vivo zebrafish. We exposed the 2,4-DCP to the zebrafish embryos and collected the samples at several selected developmental stages (70–85% epiboly/10–12 somite/prim-5) for the whole mount in situ hybridization. The staining is used to investigate the ventral patterning, presumptive neural formation, and brain development. Results suggested that the 2,4-DCP exposure (up to 2.5 mg/L) did not affect the tested developmental processes in the survived embryos. Further experiments on lipid accumulation and oxidative stress were carried out at 5 days post fertilization larvae. Results showed the accumulation of oil droplets and induction of reactive oxygen species (ROS) in the larvae after the highest dosage exposure (2.5 mg/L). The real-time qPCR results suggested that the alternation of lipid metabolism was due to the reduced mRNA expressions of proliferator-activated receptor alpha (ppar-α) and acetyl-CoA carboxylase (acc); while the suppressed glutathione peroxidase (gpx) mRNA expression was responsible for the induction of the ROS. To conclude, the study provided scientific merits of understanding 2,4-DCP toxicity, and suggested the possible underlying mechanism of the defects.

DOI： 10.1016/j.ecoenv.2021.113133

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Publisher：Biomolecules  

Unlike mammals, zebrafish are capable to regenerate many of their organs, however, the response of tissue damage varies across tissues. Understanding the molecular mechanism behind the robust regenerative capacity in a model organism may help to identify and develop novel treatment strategies for mammals (including humans). Hence, we systematically analyzed the current literature on the proteome profile collected from different regenerated zebrafish tissues. Our analyses underlining that several proteins and protein families responsible as a component of cytoskeleton and structure, protein synthesis and degradation, cell cycle control, and energy metabolism were frequently identified. Moreover, target proteins responsible for the initiation of the regeneration process, such as inflammation and immune response were less frequently detected. This highlights the limitation of previous proteomic analysis and suggested a more sensitive modern proteomics analysis is needed to unfold the mechanism. This brief report provides a list of target proteins with predicted functions that could be useful for further biological studies.

DOI： 10.3390/biom12010035

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/lam.13539

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmars.2021.618489

Language：English   Publishing type：Research paper (scientific journal)  

Keng Po Lai, Zhiyuan Gong, William Ka Fai Tse

DOI： 10.1016/j.aquatox.2021.105813

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.marpolbul.2021.112090

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/cancers12051288

Language：English   Publishing type：Research paper (scientific journal)  

Deubiquitinases (DUBs) are involved in various cellular functions. They deconjugate ubiquitin (UBQ) from ubiquitylated substrates to regulate their activity and stability. Studies on the roles of deubiquitylation have been conducted in various cancers to identify the carcinogenic roles of DUBs. In this review, we evaluate the biological roles of DUBs in cancer, including proliferation, cell cycle control, apoptosis, the DNA damage response, tumor suppression, oncogenesis, and metastasis. This review mainly focuses on the regulation of different downstream effectors and pathways via biochemical regulation and posttranslational modifications. We summarize the relationship between DUBs and human cancers and discuss the potential of DUBs as therapeutic targets for cancer treatment. This review also provides basic knowledge of DUBs in the development of cancers and highlights the importance of DUBs in cancer biology.

DOI： 10.3390/ijms21072548

Language：English   Publishing type：Research paper (scientific journal)  

Perfluorooctanoic acid (PFOA) is a dispersive persistent organic pollutant in the environment. Accumulating reports suggest that PFOA is toxic to human lymphocytes; however, the toxicological effects of PFOA on these cells remain largely unclear. In this study, ultra-performance liquid chromatography (UPLC)-based metabolomic analysis was employed to identify metabolites in human peripheral blood lymphocytes and to assess the metabolic alterations caused by PFOA exposure. Our comparative metabolomic analysis results demonstrated that PFOA treatment could increase the level of organic acids and reduce the level of lipid molecules. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation further highlighted the fact that the PFOA treatment interfered with the metabolism of amino acids, carbohydrates and lipids, which may lead to disruption of the immune system.

DOI： 10.1016/j.chemosphere.2019.124810

Language：English   Publishing type：Research paper (scientific journal)  

Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.

DOI： 10.1096/fj.201902557RRR

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Treacher Collins syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects. Recently, the authors' group unfolded the pathogenesis of polr1c Type 3 TCS by using the zebrafish model. Facial development depends on the neural crest cells, in which polr1c plays a role in regulating their expression. In this study, the authors aimed to identify the functional time window of polr1c in TCS by the use of photo-morpholino to restore the polr1c expression at different time points. Results suggested that the restoration of polr1c at 8 hours after fertilization could rescue the TCS facial malformation phenotype by correcting the neural crest cell expression, reducing the cell death, and normalizing the p53 mRNA expression level in the rescued morphants. However, such recovery could not be reproduced if the polr1c is restored after 30 hours after fertilization.

DOI： 10.1016/j.ajpath.2017.10.004

Language：English   Publishing type：Research paper (scientific journal)  

The changes in ambient salinity influence ion and water homeostasis, hormones secretion, and immune response in fish gills. The physiological functions of hormones and ion transporters in the regulation of gill-osmoregulation have been widely studied, however the modulation of immune response under salinity changes is not determined. Using transcriptome sequencing, we obtained a comprehensive profile of osmo-responsive genes in gill cells of Japanese eel (Anguilla japonica). Herein, we applied bioinformatics analysis to identify the immune-related genes that were significantly higher expressed in gill pavement cells (PVCs) and mitochondrial-rich cells (MRCs) in freshwater (FW) than seawater (SW) adapted fish. We validated the data using the real-time qPCR, which showed a high correlation between the RNA-seq and real-time qPCR data. In addition, the immunohistochemistry results confirmed the changes of the expression of selected immune-related genes, including C-reactive protein (CRP) in PVCs, toll-like receptor 2 (TLR2) in MRCs and interleukin-1 receptor type 2 (IL-1R2) in both PVCs and MRCs. Collectively our results demonstrated that those immune-related genes respond to salinity changes, and might trigger related special signaling pathways and network. This study provides new insights into the impacts of ambient salinity changes on adaptive immune response in fish gill cells.

