GC/HRMS Analysis of E-Liquids Complements In Vivo Modeling Methods and can Help to Predict Toxicity.

Tobacco smoking is a major risk factor for disease development, with the user inhaling various chemicals known to be toxic. However, many of these chemicals are absent before tobacco is "burned". Similar, detailed data have only more recently being reported for the e-cigarette with regards to chemicals present before and after the e-liquid is "vaped." Here, zebrafish were dosed with vaped e-liquids, while C57-BL/6J mice were vaped using nose-cone only administration. Preliminary assessments were made using e-liquids and GC/HRMS to identify chemical signatures that differ between unvaped/vaped and flavored/unflavored samples. Oxidative stress and inflammatory immune cell response assays were then performed using our in vivo models. Chemical signatures differed, e.g., between unvaped/vaped samples and also between unflavored/flavored e-liquids, with known chemical irritants upregulated in vaped and unvaped flavored e-liquids compared with unflavored e-liquids. However, when possible respiratory irritants were evaluated, these agents were predominantly present in only the vaped e-liquid. Both oxidative stress and inflammatory responses were induced by a menthol-flavored but not a tobacco-flavored e-liquid. Thus, chemical signatures differ between unvaped versus vaped e-liquid samples and also between unflavored versus flavored e-liquids. These flavors also likely play a significant role in the variability of e-liquid characteristics, e.g., pro-inflammatory and/or cytotoxic responses.

Deficiency of spns1 exacerbates per- and polyfluoroalkyl substances mediated hepatic toxicity and steatosis in zebrafish (Danio rerio).

Per- and polyfluoroalkyl substances (PFAS) are man-made long-lasting chemical compounds that are found in everyday household items. Today they occur in the environment as a major group of pollutants. These compounds are broadly used in commercial product preparation such as, for food packaging, nonstick coatings, and firefighting foam. In humans, PFAS can cause immune disorders, impaired fetal development, abnormal skeletal tissue development, osteoarthritis, thyroid dysfunctions, cholesterol changes, affect insulin regulation and lipid metabolism, and are also involved in the development of fatty liver disease. In the current study, we investigated the effect of low, but physiologically relevant, concentrations of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA) on gene expression markers of an inflammatory response (tnfa, il-1b, il-6, rplp0, edem1, and dnajc3a), unfolded protein response (UPR) (bip, atf4a, atf6, xbp1, and ddit3), senescence (p21, pai1, smp30, mdm2, and baxa), lipogenesis (scd1, acc, srebp1, pparγ, and fasn) and autophagy (p62, atg3, atg7, rab7, lc3b, and becn1) in AB wild-type (+/+), spns1-wt sibling (+/+), (+/-) and spns1 homozygous mutant (-/-) zebrafish embryos. Exposure to PFOA and HFBA (50 and 100 nM) specifically modulated inflammatory, UPR, senescence, lipogenic, and autophagy signaling in spns1-wt (+/+), (+/-), and spns1-mutant (-/-) zebrafish embryos. Furthermore, PFOA, but not HFBA, upregulated lipogenic-related gene expression and enhanced hepatic steatosis in spns1-wt (+/+), (+/-) zebrafish embryos. Combined exposure to PFOA, HFBA, and PFTA differentially expressed inflammatory, senescence, lipogenic, and autophagy-associated gene expression in spns1-mutant (-/-) zebrafish embryos compared with spns1-wt (+/+), (+/-) and AB-wt (+/+) zebrafish embryos. In addition, chronic exposure (∼2 months) to PFOA (120-600 nM) upregulated the expression of hepatic lipogenic and steatosis biomarkers in AB-wt (+/+) zebrafish. Collectively, our data suggest that acute/chronic physiologically relevant concentrations of PFOA upregulate inflammatory, UPR, senescence, and lipogenic signaling in spns1-wt (+/+), (+/-) and spns1-mutant (-/-) zebrafish embryos as well as in two-month-old AB-wt zebrafish, by targeting autophagy and hence induces toxicity that could promote nonalcoholic fatty liver disease.

An assessment of vaping-induced inflammation and toxicity: A feasibility study using a 2-stage zebrafish and mouse platform.

