Development a high-throughput zebrafish embryo acute toxicity testing method based on OECD TG 236.

The Organization for Economic Co-operation and Development (OECD）Test Guideline (TG) 236 for zebrafish embryo acute toxicity testing was adopted for chemical toxicity assessment in 2013. Due to the increasing demand for prediction and evaluation of the acute toxicity using zebrafish embryos, we developed a method based on OECD 236 test guideline with the aim to improve the testing efficiency. We used 4-128 cell stage zebrafish embryos and performed an exposure assay in a 96-well microtiter plate, observing the lethality endpoints of embryos at 48-h postexposure. A total of 32 chemicals (two batches) were used in the comparison study. Our results indicated that the logarithmic LC50 (half lethal concentration) obtained by the modified method exhibited good correlation with that obtained by the OECD 236 testing method, and the R2 of the linear regression analysis was 0.9717 (0.9621 and 0.9936 for the two batches, respectively). Additionally, the intra- and inter-laboratory coefficient of variation (CVs) for the LC50 from the testing chemicals (17 chemicals in second batch) was less than 30%, except for CuSO4. Therefore, the developed method was less time-consuming and demonstrated a higher throughput for toxicity testing compared to the prior method. We argue the developed method could be used as an additional choice for high-throughput zebrafish embryo acute toxicity test.

Metformin accelerates zebrafish heart regeneration by inducing autophagy.

Metformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Spectroscopic and microscopic examination of teeth exposed to green tea at different temperatures.

Tea is a popular beverage consumed at different temperatures. The effect of tea on teeth at different temperatures has not been studied previously. The present study used an in vitro green tea immersed tooth model at different tea temperatures (hot and cold) compared to an in vivo tea administration model allowing rats to drink tea over the course of a week. The elements present in tea leaves were identified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and compared to the elements in teeth (enamel surface) using Laser-Induced Breakdown Spectroscopy (LIBS). Here, LIBS demonstrated in vivo and in vitro green tea treatments resulted in a significant increase in the mineral elements found in enamel. For the in vitro assessment, elements in enamel varied based on cold-tea and hot-tea treatment; however, hot water reduced the elements in enamel. Atomic force microscopy found the in vivo tea group had a higher roughness average (RA) compared with the in vivo water group. Cold tea and hot tea in vitro groups demonstrated lower RA than in vitro water controls. Scanning electron microscopy found hot water induced cracks more than 1.3µm in enamel while cold tea and hot tea promoted the adhering of extrinsic matter to teeth. Overall, teeth treated to high temperature lost the mineral phase leading to demineralization. Our results indicate that green tea protects enamel, but its protective action in dental structures is enhanced at cold temperature.

Exposure to phthalates impaired neurodevelopment through estrogenic effects and induced DNA damage in neurons.

Phthalates are commonly used in plastic products in daily life. The endocrine-disrupting effects of phthalates have been widely reported. Accumulating evidence from human cohorts and lab animals indicate exposure to phthalates might impair neurodevelopment. However, the direct causal relationship and mechanism between phthalates with neurodevelopment and neurotoxicity have not been firmly established. We found that phthalates (i.e. DBP, DINP, BBP) disrupted the expression of estrogen receptors (esr1, esr2a, esr2b), and impaired neurogenesis in the brain of zebrafish during embryonic development. Moreover, the abnormal expression of estrogen receptors, especially esr2a, was partly rescued in zebrafish which exposed to phthalates, with the estrogen receptor antagonist tamoxifen. Hence, impaired neurogenesis of zebrafish exposed to phthalates was partly reversed by tamoxifen treatment. Moreover, our results show that induced pluripotent stem cells (iPSC)-derived human neurons exposed to phthalates triggered double-strand DNA breaks in vitro. Overall, this study demonstrates that exposure to phthalates affects neurodevelopment in zebrafish embryos and induces neurotoxicity in human neurons partly through disrupting the expression of estrogen receptors.

Estrogen accelerates heart regeneration by promoting the inflammatory response in zebrafish.