DOI： 10.1016/j.fsi.2017.12.026

Language：English   Publishing type：Research paper (scientific journal)  

mibⁿⁿ²⁰⁰², identified from an allele screen, shows early segmentation defect and severe cell death phenotypes, which are different from those of other described mib mutant alleles. We have previously reported its defects in somitogenesis and identified its origin of mutation, a large deletion in LG2. The report here is a continuous study, where we applied the bioinformatics analysis to profile the genetic background of mibⁿⁿ²⁰⁰² mutants. By comparing the transcriptomic data of mibⁿⁿ²⁰⁰² mutants with those of AB wild-type, a total of 1945 differentially expressed genes were identified, including 685 up- and 1260 down-regulated genes. The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis and Ingenuity Pathway Analysis (IPA) identified the enriched pathways and their related biological functions. Our data further demonstrated that the defects in the somitogenesis were related to the down-regulated segmentation genes, such as foxc1a, smyhc1, myod1 and mylpfa.

DOI： 10.1016/j.gene.2017.11.016

Language：English   Publishing type：Research paper (scientific journal)  

Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca²⁺ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca²⁺ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca²⁺ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.

DOI： 10.1038/s41598-018-19327-9

Language：English   Publishing type：Research paper (scientific journal)  

Deubiquitinases (DUBs) deconjugate ubiquitin (UBQ) from ubiquitylated substrates to regulate their activity and stability. They play different cellular functions such as cell cycle regulation, DNA repair, and early embryogenesis. Additionally, studies have demonstrated that some DUBs are the signaling targets of cellular stress such as oxidative stress. Reactive oxygen species are generated during normal mitochondrial oxidative metabolism and proper cellular mechanism could protect the cell from the oxidative stress. However, there are limited studies that specifically focus on the role of DUBs in oxidative stress, and thus the underlying protective mechanism by DUBs is not yet known. The report here, for the first time, applied the mouse-specific DUB RT² Profiler PCR array to identify DUBs that are responsive to oxidative stress. Out of the tested 83 DUBs, 15 of them were found to be differentially expressed. Among them, Usp18 was found to be induced with a dose- and time-dependent manner of oxidative stress. In functional studies, depletion of Usp18 could stimulate the p53 and caspase 3 protein levels. In addition, knockdown of Usp18 could lead to the reduced cell viability and increased in apoptotic cell death under oxidative stress. Collectively, Usp18 protects the cells from oxidative stress-induced apoptosis which may be through the regulation of p53 and caspase 3.

DOI： 10.1002/cbin.10799

Language：English   Publishing type：Research paper (scientific journal)  

Deconjugation of ubiquitin and/or ubiqutin-like modified substrates is essential to maintain a sufficient free ubiquitin within the cell. Deubiquitinases (DUBs) play a key role in the process. Besides, DUBs also play several important regulatory roles in cellular processes. However, our knowledge of their developmental roles are limited. The report here aims to study their potential roles in craniofacial development. Based on the previous genome-wide study in 2009, we selected 36 DUBs to perform the morpholino (MO) knockdown in this study, followed by the Alcian blue cartilage staining at 5 days post-fertilization (dpf) larvae to investigate the facial development. Results classified the tested DUBs into three groups, in which 28% showed unchanged phenotype (Class 1); 22% showed mild changes on the branchial arches (Class 2A); 31% had malformation on branchial arches and ethmoid plate (Class 2B); and 19% had severe changes in most of the facial structures (Class 3). Lastly, we used uchl3 morphant as an example to show that our screening data could be useful for further functional studies. To summarize, we identified new craniofacial developmental role of 26 DUBs in the zebrafish.

DOI： 10.1016/j.bbrc.2017.04.132

Language：English   Publishing type：Research paper (scientific journal)  

Benozophenone (BP) type UV filters are extensively used in the personal care products to provide protection against the harmful effects of UV radiation. BPs are one of the primary components in the UV filter family, in which benophenone-2 (BP2) is widely used as a UV filter reagent in the sunscreen. Humans used these personal care products directly on skin and the chemicals will be washed away to the water system. BP2 has been identified as one of the endocrine disruptor chemicals, which can inference the synthesis, metabolism, and action of endogenous hormones. Environmentally, it has been found to contaminate water worldwide. In this study, we aimed to unfold the possible developmental toxicology of this chemical. Zebrafish are used as the screening model to perform in situ hybridization staining to investigate the effects of BP2 on segmentation, brain regionalization, and facial formation at four developmental stages (10–12 somite, prim-5, 2 and 5 days post-fertilization). Results showed 40 μM (9.85 mg L⁻¹) or above BP2 exposure in zebrafish embryos for 5 days resulted in lipid accumulation in the yolk sac and facial malformation via affecting the lipid processing and the expression of cranial neural crest cells respectively. To conclude, the study alarmed its potential developmental toxicities at high dosage exposure.

DOI： 10.1016/j.chemosphere.2016.08.073

Language：English   Publishing type：Research paper (scientific journal)  

Treacher Collins syndrome (TCS, OMIM: 154500), an autosomal-dominant craniofacial developmental syndrome that occurs in 1 out of every 50,000 live births, is characterized by craniofacial malformation. Mutations in TCOF1, POLR1C, or POLR1D have been identified in affected individuals. In addition to established mouse models, zebrafish models have recently emerged as an valuable method to study facial disease. In this report, we summarized the two updated articles working on the pathogenesis of the newly identified polr1c and polr1d TCS mutations (Lau et al., 2016; Noack Watt et al., 2016) and discussed the possibility of using the anti-oxidants to prevent or rescue the TCS facial phenotype (Sakai et al., 2016). Taken together, this article provides an update on the disease from basic information to pathogenesis, and further summarizes the suggested therapies from recent laboratory research.

DOI： 10.1016/j.biocel.2016.10.016

Language：English   Publishing type：Research paper (scientific journal)  

Perfluorooctane sulfonate (PFOS), a hepato-toxicant and potential non-genotoxic carcinogen, was widely used in industrial and commercial products. Recent studies have revealed the ubiquitous occurrence of PFOS in the environment and in humans worldwide. The widespread contamination of PFOS in human serum raised concerns about its long-term toxic effects and its potential risks to human health. Using fatty liver mutant foie gras (fgr^-/-)/transport protein particle complex 11 (trappc11^-/-) and PFOS-exposed wild-type zebrafish embryos as the study model, together with RNA sequencing and comparative transcriptomic analysis, we identified 499 and 1414 differential expressed genes (DEGs) in PFOS-exposed wild-type and trappc11 mutant zebrafish, respectively. Also, the gene ontology analysis on common deregulated genes was found to be associated with different metabolic processes such as the carbohydrate metabolic process, glycerol ether metabolic process, mannose biosynthetic process, de novo' (Guanosine diphosphate) GDP-l-fucose biosynthetic process, GDP-mannose metabolic process and galactose metabolic process. Ingenuity Pathway Analysis further highlighted that these deregulated gene clusters are closely related to hepatitis, inflammation, fibrosis and cirrhosis of liver cells, suggesting that PFOS can cause liver pathogenesis and non-alcoholic fatty liver disease in zebrafish. The transcriptomic alterations revealed may serve as biomarkers for the hepatotoxic effect of PFOS.

