Identification of a novel interaction site between the large hepatitis delta antigen and clathrin that regulates the assembly of genotype III hepatitis delta virus.

BACKGROUND: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly. METHODS: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays. RESULTS: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV. CONCLUSIONS: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.

Investigating Potential Cardiovascular Toxicity of Two Anti-Leukemia Drugs of Asciminib and Ponatinib in Zebrafish Embryos.

BCR-ABL, a fusion protein kinase, is a druggable target exclusively expressed in patients with chronic myeloid leukemia (CML). Several anti-leukemia medicines targeting this protein have been developed in recent years. However, therapeutic options are limited for CML patients bearing multiple BCR-ABL1 mutations. Ponatinib (PON), a potent tyrosinase inhibitor, was one of the approved drugs for managing BCR-ABL1 T315I mutant disease. However, treatment of patients with PON reported severe side effects related to cardiovascular events. Asciminib (ASC) was the first allosteric inhibitor approved to target the myristoyl pocket of BCR-ABL protein to inhibit protein activity. The different mechanism of inhibition opens the possibility of co-exposure with both medicines. Reports on cardiovascular side effects due to the combination use of PON + ASC in pre-clinical and clinical studies are minimal. Thus, this study aimed to observe the potential cardiovascular-related side effect after co-exposure to ASC and PON using zebrafish as an animal model. In this study, zebrafish were acutely exposed to both compounds. The cardiovascular physiology parameters and gene expression related to cardiovascular development were evaluated. We demonstrate that combining ASC with PON at no observed effect concentration (NOEC) did not cause any significant change in the cardiac performance parameter in zebrafish. However, a significant increase in nkx2.5 expression level and a substantial decrease in blood flow velocity were recorded, suggesting that combining these compounds at NOEC can cause mild cardiovascular-related side effects.

The role of transformation in the risks of chemically exfoliated molybdenum disulfide nanosheets to the aquatic environment.

While the effects of environmental factors (e.g., coexisting organic macromolecules and solar irradiation) on the phase transformation and oxidative dissolution of chemically exfoliated molybdenum nanosheets (ceMoS2) have been recognized, the effects of environmental processes on the subsequent biological impacts of ceMoS2 are still poorly understood. In this study, the bioavailability and transitions in chemical speciation occurring during the aging process are demonstrated to be key factors causing ceMoS2 to affect aquatic organisms. The lower survival rate of embryonic zebrafish with aged (i.e., sunlight-irradiated and dark-ambient-aged) ceMoS2, compared to that with freshly prepared ceMoS2, was due to the release of ionic aging products (mainly acidic Mo species) throughout the oxidative dissolution of ceMoS2. The released soluble molybdenum interacted with natural organic matter (NOM) depending on their functionality, and this attenuated the toxicity caused by ceMoS2 to different degrees. Toxicity triggered by aged ceMoS2 under both dark and irradiated conditions was significantly reduced by Suwannee River NOM due to the formation of complexes with ionic Mo species, which was established by Mo K-edge X-ray absorption spectroscopy. The findings provide useful insights for comprehending the impacts of ceMoS2 on aquatic organisms and guidance for the prevention measures necessary in the applications of MoS2 nanosheets.

Momordica cochinchinensis Aril Ameliorates Diet-Induced Metabolic Dysfunction and Non-Alcoholic Fatty Liver by Modulating Gut Microbiota.

Obesity and its associated conditions, such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are a particular worldwide health problem at present. Momordica cochinchinensis (MC) is consumed widely in Southeast Asia. However, whether it has functional effects on fat-induced metabolic syndrome remains unclear. This study was conducted to examine the prevention effect of Momordica cochinchinensis aril (MCA) on obesity, non-alcoholic fatty liver and insulin resistance in mice. MCA protected the mice against high-fat diet (HFD)-induced body weight gain, hyperlipidemia and hyperglycemia, compared with mice that were not treated. MCA inhibited the expansion of adipose tissue and adipocyte hypertrophy. In addition, the insulin sensitivity-associated index that evaluates insulin function was also significantly restored. MCA also regulated the secretion of adipokines in HFD-induced obese mice. Moreover, hepatic fat accumulation and liver damage were reduced, which suggested that fatty liver was prevented by MCA. Furthermore, MCA supplementation suppressed hepatic lipid accumulation by activation of the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) signaling pathway in the human fatty liver HuS-E/2 cell model. Our data indicate that MCA altered the microbial contents of the gut and modulated microbial dysbiosis in the host, and consequently is involved in the prevention of HFD-induced adiposity, insulin resistance and non-alcoholic fatty liver disease.