It is currently understood that tobacco smoking is a major cause of pulmonary disease due to pulmonary/lung inflammation. However, due to a highly dynamic market place and an abundance of diverse products, less is known about the effects of e-cigarette (E-cig) use on the lung. In addition, varieties of E-cig liquids (e-liquids), which deliver nicotine and numerous flavor chemicals into the lungs, now number in the 1000s. Thus, a critical need exists for safety evaluations of these E-cig products. Herein, we employed a "2-stage in vivo screening platform" (zebrafish to mouse) to assess the safety profiles of e-liquids. Using the zebrafish, we collected embryo survival data after e-liquid exposure as well as neutrophil migration data, a key hallmark for a pro-inflammatory response. Our data indicate that certain e-liquids induce an inflammatory response in our zebrafish model and that e-liquid exposure alone results in pro-inflammatory lung responses in our C57BL/6J model, data collected from lung staining and ELISA analysis, respectively, in the mouse. Thus, our platform can be used as an initial assessment to ascertain the safety profiles of e-liquid using acute inflammatory responses (zebrafish, Stage 1) as our initial metric followed by chronic studies (C57BL/6J, Stage 2).

Zebrafish Xenograft Model to Study Human Cancer.

The zebrafish, Danio rerio, has been an important animal model for cancer research over the last decade. The capability of a high-throughput screen in zebrafish and a wide range of pharmacologically active compounds elicit physiological responses in zebrafish embryos comparable to those in mammalian systems, making zebrafish ideal for identifying clinically relevant drug targets and compounds that regulate tumor progression. The zebrafish model is suitable for patient-derived xenograft (pdx) and large-scale screening of lead compounds against specific malignancies. This established vertebrate model has many advantages, including fast response time, cost efficiency for drug testing, efficient manipulation of the host microenvironment by genetic tools, suitable for small molecule drug screening in high-throughput setting, easy maintenance, transparency for easy observation, high fecundity, and rapid generation time. The zebrafish model is a good alternative in vivo model to mammals for robust testing of drug candidates for cancer therapy.

Inhibitory Effects of Euphorbia ebracteolata Hayata Extract ECB on Melanoma-Induced Hyperplasia of Blood Vessels in Zebrafish Embryos.

Melanoma is a serious malignant form of skin cancer. Euphorbiaceae compound B (ECB, 2,4-dihydroxy-6-methoxy-3-methylacetophenone) is an acetophenone compound that is isolated from Euphorbia ebracteolata Hayata (EEH), an herbaceous perennial, and has antitumor activity. Here, we transplanted human melanoma cells into zebrafish embryos to establish a zebrafish/melanoma model. We showed that this model can be used to evaluate the therapeutic effect of EEH and ECB and discussed its potential mechanism of action. The results showed that ECB was an active ingredient of EEH in inhibiting melanoma-induced hyperplasia of blood vessels in zebrafish embryos, similar to the angiogenic inhibitor vatalanib. ECB inhibited the number and length of subintestinal veins (p < 0.05), as well as the distribution of melanoma in zebrafish embryos (p < 0.05). More importantly, unlike vatalanib, ECB only inhibited melanoma-induced abnormal and excessive growth of blood vessels in xenografts. In addition, ECB inhibited the mRNA expression of vegfr2 and vegfr3 in zebrafish. Both vegfr2 and vegfr3 are essential genes that regulate blood vessel formation and upregulate the expression of p53 and casp3a genes in zebrafish. Together, the above-mentioned results indicate that ECB has a potential antimelanoma effect in vivo, which may be mediated by inhibiting vascular endothelial growth factor receptors.

Targeted therapy of human leukemia xenografts in immunodeficient zebrafish.

Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a helpful tool for analyzing the effect of drugs against an individual's tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia.

Methylglyoxal-Induced Retinal Angiogenesis in Zebrafish Embryo: A Potential Animal Model of Neovascular Retinopathy.

Methylglyoxal (MG) is an intermediate of glucose metabolism and the precursor of advanced glycation end products (AGEs) found in high levels in blood or tissue of diabetic patients. MG and AGEs are thought to play a major role in the pathogenesis of diabetic retinopathy. In order to determine if zebrafish is valuable to help us understand more about retinopathy, we evaluate if MG induces abnormal vascular change and angiogenesis in zebrafish in a short incubation period. We also used an inhibitor of VEGFR (PTK787) to explore the mechanistic role of VEGF in MG-induced pathogenesis. A transgenic Tg(flk1:GFP) zebrafish line was used, and the embryos were incubated with MG solution and in combination with glucose (to mimic hyperglycemia). Retinal vascular structure visible with fluorescence signal was imaged using fluorescence microscopy. The percentage of vascular area was calculated and found elevated in the MG treatment groups than that in the control group (p < 0.01) which indicated increased angiogenesis induced by MG treatment. PTK787 blocked the proangiogenic effects of MG treatment. This study suggests that MG has a potential proangiogenic effect via VEGF signaling in the retina of zebrafish embryos. Therefore, this zebrafish model may be used to study neovascular retinopathy.

Rescue of hematopoietic stem/progenitor cells formation in plcg1 zebrafish mutant.