DOI： 10.1016/j.chemosphere.2016.05.060

Language：English   Publishing type：Research paper (scientific journal)  

Triclosan (TCS) is an active antimicrobial ingredient used in many household products, such as skin creams and toothpaste. It is produced in high volumes, and humans are directly exposed to it and dispose it on a daily basis. TCS has been found to contaminate water worldwide. This study aimed to understand the potential developmental and metabolic abnormalities caused by TCS exposure by using zebrafish as the experimental model. Four developmental stages (70-85% epiboly, 10-12 somite, prim-5, and 5dpf) were selected to perform in situ hybridization staining to investigate the effects of TCS on dorsal ventral patterning, segmentation, brain development, and organ formation. Results showed, in terms of developmental toxicology, that neither phenotypic nor molecular changes were found after 5 days of 250. μg/L TCS exposure. However, such dosage of TCS exposure resulted in lipid droplet accumulation in the yolk sac, which might due to the deregulated mRNA expression level of beta-oxidation transcripts. This study showed that 250. μg/L TCS exposure does not affect normal embryogenesis or organogenesis; however, there are concerns regarding possible impairment of lipid metabolism.

DOI： 10.1016/j.aquatox.2016.01.001

Language：English   Publishing type：Research paper (scientific journal)  

Background: Homeostasis of ions and water is important for the maintenance of cellular functions. The regulation of the homeostasis is particularly important in euryhaline fish that migrate between freshwater (FW) and seawater (SW) environments. The fish gill, the major tissue that forms an interface separating the extracellular fluids and external water environment, has an effective transport system to maintain and regulate a constant body osmolality. In fish gills, the two major epithelial cells, pavement cells (PVCs) and mitochondria-rich cells (MRCs), are known to play key and complementary roles in ion transport at the interface. Discovering the robust mechanisms underlying the two cell types' response to osmotic stress would benefit our understanding of the fundamental mechanism allowing PVCs and MRCs to handle osmotic stress. Owing to the limited genomic data available on estuarine species, existing knowledge in this area is slim. In this study, transcriptome analyses were conducted using PVCs and MRCs isolated from Japanese eels adapted to FW or SW environments to provide a genome-wide molecular study to unravel the fundamental processes at work. Results: The study identified more than 12,000 transcripts in the gill cells. Interestingly, remarkable differential expressed genes (DEGs) were identified in PVCs (970 transcripts) instead of MRCs (400 transcripts) in gills of fish adapted to FW or SW. Since PVCs cover more than 90% of the gill epithelial surface, the greater change in gene expression patterns in PVCs in response to external osmolality is anticipated. In the integrity pathway analysis, 19 common biological functions were identified in PVCs and MRCs. In the enriched signaling pathways analysis, most pathways differed between PVCs and MRCs; 14 enriched pathways were identified in PVCs and 12 in MRCs. The results suggest that the osmoregulatory responses in PVCs and MRCs are cell-type specific, which supports the complementary functions of the cells in osmoregulation. Conclusions: This is the first study to provide transcriptomic analysis of PVCs and MRCs in gills of eels adapted to FW or SW environments. It describes the cell-type specific transcriptomic network in different tonicity. The findings consolidate the known osmoregulatory pathways and provide molecular insight in osmoregulation. The presented data will be useful for researchers to select their targets for further studies.

DOI： 10.1186/s12864-015-2271-0

Language：English   Publishing type：Research paper (scientific journal)  

This article contains data related to the two research articles titled Transcriptomic and iTRAQ proteomic approaches reveal novel short-term hyperosmotic stress responsive proteins in the gill of the Japanese eel (Anguilla japonica) (Tse et al. [1]) and iTRAQ-based quantitative proteomic analysis reveals acute hypo-osmotic responsive proteins in the gills of the Japanese eel (Anguilla japonica) (Tse et al. [2]). The two research articles show the usefulness of combining transcriptomic and proteomic approaches to provide molecular insights of osmoregulation mechanism in a non-model organism, the Japanese eel. The information presented here combines the raw data from the two studies and provides an overview on the physiological functions of fish gills.

DOI： 10.1016/j.dib.2015.02.012

Language：English   Publishing type：Research paper (scientific journal)  

mib nn2002, found from an allele screen, showed early segmentation defect and severe cell death phenotypes, which are different from previously known mib mutants. Despite distinct morphological phenotypes, the typical mib molecular phenotypes: her4 down-regulation, neurogenic phenotype and cold sensitive dlc expression pattern, still remained. The linkage analysis also indicated that mib nn2002 is a new mib allele. Failure of specification in anterior 7-10 somites is likely due to lack of foxc1a expression in mib nn2002 homozygotes. Somites and somite markers gradually appeared after 7-10 somite stage, suggesting that foxc1a is only essential for the formation of anterior 7-10 somites. Apoptosis began around 16-somite stage with p53 up-regulation. To find the possible links of mib, foxc1a and apoptosis, transcriptome analysis was employed. About 140 genes, including wnt3a, foxc1a and mib, were not detected in the homozygotes. Overexpression of foxc1a mRNA in mib nn2002 homozygotes partially rescued the anterior somite specification. In the process of characterizing mib nn2002 mutation, we integrated the scaffolds containing mib locus into chromosome 2 (or linkage group 2, LG2) based on synteny comparison and transcriptome results. Genomic PCR analysis further supported the conclusion and showed that mib nn2002 has a chromosomal deletion with the size of about 9.6 Mbp.