Sexual differences have been observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we found that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and is suppressed by the estrogen-antagonist tamoxifen. Injuries to the zebrafish heart, but not other tissues, increased plasma estrogen levels and the expression of estrogen receptors, especially esr2a. The resulting endocrine disruption induces the expression of the female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered pronounced immune and inflammatory responses in females. These responses, previously shown to elicit heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in females. Furthermore, a prior exposure to estrogen preconditioned the zebrafish heart for an accelerated regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to cardiac injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provide a new model system for the study of sexual differences in human cardiac repair.

Precise Drug Delivery by Using PLGA-Based Microspheres and Optical Manipulators.

The accurate delivery of precise amounts of drugs to a specific location can considerably affect various clinical applications. The precise control of drug amount and position is crucial to a successful drug delivery. This paper proposes the use of poly(lactide-co-glycolicacid) (PLGA)-based microspheres to contain precise amounts of drugs and an optical tweezer manipulator to transport these drug-containing microspheres to their targeted sites in vivo. The drugs were delivered by the PLGA-based microspheres to the yolk sac of zebrafish embryos, and a sustained drug release was observed to examine the anti-angiogenesis and angiogenesis activities. The PLGA-based microspheres degraded in zebrafish, thereby verifying that these microspheres can be used as drug carriers in vivo to ensure good biocompatibility and biodegradation. The proposed precise drug delivery approach can be used in protein tests and drug property characterization in vivo.

Prolonged neutrophil retention in the wound impairs zebrafish heart regeneration after cryoinjury.

Neutrophils are the first line defenders in the innate immune response, and rapidly migrate to an infected or injured area. Recently, bidirectional migration of neutrophils to the wound and the corresponding functions have become popular research pursuits. In zebrafish larvae, CXCR1/CXCL8 is the predominant chemoattractant pathway to recruit neutrophil to wound, while CXCR2/CXCL8 pathway mediate neutrophil dispersal in wound after injury. Here, we found that both CXCR1/CXCL8 and LTB4/BLT1 signals are activated in zebrafish heart after cryoinjury. And with a CXCR1/2 selective inhibitor (SB225002) treatment, the recruitment of neutrophils was not affected, but reverse migration of neutrophils was inhibited after cryoinjury of heart. We suggested that the neutrophil recruitment to cryoinjured area might be mediated by LTB4/BLT1 signals at the presence of SB225002. Therefore, SB225002 treatment resulted more accumulation and long retention of neutrophils in the injured heart. The long retention of neutrophils in the wound promoted revascularization in the injured heart; however, the AKT/mTOR pathway was inhibited and the regeneration was impaired. Our findings suggest that retention of neutrophils is a well-orchestrated process and might regulate regeneration by the AKT/mTOR pathway.

Excessive inflammation impairs heart regeneration in zebrafish breakdance mutant after cryoinjury.

Inflammation plays a crucial role in cardiac regeneration. Numerous advantages, including a robust regenerative ability, make the zebrafish a popular model to study cardiovascular diseases. The zebrafish breakdance (bre) mutant shares several key features with human long QT syndrome that predisposes to ventricular arrhythmias and sudden death. However, how inflammatory response and tissue regeneration following cardiac damage occur in bre mutant is unknown. Here, we have found that inflammatory response related genes were markedly expressed in the injured heart and excessive leukocyte accumulation occurred in the injured area of the bre mutant zebrafish. Furthermore, bre mutant zebrafish exhibited aberrant apoptosis and impaired heart regenerative ability after ventricular cryoinjury. Mild dosages of anti-inflammatory or prokinetic drugs protected regenerative cells from undergoing aberrant apoptosis and promoted heart regeneration in bre mutant zebrafish. We propose that immune or prokinetic therapy could be a potential therapeutic regimen for patients with genetic long QT syndrome who suffers from myocardial infarction.

Spectroscopic examination of enamel staining by coffee indicates dentin erosion by sequestration of elements.