Nanoplastics Cause Neurobehavioral Impairments, Reproductive and Oxidative Damages, and Biomarker Responses in Zebrafish: Throwing up Alarms of Wide Spread Health Risk of Exposure.

Plastic pollution is a growing global emergency and it could serve as a geological indicator of the Anthropocene era. Microplastics are potentially more hazardous than macroplastics, as the former can permeate biological membranes. The toxicity of microplastic exposure on humans and aquatic organisms has been documented, but the toxicity and behavioral changes of nanoplastics (NPs) in mammals are scarce. In spite of their small size, nanoplastics have an enormous surface area, which bears the potential to bind even bigger amounts of toxic compounds in comparison to microplastics. Here, we used polystyrene nanoplastics (PS-NPs) (diameter size at ~70 nm) to investigate the neurobehavioral alterations, tissue distribution, accumulation, and specific health risk of nanoplastics in adult zebrafish. The results demonstrated that PS-NPs accumulated in gonads, intestine, liver, and brain with a tissue distribution pattern that was greatly dependent on the size and shape of the NPs particle. Importantly, an analysis of multiple behavior endpoints and different biochemical biomarkers evidenced that PS-NPs exposure induced disturbance of lipid and energy metabolism as well as oxidative stress and tissue accumulation. Pronounced behavior alterations in their locomotion activity, aggressiveness, shoal formation, and predator avoidance behavior were exhibited by the high concentration of the PS-NPs group, along with the dysregulated circadian rhythm locomotion activity after its chronic exposure. Moreover, several important neurotransmitter biomarkers for neurotoxicity investigation were significantly altered after one week of PS-NPs exposure and these significant changes may indicate the potential toxicity from PS-NPs exposure. In addition, after ~1-month incubation, the fluorescence spectroscopy results revealed the accumulation and distribution of PS-NPs across zebrafish tissues, especially in gonads, which would possibly further affect fish reproductive function. Overall, our results provided new evidence for the adverse consequences of PS-NPs-induced behavioral dysregulation and changes at the molecular level that eventually reduce the survival fitness of zebrafish in the ecosystem.

Development of a Modified Three-Day T-maze Protocol for Evaluating Learning and Memory Capacity of Adult Zebrafish.

A T-maze test is an experimental approach that is used in congenital research. However, the food reward-based protocol for the T-maze test in fish has low efficiency and a long training period. The aim of this study is to facilitate the T-maze conditions by using a combination of the principles of passive avoidance and a spatial memory test. In our modified T-maze settings, electric shock punishment (1-2 V, 0.3-0.5 mA) is given at the left arm, with a green cue at the right arm. Also, the depth of both arms of the T-maze was increased. The parameters measured in our T-maze design were latency, freezing time, and time spent in different areas of the T-maze. We validated the utility of our modified T-maze protocol by showing the consistent finding of memory impairment in ZnCl2-treated fish, which has been previously detected with the passive avoidance test. In addition, we also tested the spatial memory performance of leptin a (lepa) mutants which displayed an obesity phenotype. The results showed that although the learning and memory performance for lepa KO fish were similar to control fish, they displayed a higher freezing behavior during the training phase. In conclusion, we have established a modified T-maze protocol that can be used to evaluate the anxiety, learning, and memory capacity of adult zebrafish within three days, for the first time.

Metabolomics profiling of haloperidol and validation of thromboxane-related signaling in the early development of zebrafish.