DOI： 10.1038/srep10673

Language：English   Publishing type：Research paper (scientific journal)  

Physiological studies of a unique endocrine gland in fish, named corpuscles of Stannius (CS), described a Ca2 ⁺ -regulatory function for this gland mediated by stanniocalcin-1, a hypocalcemic polypeptide hormone. However, to date, the endocrine functions of the glands have not been completely elucidated.We hypothesized that other unidentified active principles in the glands are involved in the regulation of plasma ion (Na ⁺, Ca2 ⁺) and/or blood pressure. In this study, transcriptome sequencing of CS glands was performed using Japanese eels (Anguilla japonica) adapted to freshwater (FW) or seawater (SW) to reveal the presence and differential expression of genes encoding proteins related to the ion-osmoregulatory and pressor functions. We acquired a total of 14.1 Mb and 12.1 Mb quality-trimmed reads from the CS glands collected from FW and SW adapted eels, respectively. The de novo assembly resulted in 9254 annotated genes. Among them, 475 genes were differentially expressed with 357 up- and 118 down-regulated in the SW group. Gene ontology analysis further demonstrated the presence of natriuresis and pressor related genes. In summary, ours is the first study using high-throughput sequencing to identify gene targets that could explain the physiological importance of the CS glands.

DOI： 10.1038/srep09836

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: The marine medaka Oryzias melastigma has been demonstrated as a novel model for marine ecotoxicological studies. However, the lack of genome and transcriptome reference has largely restricted the use of O. melastigma in the assessment of in vivo molecular responses to environmental stresses and the analysis of biological toxicity in the marine environment. Although O. melastigma is believed to be phylogenetically closely related to Oryzias latipes, the divergence between these two species is still largely unknown. Using Illumina high-throughput RNA sequencing followed by de novo assembly and comprehensive gene annotation, we provided transcriptomic resources for the brain, liver, ovary and testis of O. melastigma. We also investigated the possible extent of divergence between O. melastigma and O. latipes at the transcriptome level.
RESULTS: More than 14,000 transcripts across brain, liver, ovary and testis in marine medaka were annotated, of which 5880 transcripts were orthologous between O. melastigma and O. latipes. Tissue-enriched genes were identified in O. melastigma, and Gene Ontology analysis demonstrated the functional specificity of the annotated genes in respective tissue. Lastly, the identification of marine medaka-enriched transcripts suggested the necessity of generating transcriptome dataset of O. melastigma.
CONCLUSIONS: Orthologous transcripts between O. melastigma and O. latipes, tissue-enriched genes and O. melastigma-enriched transcripts were identified. Genome-wide expression studies of marine medaka require an assembled transcriptome, and this sequencing effort has generated a valuable resource of coding DNA for a non-model species. This transcriptome resource will aid future studies assessing in vivo molecular responses to environmental stresses and those analyzing biological toxicity in the marine environment.

DOI： 10.1186/s12864-015-1325-7

Language：English   Publishing type：Research paper (scientific journal)  

Osmoregulation in fish has been a classical research topic for several decades. Salmon and eels are the widely used model animals because of their wide distribution in different geographical locations and spawning migration between fresh- and salt-water habitats. Numerous fish osmoregulatory hormones and ion transporters were identified for their essential roles in acclimation and adaptation to waters of different salinities. Because of the lack of a genomic database, the scope of most studies, however, is very limited. Recently, our group reported the first high-throughput transcriptomic and proteomic studies to identify hyperosmotic-responsive genes/proteins in gills of Japanese eels. In this study, we aimed to decipher changes in hypo-osmotic-responsive proteins in fish acclimating from seawater (SW) to freshwater (FW) conditions. We collected gill samples from SW-adapted and SW-to-FW-acclimating fish. The respective gill proteins were extracted and labeled using isobaric tags for relative and absolute quantitation (iTRAQ) and analyzed using a high-resolution mass spectrometer. In the short-term transfer from SW to FW, 51 hypo-responsive proteins were detected, and 24 unique hypo-osmotic-responsive proteins were identified (15 up-regulated and nine down-regulated proteins). Our data support the use of an omics approach to facilitate the application of functional genomics in non-model organisms. Biological significance: By combining transcriptomic and proteomic approaches, the study has provided the most comprehensive, targeted investigation of eel gill hypo-osmotic responsive proteins that provides molecular insights of osmoregulation mechanisms in a non-model organism, eel. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

DOI： 10.1016/j.jprot.2014.01.025

Language：English  

Zebrafish have been widely used in developmental studies as an excellent in vivo, high-throughput and scalable system for decades, due to the following advantages. Its small size (< 5mm at 7 days post-fertilization (dpf)) and large number of rapidly developing transparent embryos (most organs are developed within 5dpf) enable researchers to undergo whole mount in situ hybridization studies, real-time fate-mapping experiments, and visualizing developing organ morphology. Furthermore, the zebrafish has a well-established genome database that provides information for genomic studies, ranging from microarrays to more advance deep-sequencing studies. Moreover, its highly conserved developmental signalling pathways position it as a good model to correlate with other mammalian studies. In the field of environmental toxicology, zebrafish is used to understand the common and standard toxicology end point, such as phenotypic changes and lethal concentrations (LC50). However, these studies do not fully utilize the features of zebrafish. This chapter aims to provide our experiences and opinion on using zebrafish to understand early developmental defects caused by toxicant exposures. A standard whole-mount in situ hybridization screening protocol was used to monitor three critical early developmental stages (60-75% epiboly, 8-10 somite, and prim-5). The screening provided first-hand information of whether the toxicant affects the dorsoventral (DV) patterning, segmentation, and brain development in zebrafish embryos within 24 hours exposure of toxicants. Since tracing the continuous developmental changes at different stages of mammalian embryos is not easy due to in utero development; the zebrafish provides an excellent alternative way to understand the developmental toxicology of different toxicants.

Language：English   Publishing type：Research paper (scientific journal)  

Osmotic stress transcription factor 1 (Ostf1) was first discovered by subtractive hybridization in the gills of Mozambique tilapia (Oreochromis mossambicus) transferred from fresh water (FW) to seawater (SW). It is a putative transcriptional regulator and the "early hyperosmotic regulated protein". In the 2 hours after FW to SW transfer, ostf1 mRNA levels increase six fold. It is believed that, as a fast-response gene, Ostf1 plays a critical role in fish osmoregulation. Since its discovery, numerous studies have been performed to understand the nature and osmoregulatory mechanism of Ostf1. A decade has passed since the discovery of Ostf1, and it is a good time to summarize our current understanding of this gene. Different fish models have been used to study Ostf1, which is not limited to the traditional euryhaline fishes, such as eels and tilapia. Ostf1 can be found in modern fish models such as medaka and zebrafish. This review covers and summarizes the findings from different fishes, and provides a perspective for future Ostf1 studies.