The mechanism of coffee eliciting erosion on teeth is unclear as few studies have investigated the direct effect of coffee on enamel and dentin structures. The present study identified how coffee, the most popular beverage worldwide, induces staining and erosion on teeth. We show the grade of erosion of molars and incisors in Sprague Dawley rats from two different age groups, young (four weeks) and old (six months). We quantified the concentration of metals contained in coffee by mass spectrometry (ICP-MS). To determine elemental content in enamel (i.e. superficial) and dentin (i.e. substructure), we used Laser-induced Breakdown Spectroscopy (LIBS) and X-ray fluorescence (XRF) spectroscopy, respectively. For LIBS, a significant decrease of Ca, P, and Na was observed in the young coffee group relative to age-matched controls, whereas a significant increase in Mn, Fe, and K was observed. In the old coffee group, a significant increase of Mg, Fe, and K was observed along with a decrease of Mg, Ca, P, Na, Sr and Zn. For XRF, a significant decrease of the Ca/P ratio in the coffee group was observed. Spectroscopy results were correlated with scanning electron microscopy (SEM) and histological analysis. The SEM analysis showed pores and open spaces between young and old coffee groups, respectively. Thinning of enamel layers, loss of continuity in the enamel-dentin-junction, and wide spaces in dentin tubules with coffee use was found histologically. Coffee induces decalcification of teeth that corresponds to erosion, exposing the dentin structure by reducing enamel. Coffee immersion demonstrated an intrinsic staining in dentin by metal deposition.

Matrix metalloproteinases (MMPs) mediate leukocyte recruitment during the inflammatory phase of zebrafish heart regeneration.

In zebrafish, the role of matrix metalloproteinases (MMPs) in the inflammatory phase of heart regeneration following cryoinjury remains poorly understood. Here, we demonstrated an increase in MMP enzymatic activity and elevated expression of mmp9 and mmp13 in the injured area (IA) of hearts from as early as 1 day post-cryoinjury (dpc). Treatment with the broad-spectrum MMP inhibitor, GM6001, during the first week after cryoinjury resulted in impaired heart regeneration, as indicated by the larger scar and reduced numbers of proliferating cardiomyocytes. GM6001 also significantly reduced the number of leukocytes to the IA at 0.5 dpc to 4 dpc. Specific inhibition of both MMP-9 and MMP-13 also resulted in impaired regeneration and leukocyte recruitment. However, chemokine rescue with recombinant CXCL8 and CCL2 restored the recruitment of macrophages and the cardiac regenerative capability in GM6001-treated fish. MMP-9 and MMP-13 cleaved zebrafish CXCL8 at the same site, and the truncated form was more chemotactic than the intact form. In contrast, CCL2 did not have an MMP-9 or MMP-13 cleavage site. Together, these data suggest that MMPs might play a key role in the inflammatory phase of heart regeneration in zebrafish, by mediating leukocyte recruitment via the activation of chemokines.

Mechanistic Study of Bakuchiol-Induced Anti-breast Cancer Stem Cell and in Vivo Anti-metastasis Effects.

Cancer stem cells are involved in cancer establishment, progression, and resistance to current treatments. We demonstrated the in vitro and in vivo anti-breast cancer effect of bakuchiol in a previous study. However, the ability of bakuchiol to target breast cancer stem cells (BCSCs) and inhibit breast cancer metastasis remains unknown. In the current study, we used the cell surface markers CD44 and CD24 to distinguish BCSCs from MCF-7 cells. Bakuchiol inhibited mammosphere formation and aldehyde dehydrogenase activity in BCSCs. Moreover, bakuchiol induced apoptosis and suppressed the mitochondrial membrane potential of BCSCs. Bakuchiol upregulated the expression levels of pro-apoptotic genes, BNIP3 and DAPK2. Bakuchiol induced oxidative stress and altered lipogenesis in BCSCs. In zebrafish xenografts, bakuchiol inhibited breast cancer cell metastasis in vivo. In addition, bakuchiol altered the expression levels of metastasis-related genes through upregulating CK18 and downregulating Notch3, FASN, TGFBR1, and ACVR1B. Our study provides evidence for the anti-breast cancer potential of bakuchiol.