Haloperidol is a common butyrophenone-derivative antipsychotic drug that is used clinically to treat schizophrenia and to control Tourette disorder. Haloperidol has been shown to be an embryonic toxicant and to cause a variety of adverse effects that affect human embryonic development. However, the pathway impaired by haloperidol during the developmental stages remains unclear. To elucidate the innate toxicological pathway of haloperidol, we investigated the lethality of haloperidol during the embryonic development of zebrafish. We observed that haloperidol caused serious morphological changes, with an LD50 of 9.7 x 10-6 ± 2.4 x 10-6 µg/L. Next, we established a systematic approach to perform metabolite profiling in embryonic zebrafish with various concentrations of haloperidol and analyzed the metabolites using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). A total of 304 metabolites were identified and 86 metabolites were chosen to predict potential pathways. Among the metabolites, we found through prediction that numerous metabolomics-biological pathways are associated with haloperidol, including peroxisome-proliferator-activated receptor (ppar), thromboxane, and mTOR signaling. Quantitative real time-qPCR was then used to validate the gene expression potentially associated with the thromboxane, which is a metabolic product of arachidonic acid and considered to be important for cell proliferation and the inflammatory response. To sum up, analysis of metabolites in the zebrafish model provides a system for mining biomarkers that reflect biological significance and highlight the therapeutic potency in humans. In addition, it may show potential for application to other pharmaceuticals to identify their various activities and clarify functional mechanisms in the future.

Helminthostachys zeylanica alleviates hepatic steatosis and insulin resistance in diet-induced obese mice.

BACKGROUND: Obesity and its associated health conditions, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are worldwide health problems. It has been shown that insulin resistance is associated with increased hepatic lipid and causes hepatic steatosis through a myriad of mechanisms, including inflammatory signaling. METHODS: Helminthostachys zeylanica (HZ) is used widely as a common herbal medicine to relieve fever symptoms and inflammatory diseases in Asia. In the present study, we evaluated whether HZ has therapeutic effects on obesity, NAFLD and insulin resistance. The protective effects of HZ extract were examined using free fatty acid-induced steatosis in human HuS-E/2 cells and a high-fat diet-induced NAFLD in mice. RESULTS: The major components of the HZ extract are ugonins J and K, confirmed by HPLC. Incubation of human hepatocytes, HuS-E/2 cells, with palmitate markedly increased lipid accumulation and treatment with the HZ extract significantly decreased lipid deposition and facilitated AMPK and ACC activation. After 12 weeks of a high-fat diet with HZ extract treatment, the HFD mice were protected from hyperlipidemia and hyperglycemia. HZ extract prevented body weight gain, adipose tissue expansion and adipocyte hypertrophy in the HFD mice. In addition, fat accumulation was reduced in mice livers. Moreover, the insulin sensitivity-associated index, which evaluates insulin function, was also significantly restored. CONCLUSIONS: These results suggest that HZ has a promising pharmacological effect on high-fat diet-induced obesity, hepatic steatosis and insulin resistance, which may have the potential for clinical application.

Overexpression of Notch Signaling Induces Hyperosteogeny in Zebrafish.

Notch signaling is one of the evolutionarily conserved signaling pathways in multicellular organisms. It plays an important role in embryonic development. During skeletal development of vertebrates, it regulates bone homeostasis by manipulating both osteoblastogenesis and osteoclastogenesis through different mechanisms. However, due to the different nature of Notch signaling in mesenchymal stem cell and osteoblast, regulation of Notch signaling in bone-related diseases remains unsettled. Previous studies by cell culture and mouse models showed contradictory results regarding the role of Notch signaling in bone homeostasis. To clarify the role of Notch signaling in osteogenesis, we established a zebrafish model, in which Notch1a intracellular domain (N1aICD) was specifically expressed in the osteoblasts. We found that overexpression of N1aICD in osteoblasts caused hyperosteogeny in the column region of zebrafish with the morphology of narrowed neural/hemal canals. Moreover, increased metabolic activity of osteoblasts instead of augmenting osteoblast number led to hyperosteogeny in N1aICD-overexpressed zebrafish. In summary, we successfully established a transgenic zebrafish line overexpressing N1aICD to clarify the in-vivo function of Notch signaling during osteoblastogenesis. In the future, this fish line can serve as a valuable tool to test the therapeutic drugs for hyperosteogeny.

Peach Kernel Oil Downregulates Expression of Tissue Factor and Reduces Atherosclerosis in ApoE knockout Mice.