DOI： 10.1186/s12983-014-0086-5

Language：English   Publishing type：Research paper (scientific journal)  

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/. frzb/. fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn-/- mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/. frzb/. fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/. frzb/. fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.

DOI： 10.1016/j.ydbio.2013.06.012

Language：English   Publishing type：Research paper (scientific journal)  

Osmoregulation is critical for the survival of fishes that migrate between freshwater (FW) and seawater (SW). The eel, as a catadromous fish, has been studied for decades to reveal the mechanisms of osmoregulation. These studies, however, have been limited by the lack of a genomic database to decipher the mechanism of osmoregulation at a molecular level. In this study, using high-throughput transcriptomic and proteomic technologies, we have provided the first genome-wide study to identify hyperosmotic responsive proteins in the gills of the Japanese eel. Deep sequencing using the 454 platform produced over 660,000 reads with a mean length of 385. bp. For the proteomic study, we collected gill samples from three different treatment groups of fish that had fully adapted to FW/SW or were transferred from FW to SW for 6. h. The respective group of gill proteins were extracted and labeled using an isobaric tag for relative and absolute quantitation (iTRAQ) using LTQ-Orbitrap, a high resolution mass spectrometer. Among the 1519 proteins identified from the gill samples, 96 proteins were differentially expressed between FW and SW adapted fish. Nineteen hyperosmotic responsive proteins were detected (10 up-regulated and 9 down-regulated proteins) after 6. h post FW to SW transfer. Biological significance: The study has provided the most comprehensive, targeted investigation of eel gill proteins to date, and shown the powerfulness of combining transcriptomic and proteomic approaches to provide molecular insights of osmoregulation mechanisms in a non-model organism, eel.

DOI： 10.1016/j.jprot.2013.05.026

Language：English   Publishing type：Research paper (scientific journal)  

Background Osmotic stress transcription factor 1/transforming growth factor-β-stimulated clone 22 domain 3 (Ostf1/Tsc22d3) is a transcription factor that plays an osmoregulatory role in euryhaline fishes. Its mRNA and protein levels are up-regulated under hyperosmotic stress. However, its osmoregulatory and developmental functions have not been studied in any stenohaline freshwater fishes. Zebrafish is an excellent model to perform such study to unfold the functional role of Tsc22d3. Methods We identified the zebrafish Tsc22d3 and performed knockdown studies using morpholino antisense oligonucleotide (MO). Results Zebrafish Tsc22d3 did not response to hypertonic stress and ts22d3 knockdown or overexpression by injecting MO or capped RNA did not change the transcriptional levels of any of the known ionocyte markers. To reveal the unknown function of zebrafish Tsc22d3, we performed several in situ molecular marker studies on tsc22d3 morphants and found that Tsc22d3 plays multi-functional roles in dorsoventral (DV) patterning, segmentation, and brain development. We then aimed to identify the mechanism of Tsc22d3 in the earliest stages of DV patterning. Our results demonstrated that tsc22d3 is a ventralizing gene that can stimulate the transcription of bone morphogenetic protein 4 (bmp4) and, thus, has a positive effect on the Bmp signaling pathway. Furthermore, we showed that Tsc22d3 interacts with deubiquitylating enzymes, ubiquitin-specific protease 15 (Usp15) and ovarian tumor domain containing protein 4 (Otud4). In addition, the interruption of Bmp4 signaling by double knockdown of usp15 and otud4 reduced the ventralized effects in tsc22d3-overexpressing embryos. Conclusions This is the first study to identify new developmental functions of Tsc22d3 in zebrafish. General significance Zebrafish tsc22d3 is a ventralizing gene and plays a role in early embryogenesis.

DOI： 10.1016/j.bbagen.2013.05.006

Language：English   Publishing type：Research paper (scientific journal)  

Osmosensing and osmoregulatory processes undertaken in gills of euryhaline fish are coordinated by integrative actions of various signaling molecules/transcriptional factors. Considerable numbers of studies report the hyper- and hypoosmoregulatory functions of fish gills, by illustrating the process of gill cell remodeling and the modulation of the expression of ion channels/transporters. Comparatively mechanistic information relayed from signal integration to transcriptional regulation in mediating gill cell functions has not yet been elucidated. In this study we demonstrate the functional links from cortisol stimulation, to Akt activation, to the expression of the transcriptional factor, Ostf1. Using the synthetic glucocorticoid receptor agonist, dexamethasone (DEX), Ostf1 expression is found to be activated via glucocorticoid receptor (GR) and mediated by the Akt-GSK3β signaling pathway. Pharmacological experiments using kinase inhibitors reveal that the expression of Ostf1 is negatively regulated by Akt activation. The inhibition of PI3K or Akt activities, by the specific kinase inhibitors (wortmannin, LY294002 or SH6), stimulates Ostf1 expression, while a reduction of GSK3β activity by LiCl reduces Ostf1 expression. Collectively, our report for the first time indicates that DEX can induce Ostf1 via GR, with the involvement of the Akt-GSK3β signaling pathway in primary eel gill cell cultures. The data also suggest that Ostf1 may play different roles in gill cell survival during seawater acclimation.

DOI： 10.1242/bio.20134135

Language：English   Publishing type：Research paper (scientific journal)  

Exposure of a developing embryo or fetus to endocrine disrupting chemicals (EDCs) has been hypothesized to increase the propensity of an individual to develop a disease or dysfunction in his/her later life. Although it is important to understand the effects of EDCs on early development in animals, sufficient information about these effects is not available thus far. This is probably because of the technical difficulties in tracing the continuous developmental changes at different stages of mammalian embryos. The zebrafish, an excellent model currently used in developmental biology, provides new insights to the field of toxicological studies. We used the standard whole-mount in situ hybridization screening protocol to determine the early developmental defects in zebrafish embryos exposed to the ubiquitous pollutant, bisphenol A (BPA). Three stages (60-75% epiboly, 8-10 somite, and prim-5) were selected for in situ screening of different molecular markers, whereas BPA exposure altered early dorsoventral (DV) patterning, segmentation, and brain development in zebrafish embryos within 24 hours of exposure.