Toxicity and estrogenic endocrine disrupting activity of phthalates and their mixtures.

Phthalates, widely used in flexible plastics and consumer products, have become ubiquitous contaminants worldwide. This study evaluated the acute toxicity and estrogenic endocrine disrupting activity of butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), bis(2-ethylhexyl) phthalate (DEHP), diisodecyl phthalate (DIDP), diisononyl phthalate (DINP), di-n-octyl phthalate (DNOP) and their mixtures. Using a 72 h zebrafish embryo toxicity test, the LC50 values of BBP, DBP and a mixture of the six phthalates were found to be 0.72, 0.63 and 0.50 ppm, respectively. The other four phthalates did not cause more than 50% exposed embryo mortality even at their highest soluble concentrations. The typical toxicity symptoms caused by phthalates were death, tail curvature, necrosis, cardio edema and no touch response. Using an estrogen-responsive ChgH-EGFP transgenic medaka (Oryzias melastigma) eleutheroembryos based 24 h test, BBP demonstrated estrogenic activity, DBP, DEHP, DINP and the mixture of the six phthalates exhibited enhanced-estrogenic activity and DIDP and DNOP showed no enhanced- or anti-estrogenic activity. These findings highlighted the developmental toxicity of BBP and DBP, and the estrogenic endocrine disrupting activity of BBP, DBP, DEHP and DINP on intact organisms, indicating that the widespread use of these phthalates may cause potential health risks to human beings.

[Cloning and expression of D-like aspartic protease of Anisakis simplex].

OBJECTIVE: To clone and express the full length of D-like aspartic protease gene (AsAP) of the third stage larvae of Anisakis simplex. METHODS: According to the partial information of D-like aspartic protease encoding gene of A. simplex from GenBank, specific primers were designed to amplify 3'end and 5' end of AsAP gene using rapid amplification of cDNA ends (RACE), and the full length of the D-like aspartic protease gene was obtained. Using total RNA of the third-stage larvae of A. simplex, coding sequence of the AsAP gene was amplified by reverse transcription-PCR (RT-PCR). The PCR product was digested by EcoR I and Sal I, and cloned into pET32 vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into E. coli BL21 (DE3). Expression of the protein induced by IPTG under gradient concentration and different time was conducted. RESULT: A 1 753 bp full length of AsAP was obtained, which contained 30 bp 5'UTR, 361 bp 3'UTR and a 1 362 bp open reading frame (ORF) encoding 453 amino acids with a predicted molecular mass of M(r) 50 726. It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum. The predicted amino acid sequence contains two conserved catalytic motif, an active site flap, an S2 subsite and an S3 subsite. A 20 amino acids signal peptide was found in the N-terminus, with significant hydrophobic property. Different concentration of the IPTG (0.2-1.6 mmol/L) showed little effect on the expression, and the production of the protein was up to maximum after 2 hours induction. CONCLUSION: The AsAP gene has been cloned and expressed.

Anisakis pegreffii: a quantitative fluorescence PCR assay for detection in situ.

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.

[Detection of anisakid nematodes by an SYBR green I real-time PCR].

OBJECTIVE: To establish an SYBR Green I real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait. METHODS: Anisakid larvae of six species (Anisakis simplex, A. physeteris, Raphidascaris trichiura, Contracaecum aduncum, C. muraenesoxi, and Contracaecum sp., a predominant species in fishes in the strait) were obtained from the guts of marine fishes and identified chiefly based on the morphological features. The ITS-2 rDNA sequences from the larvae were amplified by PCR using universal primers, then cloned and bidirectionally sequenced. According to these sequences, six specific forward primers were designed and synthesized. Specificity was determined by a series of conventional PCR respectively, the ITS-2 sequences amplified above were cloned into T vector which was subsequently transformed into E. coli DH5alpha. Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve. Sensitivity and reproducibility were determined. RESULTS: All the 6 standard curves established by the recombinant plasmids showed adequate linear relationship between threshold cycle (Ct) and template concentration. Melt curves were specific and all the 6 correlation coefficients were above 0.998. In the reproducibility test, the coefficients of variation (cv) of Ct values for detection of the 6 nematodes ranged between 0.18% and 2.80%, and the cv of the inter-assay ranged between 0.55% and 1.94%. The sensitivity of the real-time PCR was 1 x 10(2) copies/microl, about 100 times higher than the conventional PCR assays. The real-time quantitative PCR detection needed only 3.5 hours from the sample treatment to result report. CONCLUSION: An SYBR Green I fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.