Atherosclerosis is the pathological process in arteries due to the plaque formation that is responsible for several diseases like heart disease, stroke and peripheral arterial disease. In this study, we performed in vitro and in vivo assays to evaluate the potential anti-atherosclerosis activity of peach kernel oil. For the in vitro assay, we incubated human umbilical vein endothelial cells (HUVEC) with tumor necrosis factor-α (TNF-α) to induce tissue factors (TF, an essential mediator of hemostasis and trigger of thrombosis) elevation. We found that TNF-α-induced TF elevation was suppressed by peach kernel oil in a dose-dependent manner at both mRNA and protein levels. Peach kernel oil can significantly improve HUVEC viability, protect the endothelial cells, which achieved the goal of prevention of thrombotic diseases. For the in vivo assay, we investigated the effect and mechanism of peach kernel oil on preventing atherosclerotic lesion formation in ApoE knockout mice. Results show that peach kernel oil could reduce total cholesterol, triglyceride, low-density lipoprotein cholesterol levels, elevate the high-density lipoprotein cholesterol level in serum, and reduce the area of the aortic atherosclerotic lesions in high-fat diet fed ApoE knockout mice. Moreover, peach kernel oil treatment can significantly down regulate the expression of TF protein to inhibit the formation of atherosclerotic plaque. In conclusion, peach kernel oil may be a potential health food to prevent atherosclerosis in cardiovascular diseases.

Evaluation of collagen mixture on promoting skin wound healing in zebrafish caused by acetic acid administration.

The aim of this study is to use zebrafish embryos as a quick platform for wound healing studies. At beginning, we optimized a protocol to induce skin lesion by acetic acid injection. The acetic acid injection induced regional inflammation wound hyperpigmentation by recruiting pigment cells to the wound area. Later, we applied established platform to evaluate the effect of tilapia's collagen peptide mixtures, including demonstration on promoting skin wound healing and eliminating inflammatory response. Results showed that after treating TY001, one of the above fish collagen peptide mixtures, not only repair and proliferation were induced, but also death and apoptosis cells were cleared within cutaneous lesion. Moreover, inflammatory response was suppressed along with collagen mixture treatment. Finally, the TY001-associated signaling was validated by real time-PCR, and numbers of gene associated with tissue repair and vessel proliferation were induced. To sum up, our findings provided a permissive model that may apply to generate a platform for further screening on repair and restoration technology. In addition, the tilapia fish collagen peptide mixture we applied on our model has great potential on developing clinical application on wound healing.

Epigallocatechin gallate inhibits hepatitis B virus infection in human liver chimeric mice.

BACKGROUND: Persistent hepatitis B virus (HBV) infection causes liver cirrhosis and hepatocellular carcinoma and constitutes a major worldwide health problem. Currently, anti-HBV drugs are limited to peginterferon and nucleos(t)ide analogs, which are costly and have considerable side effects; the development of novel, effective anti-HBV agents is crucial. METHODS: Catechins are a major group of compounds found in green tea extract and epigallocatechin gallate (EGCG) has been shown to have antiviral properties, including inhibition of cellular entry by HBV. FRG (Fah-/-/ Rag2-/-/ IL-2Rγ/-) mice were used in this study to generate chimeras carrying human primary hepatocytes, to facilitate investigation of the inhibitory effect of EGCG on HBV infection. RESULTS: Here, we show the inhibitory effect of EGCG on HBV infection and replication in HuS-E/2 cells. The inhibitory effect of EGCG on HBV infection in vivo was confirmed by monitoring HBV DNA and HBsAg in serum and immunostaining the liver tissues of the human liver chimeric mice. CONCLUSIONS: The effects of EGCG suggest a robust strategy for the treatment of HBV infection and EGCG may have therapeutic potential for the treatment of HBV-associated liver diseases.

The Power of Fish Models to Elucidate Skin Cancer Pathogenesis and Impact the Discovery of New Therapeutic Opportunities.