DOI： 10.1242/bio.20134283

Language：English   Publishing type：Research paper (scientific journal)  

Osmoregulation is an essential mechanism for euryhaline fish. Gill cells undergo rapid mechanism to maintain the cellular homeostasis during osmotic stress. Reports have suggested that gill cells may be able to migrate between primary filament and secondary lamella during seawater acclimination. However, the factor that can trigger such process is not well-known. Previously, we identified the osmotic stress transcription factor 1b (Ostf1b) in medaka and found that it is an early hypertonic responsive gene and can activate the c-Jun N-terminal kinase (JNK) pathway. In this report, we aim to know if Ostf1b plays the role in the migration. Ostf1b was ectopic expressed in the human embryonic kidney cell line (HEK293) to understand the Ostf1b function. Results clearly demonstrated that Ostf1b could constitutively activate the Rho kinase 1 (ROCK1) and myosin light chain 2 (MLC2) signalling pathway that promotes cell migration, epithelial mesenchymal transition (EMT) and cytoskeletal dynamics through stress fibre formation. The study supports the notion of cell migration and cytoskeleton rearrangement theories in osmoregulation.

DOI： 10.1016/j.biocel.2013.05.023

Language：English   Publishing type：Research paper (scientific journal)  

Background: Osmotic stress transcriptional factor 1 (Ostf1) was firstly identified in tilapia in 2005. Then numerous studies have investigated its regulation and expression profile in fish gill tissues in related to osmoregulation. Generally, hyperosmotic stress induced ostf1 mRNA expression level, however there is no report studying the cellular localization of Ostf1 expression in any osmoregulatory tissue. In this study immunohistochemical (IHC) approach was used to study the cellular localization of Ostf1 in gill cells of Japanese eels.Findings: Ostf1 protein was found to be localized in branchial mitochondria-rich/chloride cell (MRC/CC) as revealed by Naα5 and CFTR co-localization. The protein was detectable at day 3 after fresh water to seawater transfer and was mainly localized in MRCs. Moreover, elevated levels of extracellular signal regulated kinase (ERK) phosphorylation was observed at day 3 of the transfer and was co-localized with MRCs.Conclusions: Our data identified Ostf1 expression in gill MRCs. The observation supports the role of Ostf1 in osmosensing and/or osmoregulation in fish gills, particularly its functional relationship with MRCs. The observation of the co-expression of pERK and Ostf1 in MRCs suggests a cross-talk mechanism between the mitogen-activated protein kinases (MAPKs) and Ostf1 in response to hyperosmotic challenge. To summarize, this report has addressed the cellular localization of Ostf1 and provides evidence to illustrate the involvement of Ostf1 and ERK on osmosensing and osmoregulatory function of gill MRCs.

DOI： 10.1186/1742-9994-9-3

Language：English  

In order to unfold the function of genes, solely performing mRNA over-expression is not enough nowadays. Traditional protein expression experiments, such as Western blotting and immunohistochemical staining, could only provide researchers the changes of expression levels and/or location of their targets. To make a more strong and convincing statement about gene function, it is necessary to perform both "gain-of-function" and "loss-of-function" studies. Both assays can be performed easily by transfecting DNA plasmid and siRNA in cell culture system; while in zebrafish, mRNA and morpholino (MO) microinjection can serve similar purposes. It is common for the zebrafish community to carry out microinjection experiments to explore a gene function. Instead of making a single knockdown/over-expression of a gene, we foresee that more and more large-scale screens on certain protein families will be performed in the future. Here, based on our previous experience in zebrafish "loss-of-function" screening on deubiquitylating enzymes, we describe a general work flow, from morpholino designation, in situ hybridization, to data analysis, as a reference for researchers who may be interested in a similar screen.

DOI： 10.1007/978-1-61779-424-7_24

Language：English  

Eels are euryhaline fishes that their life cycles involve the stay in fresh water and seawater. In order to compensate the osmotic challenges in fresh water (ion loss and water gain) and in seawater habitat (ion gain and water loss), eels have developed specific osmoregulatory mechanisms to acclimate rapidly and effectively in such two extreme aquatic environments. Fish gill is the outermost tissue that is in direct contact with the external media. In the gill tissue, two specific osmoregulatory cell types (pavement cells (PVCs) and mitochondria-rich/chloride cells (MRCs/CCs)) have been identified and characterized. It is generally believed that MRCs, which we will focus on, are the essential ion-regulatory cell type in the eel gill. With the recent advances in molecular biology, together with the immunohistochemical staining and electrophysiology studies, more transcriptional and translational profiling data are generated to unfold the molecular nature of the branchial osmoregulatory mechanisms. In this review, we will begin with the general description of eel gill structure and cell types (PVCs and MRCs), followed by the discussion on the differential mRNA expressions of different ion transporters in MRCs under hyper/ hypo- tonic stress conditions. Lastly, we will provide an opinion on the use of recent advanced genomic transcriptome data to form a public accessible database. To summarize, this chapter provides essential updated information on the expressions of ion channels/ transporters in eel gills acclimated in waters of different salinities, providing new in-sight on the future direction of eel gill studies in modern molecular perspectives.

Language：English   Publishing type：Research paper (scientific journal)  

Eukaryotic cells undergo rapid regulatory processes to maintain cellular homeostasis upon osmotic stress. In fishes, gill epithelial cells play main roles in these processes. Although osmoregulatory functions of fish gills have been well studied, little is known about the underlying mechanisms, particularly the hypertonic-induced signalling pathways during osmotic stress. This study reports for the first time on the osmo-sensing signal cascade that related to the medaka osmotic stress transcription factor 1 (Ostf1), a hypertonic induced immediate early gene, under hypertonic stress. Quantitative real-time PCR showed the rapid increase of Ostf1 in gill after transfer of medaka from fresh water to 50% seawater; particularly Ostf1b whose mRNA expression increased to 4 folds at 0.5 h and reached to 10 folds at 6 h after the transfer. The in vivo knockdown of Ostf1b profoundly inhibited SEK and JNK phosphorylation, but not p38 and ERK phosphorylation in the medaka gill tissue. To further investigate the possible role of Ostf1b in the JNK pathway, Ostf1b was ectopically expressed in HEK293 cells. Results indicated that Ostf1b is a downstream target of SEK and JNK and exerts a positive feedback loop on the JNK signalling pathway via activation of GCK and/or MLK3 proteins. Additionally, MAPK inhibitors experiments suggested that activation of the JNK pathway by hypertonicity is involved in the maintenance of Ostf1b stability, which in turn provides continuous stimulation of GCK for JNK phosphorylation. Lastly, changes in transcription levels of different water/ion transporters were found in knockdown or ecoptic over-expression of Ostf1b in medaka gills and human embryonic kidney cells, suggesting the role of Ostf1b in modulation of critical water channel/ion transporters during osmotic stress.