[Cloning and expression of L-like cysteine protease of Anisakis simplex].

OBJECTIVE: To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex (AsCP). METHODS: According to L-like cysteine protease encoding gene of A. simplex from GenBank EST database, specific primers were designed to amplify 3'-end of AsCP gene using rapid-amplification of cDNA ends (RACE), and the full lenth of the L-like cysteine protease gene was obtained. Specific primers were designed according to the full length of the gene. Using total RNA of A. simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT-PCR. The PCR product was digested by EcoR I and Sal I, and cloned into pET32a(+) vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21 (DE3). Expression of the protein induced by IPTG of gradient concentration (0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L) and by the same concentration (1 mmol/L) of IPTG at different time(0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h) was conducted. The expression situation of recombinant protein was analyzed by SDS-PAGE. RESULTS: A 1211 bp of 3'-end of AsCP gene was amplified by 3'RACE, full length of the gene was 1462 bp and coding 411 amino acids. It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans. Double enzyme digestion of the constructed recombinant plasmid pET32a(+)-AsCP showed that there was about 1150 bp fragment, the constructed recombinant plasmid was then identified by sequencing. SDS-PAGE showed that the recombinant protein (Mr 60,000) was identical with the target. IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction. CONCLUSION: The AsCP gene has been cloned and expressed.

ES proteins analysis of Angiostrongylus cantonensis: products of the potential parasitism genes?

The expressed sequence tags (ESTs) of Angiostrongylus cantonensis were analyzed in an attempt to gain further insight into its genomic expression patterns. A total of 1,277 ESTs of A. cantonensis were randomly downloaded from NCBI databank. ESTs were analyzed and annotated using Blastx. The result showed that there were 60 ESTs had no match to any of the proteins and gene sequences in the published databases, and 695 ESTs score more than 80. According to the function, the identified 695 ESTs could be grouped into 13 categories related to metabolism, cellular development, immune evasion, host-parasite interactions, and so on. Among them, 65 (9.4%) were proteases and protease inhibitors, represented 19 potential proteases and protease inhibitors genes; 42 (6.0%) were allergens or antigens, represented 15 potential antigens/allergens genes. Signal P analysis was applied to the 19 putative proteases and protease inhibitors and the 15 antigens/allergens protein sequences to identify the potential signal peptides and anchors. The result demonstrated that there were ten putative proteins had N-terminal signal peptides and three had signal anchors, these putative excretory/secretory proteins might be the products of potential parasitism genes which played an important role in the adaptation of A. cantonensis to a parasitism life. These parasitism genes and proteins identified are expected to become potential targets for future research on anti-A. cantonensis drugs; moreover, the resulting genetic information is useful in elucidating the mechanisms of parasitism of A. cantonensis.

Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait.

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.

[Expressed sequence tags (ESTs) analysis of Angiostrongylus cantonensis].

A total of 1,277 ESTs of Angiostrongylus cantonensis were downloaded from GenBank and analyzed with BlastX. SignalP V3.0 analysis was applied to predict potential putative antigen or allergen relative proteins with N-terminal secreted signal peptides or signal anchors. BlastX analysis showed that there were 614 ESTs scored more than 100, of which 14 were identical with A. cantonensis, 60 ESTs did not match any proteins in the databases. The identified 614 ESTs could be grouped into 10 categories, 80 ESTs expressed 22 antigen or allergen relative proteins, in which 12 had N-terminal secreted signal peptides and 3 had signal anchors.