Animal models play important roles in investigating the pathobiology of cancer, identifying relevant pathways, and developing novel therapeutic tools. Despite rapid progress in the understanding of disease mechanisms and technological advancement in drug discovery, negative trial outcomes are the most frequent incidences during a Phase III trial. Skin cancer is a potential life-threatening disease in humans and might be medically futile when tumors metastasize. This explains the low success rate of melanoma therapy amongst other malignancies. In the past decades, a number of skin cancer models in fish that showed a parallel development to the disease in humans have provided important insights into the fundamental biology of skin cancer and future treatment methods. With the diversity and breadth of advanced molecular genetic tools available in fish biology, fish skin cancer models will continue to be refined and expanded to keep pace with the rapid development of skin cancer research. This review begins with a brief introduction of molecular characteristics of skin cancers, followed by an overview of teleost models that have been used in the last decades in melanoma research. Next, we will detail the importance of the zebrafish (Danio rerio) animal model and other emerging fish models including platyfish (Xiphophorus sp.), and medaka (Oryzias latipes) in future cutaneous malignancy studies. The last part of this review provides the recent development and genome editing applications of skin cancer models in zebrafish and the progress in small molecule screening.

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

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

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

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

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

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

Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus.

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.

Teratogenic Effects of Topiramate in a Zebrafish Model.

Topiramate is commonly used for treating epilepsy in both children and adults. Recent clinical data suggests that administration of topiramate to women during pregnancy increases the risk of oral clefts in their offspring. To better understand the potential effects of topiramate, we dosed adult female zebrafish with topiramate, and investigated the altered morphologies in adult females and their offspring. It showed that topiramate-treated female fish had reduced oocyte maturation, and the survival rates of their offspring were seriously decreased during embryogenesis. In addition, around 23% of offspring displayed cartilage malformation in the craniofacial area, such as loss of ceratobranchial cartilages as well as impaired ceratohyal, Meckel's cartilage and ethmoid plate development. Moreover, mineralization of ceratohyal, Meckel's cartilage, and vertebrae were downregulated during bone development. Taken together, we concluded that topiramate impaired oogenesis in the maternal reproductive system, and then caused offspring cartilage malformation or bone dysplasia.

A Simple Method to Decode the Complete 18-5.8-28S rRNA Repeated Units of Green Algae by Genome Skimming.

Green algae, Chlorella ellipsoidea, Haematococcus pluvialis and Aegagropila linnaei (Phylum Chlorophyta) were simultaneously decoded by a genomic skimming approach within 18-5.8-28S rRNA region. Whole genomic DNAs were isolated from green algae and directly subjected to low coverage genome skimming sequencing. After de novo assembly and mapping, the size of complete 18-5.8-28S rRNA repeated units for three green algae were ranged from 5785 to 6028 bp, which showed high nucleotide diversity (π is around 0.5-0.6) within ITS1 and ITS2 (Internal Transcribed Spacer) regions. Previously, the evolutional diversity of algae has been difficult to decode due to the inability design universal primers that amplify specific marker genes across diverse algal species. In this study, our method provided a rapid and universal approach to decode the 18-5.8-28S rRNA repeat unit in three green algal species. In addition, the completely sequenced 18-5.8-28S rRNA repeated units provided a solid nuclear marker for phylogenetic and evolutionary analysis for green algae for the first time.

Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization.

Currently, drug screening relies on cell-based experiments or on animal models to confirm biological effects. The mammalian system is considered too time-consuming, expensive and complex to perform high-throughput drug screening. There is a gap between in vitro cell-based models and the in vivo mammalian models. The zebrafish is an ideal model that could link preclinical toxicity screening with the drug development pipeline. Taking advantage of a highly conservative genomic, rapid development, large number of offspring, low cost and easy manipulation, zebrafish has been considered an excellent animal model for disease-based drug screening. In this study, zebrafish embryos were incubated with small molecular compounds that potentially affected bone mineralization in microplates. Two compounds of alendronate and dorsomorphin were used as positive and negative controls, respectively. The level of osteogenic mineralization was measured and quantified by using ImageJ software with fluorescent calcein-staining images. Among twenty-four tested compounds from the kinase inhibitor library, we identified two compounds, pentamidine and BML-267, which showed increased embryonic mineralization; while six compounds, RWJ-60475, levamisole HCL, tetramisole HCL, fenvalerate, NSC-663284, and BML-267ester, were inhibitory to bone mineralization. In addition, real time quantitative PCR (RT-qPCR) was performed to evaluate the biological pathways involved in bone metabolism at the molecular level. We confirmed that alendronate enhanced the level of bone mineralization by inhibiting osteoclast-related genes. In summary, our research established a simple method to screen potential bone metabolic drugs and to perform mechanism analysis for bone mineralization in vivo.