DOI： 10.1016/j.biocel.2011.08.013

Language：English   Publishing type：Research paper (scientific journal)  

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na⁺ uptake and to examine the effect of adjusting external Na⁺ and Cl^- ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na⁺-deficient AF, or (c) Cl^--deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na⁺-deficient AF, or Cl^--deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na⁺ and/or Cl^- levels when compared with DI-acclimated eel. Significant inductions of V-type H⁺-ATPase (V-H⁺-ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na⁺-deficient AF, mRNA expression levels of V-H⁺-ATPase, NBC1, and Na⁺/H⁺-exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl^--deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na⁺ in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na⁺ uptake.

DOI： 10.1002/jez.681

Language：English   Publishing type：Research paper (scientific journal)  

Mitochondria-rich cells (MRCs) of fish gills are well known for their roles in Na⁺ and Cl^- excretion in seawater environment. However, the level of understanding of their roles in fresh water is limited. In this study, we used the Percoll gradient technique to isolate gill MRCs from Japanese eels (Anguilla japonica Temminck and Schlegel, 1846) and measured the mRNA expression of two selected ion transporters, which were partially cloned in our laboratory: sodium bicarbonate co-transporter 1 (nbce1) and vacuolar proton pumps (H⁺-atpase). In addition, protein expressions of H⁺-ATPase and NBCe1 in whole gill were studied using Western blotting. The data indicated that both NBCe1 and H⁺-ATPase mRNA and protein expressions increased after seawater to freshwater transfer. This is the first report of changes in mRNA expression for these two ion transporters in MRCs of Japanese eels during seawater to freshwater transfer, confirming the roles of MRCs in osmoregulation in fresh water.

DOI： 10.1139/z11-009

Language：English   Publishing type：Research paper (scientific journal)  

Zebrafish cops6 encodes a putative deubiquitylating enzyme (DUB) that belongs to the JAMM family. It consists of 297 amino acids and includes the Mov34/MPN/PAD-1 (PF01398) domain. Ubiquitylation is involved in many cellular processes and deconjugation of ubiquitinmodified substrates is important to maintain a sufficient amount of free ubiquitin in the cell. Here, we report our findings regarding the general function of the cops6 gene, as a continuation of our previous studies involving DUB knockdown screening. We have found that cops6 plays different roles in early embryonic development in the zebrafish, including dorsoventral patterning, convergent extension movement and brain formation. In addition, our findings indicate that cops6 plays an anti-apoptotic role during segmentation. Overall, the present study that consolidates our previous work on zebrafish DUB genes, corroborates the hypothesis of multi-functional roles for DUB genes during development.

DOI： 10.1387/ijdb.103089wt

Language：English   Publishing type：Research paper (scientific journal)  

Background: Deconjugation of ubiquitin and/or ubiquitin-like modified protein substrates is essential to modulate protein-protein interactions and, thus, signaling processes in cells. Although deubiquitylating (deubiquitinating) enzymes (DUBs) play a key role in this process, however, their function and regulation remain insufficiently understood. The "loss-of-function" phenotype studies can provide important information to elucidate the gene function, and zebrafish is an excellent model for this goal.Results: From an in silico genome-wide search, we found more than 90 putative DUBs encoded in the zebrafish genome belonging to six different subclasses. Out of them, 85 from five classical subclasses have been tested with morpholino (MO) knockdown experiments and 57 of them were found to be important in early development of zebrafish. These DUB morphants resulted in a complex and pleiotropic phenotype that, regardless of gene target, always affected the notochord. Based on the huC neuronal marker expression, we grouped them into five sets (groups I to V). Group I DUBs (otud7b, uchl3 and bap1) appear to be involved in the Notch signaling pathway based on the neuronal hyperplasia, while group IV DUBs (otud4, usp5, usp15 and usp25) play a critical role in dorsoventral patterning through the BMP pathway.Conclusion: We have identified an exhaustive list of genes in the zebrafish genome belonging to the five established classes of DUBs. Additionally, we performed the corresponding MO knockdown experiments in zebrafish as well as functional studies for a subset of the predicted DUB genes. The screen results in this work will stimulate functional follow-up studies of potential DUB genes using the zebrafish model system.

DOI： 10.1186/1471-2164-10-637

Language：English   Publishing type：Research paper (scientific journal)  

In the present study, we aimed to clone an osmotic stress transcriptional factor (Ostf) from gill cells of Japanese eels. In addition, we measured its expression in Percoll™-gradient-isolated gill chloride (CC) and pavement (PVC) cells and determined the regulation of its expression in primary gill cell culture. Using degenerative primers and RACE techniques, we cloned a cDNA of 615 bp, encompassing the coding sequence of Ostf (204 amino acids). The cloned Ostf1 DNA sequence shared 84% DNA homology with the Ostf1 of tilapia. In general, the basal Ostf expression level was found to be significantly higher in CCs than in PVCs. In the direct transfer of fish from freshwater to seawater, a significant but transient induction of Ostf mRNA in CCs and PVCs was measured after 6 h of acclimation. Compared with gill CCs, the level of induction measured at PVCs was lower. In the seawater-to-freshwater transfer, no significant change in Ostf transcript levels was detected in either CCs or PVCs. To decipher the regulatory mechanism of Ostf expression, we conducted experiments using primary gill cell culture to specifically address the involvement of two putative osmosensors (i.e. intracellular ion strength/macromolecular crowding and cytoskeleton) in the regulation of Ostf expression. Hypertonic treatment using impermeable solutes (i.e. NaCl, 500 mOsmol l^-1) induced Ostf mRNA expression in 6 h, but no noticeable effect was measured using permeable solute (i.e. urea, 500 mOsmol l ^-1). The induction was transcriptionally regulated and was abolished by the addition of organic osmolytes (i.e. betaine, inositol or taurine) into the culture media. Addition of colchicine (an inhibitor of microtubule polymerization) to hypertonic (with added NaCl, 500 mOsmol l^-1) cells reduced Ostf mRNA expression, suggesting that an increase in intracellular ionic strength and the integrity of the cytoskeleton are involved in the activation of Ostf mRNA expression in the cells. Collectively, the results of this study reveal, for the first time, the differential expression of Ostf in isolated CCs and PVCs. The resulting knowledge can shed light on how Ostf participates in hyperosmotic adaptation in fish gills.

DOI： 10.1242/jeb.017368

Language：English   Publishing type：Research paper (scientific journal)  

It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l^-1) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na⁺/K⁺-ATPase, Na⁺/K⁺/2Cl^- cotransporter (NKCC) and Na ⁺/H⁺ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, a and β subunits of Na⁺/K ⁺-ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T ₃) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions.

DOI： 10.1242/jeb.004101

Language：English   Publishing type：Research paper (scientific journal)  

Ion channels and transporters (i.e. cystic fibrosis transmembrane regulator (CFTR), inward rectifier potassium channel (eKir), Na/K-ATPase, Na/K/Cl₂ co-transporter (NKCC), aquaporin-3 (AQP-3), and Na/H exchanger-1 (NHE-1)) are known to be expressed in gill epithelia of teleost fish. Owing to the anatomical complexity of gill structures, their temporal expression profile in seawater acclimating gill pavement (PVCs) and chloride cells (CCs) are limited. In this study, we isolated the gill PVCs and CCs from seawater acclimating Japanese eels to address the issue. In the gill epithelia of freshwater adapted eels, CCs expressed the highest mRNA and/or protein levels of Na/K-ATPase, NKCC, and eKir as demonstrated by real-time PCR and/or immunohistochemical staining. AQP-3 mRNA was highly expressed in freshwater PVCs and its protein was in general expressed in all gill cells. The NHE-1 transcripts were expressed in similar levels in both PVCs and CCs. CFTR mRNA transcript was almost undetectable in all the freshwater gill cell samples. Seawater acclimation induced the transcript and/or protein levels of Na/K-ATPase, NKCC, CFTR, and eKir in CCs. The upregulation and the coexpression of these transporters in CCs suggested their cohort function in mediating Na⁺, K⁺, and Cl^- transport. The expression of CFTR was found to be tightly regulated as its expression was restricted only in "seawater CCs". AQP-3 transcript and protein levels in PVCs reduced significantly during the acclimation. Interestingly immunocytochemical (ICC) staining of seawater gill epithelia revealed that AQP-3 immunoreactivities were mainly localized in seawater CCs. In the acclimation, there was no significant reduction of NHE-1 mRNA in both PVCs and CCs, however its protein level dropped significantly in the seawater condition. The present study is the first to demonstrate the activation of the mRNA transcripts for the ion channels and transporters in isolated gill CCs during seawater acclimation. The activating mechanism is found to be confined primarily in CCs. These results indicated that in addition to the increase in size and number of CCs, the molecular remodeling and the functional plasticity of CCs were essential in the ion transport process during seawater acclimation.

DOI： 10.1016/j.bbrc.2006.06.028

Language：English   Publishing type：Research paper (scientific journal)  

Changes in cellular energy and redox states in the C6 glioma cells exposed to increasing concentrations of either Zn or Se were studied to examine whether different elements cause different patterns of changes in cellular metabolism. Following a 3-h exposure, both Zn and Se⁺⁴ caused dose-dependent decreases in cell viability and total adenosine nucleotides (TAN = ATP + ADP + AMP). In addition, Zn caused a dose-dependent increase in cellular ATP/TAN and a decrease in the ADP/TAN and AMP/TAN. These changes resulted in a significant increase in energy charge potential (ECP = [ATP + 0.5ADP]/TAN). Se⁺⁴, on the other hand, caused a dose-dependent decrease in ATP/TAN but an increase in both ADP/TAN and AMP/TAN, resulting in a dose-dependent decrease in ECP. Both Zn and Se⁺⁴ caused a dose-dependent decrease in GSH/GSSG and an increase in GSH + GSSG when compared to TAN. In contrast to Zn and Se ⁺⁴, the nontoxic Se⁺⁶ caused no significant changes in cellular energy states but reduced the GSH/GSSG ratio from 3.14 ± 0.49 to 2.05 ± 0.29, which could be explained by the effect of Se on enzymes responsible for GSH metabolism. As the cellular ATP level has been considered an important element that mediates the mode of cell death, it was suggested that a significant increase in ATP/TAN upon exposure to Zn would indicate that cell death occurred via apoptosis, while Se⁺⁴ caused a different pattern of cell death. This was confirmed by the appearance of cells with fragmented nucleus in cells treated with Zn, but not Se⁺⁴ and Se⁺⁶. The results demonstrated that different chemicals caused different patterns of metabolic changes. The correlation between metabolic changes and the mode of cell death was discussed.

DOI： 10.1080/15376520500194692

Event date： 2019.8

Language：English  

Venue：Suzhou   Country：China  

Event date： 2019.5

Language：English  

Venue：Osaka   Country：Japan  

Event date： 2018.8

Language：English  

Venue：Nagoya   Country：Japan  

Event date： 2018.6

Language：English  

Venue：Tokyo   Country：Japan  

Event date： 2017.8

Language：English  

Venue：Yamanashi Prefectural Library Hall   Country：Japan  

Event date： 2017.5

Language：English  

Venue：Tokyo   Country：Japan  

Event date： 2015.7

Language：English   Presentation type：Oral presentation (general)  

Venue：Li Ka Shing Faculty of Medicine, The University of Hong Kong  

Event date： 2015.6

Language：English  

Venue：Tsukuba   Country：Japan  

Event date： 2015.6

Language：English  

Venue：Tsukuba   Country：Japan  

Event date： 2014.5

Language：English  

Venue：Nagoya   Country：Japan  

Event date： 2014.5

Language：English  

Venue：Nagoya   Country：Japan  

Event date： 2013.5

Language：English   Presentation type：Oral presentation (general)  

Venue：Shimane   Country：Japan  

Event date： 2012.11

Language：English  

Event date： 2009.7

Language：English  

Venue：San Francisco   Country：United States  

Event date： 2009.5

Language：English  

Venue：Niigata   Country：Japan  

Event date： 2008.6

Language：English  

Venue：Madison   Country：United States  

Event date： 2006.7

Language：English  

Venue：St. John’s, Newfoundland   Country：Canada  

Event date： 2006.2

Language：English   Presentation type：Oral presentation (general)  

Venue：Bangkok   Country：Thailand  

Event date： 2005.3

Language：English  

Venue：New Orleans   Country：United States  

Type：Peer review 

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Number of peer-reviewed articles in foreign language journals：30

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Type：Competition, symposium, etc. 

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Number of peer-reviewed articles in foreign language journals：50

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Number of peer-reviewed articles in foreign language journals：26

Type：Academic society, research group, etc. 

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Number of peer-reviewed articles in foreign language journals：24

Type：Other

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