Hematopoietic stem/progenitor cells (HSPC) in zebrafish emerge from the aortic hemogenic endothelium (HE) and migrate towards the caudal hematopoietic tissue (CHT), where they expand and differentiate during definitive hematopoiesis. Phospholipase C gamma 1 (Plcγ1) has been implicated for hematopoiesis in vivo and in vitro and is also required to drive arterial and HSPC formation. Genetic mutation in plcg1-/- (y10 allele) completely disrupts the aortic blood flow, specification of arterial fate, and HSPC formation in zebrafish embryos. We previously demonstrated that ginger treatment promoted definitive hematopoiesis via Bmp signaling. In this paper, we focus on HSPC development in plcg1-/- mutants and show that ginger/10-gingerol (10-G) can rescue the expression of arterial and HSPC markers in the HE and CHT in plcg1-/- mutant embryos. We demonstrate that ginger can induce scl/runx1 expression, and that rescued HE fate is dependent on Bmp and Notch. Bmp and Notch are known to regulate nitric oxide (NO) production and NO can induce hematopoietic stem cell fate. We show that ginger produces a robust up-regulation of NO. Taken together, we suggest in this paper that Bmp, Notch and NO are potential players that mediate the effect of ginger/10-G for rescuing the genetic defects in blood vessel specification and HSPC formation in plcg1-/- mutants. Understanding the molecular mechanisms of HSPC development in vivo is critical for understanding HSPC expansion, which will have a positive impact in regenerative medicine.

An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide.

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be "self-cleaved" into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd -values of 90.13-149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants.

Synthesis, evaluation, and metabolism of novel [6]-shogaol derivatives as potent Nrf2 activators.

Oxidative stress is a central component of many chronic diseases. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 p45-related factor 2 (Nrf2) system is a major regulatory pathway of cytoprotective genes against oxidative and electrophilic stress. Activation of the Nrf2 pathway plays crucial roles in the chemopreventive effects of various inducers. In this study, we developed a novel class of potent Nrf2 activators derived from ginger compound, [6]-shogaol (6S), using the Tg[glutathione S-transferase pi 1 (gstp1):green fluorescent protein (GFP)] transgenic zebrafish model. Investigation of structure-activity relationships of 6S derivatives indicates that the combination of an α,ß-unsaturated carbonyl entity and a catechol moiety in one compound enhances the Tg(gstp1:GFP) fluorescence signal in zebrafish embryos. Chemical reaction and in vivo metabolism studies of the four most potent 6S derivatives showed that both α,ß-unsaturated carbonyl entity and catechol moiety act as major active groups for conjugation with the sulfhydryl groups of the cysteine residues. In addition, we further demonstrated that 6S derivatives increased the expression of Nrf2 downstream target, heme oxygenase-1, in both a dose- and time-dependent manner. These results suggest that α,ß-unsaturated carbonyl entity and catechol moiety of 6S derivatives may react with the cysteine residues of Keap1, disrupting the Keap1-Nrf2 complex, thereby liberating and activating Nrf2. Our findings of natural product-derived Nrf2 activators lead to design options of potent Nrf2 activators for further optimization.

[10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos.

Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is still unclear if the metabolites of 10G retain the efficacy of the parent compound toward pathological anemia treatment. In the present study, we first investigated the metabolism of 10G in zebrafish embryos and then explored the biotransformation of 10G in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and humans, in which two major metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MS(n) spectra and comparison to authentic standards that we synthesized. After 24 h of treatment of zebrafish embryos, 10G was mostly converted to its metabolites. Our results clearly indicate that the reductive pathway is a major metabolic route for 10G in both zebrafish embryos and humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar hematopoietic effects as 10G in zebrafish embryos.

G protein-coupled receptor kinase 6 deficiency promotes angiogenesis, tumor progression, and metastasis.

G protein-coupled receptor kinases (GRKs) phosphorylate the activated form of G protein-coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6(-/-)) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6(+/+)). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because ß-arrestin-2-deficient (ßarr2(-/-)) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6(-/-), we developed a double GRK6(-/-)/ßarr2(-/-) mouse model. Surprisingly, GRK6(-/-)/ßarr2(-/-) mice exhibited faster tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6(-/-) and GRK6(-/-)/ßarr2(-/-) animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6(-/-)/ßarr2(-/-) showed greater tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.

Cinnamon extract inhibits angiogenesis in zebrafish and human endothelial cells by suppressing VEGFR1, VEGFR2, and PKC-mediated MAP kinase.

Angiogenesis is a process of new blood vessel generation and under pathological conditions, lead to tumor development, progression, and metastasis. Many bioactive components have been studied for its antiangiogenic properties as a preventive strategy against tumor development. This study is focused on the effects of cinnamon extract in modulating the pathway involved in angiogenesis. Human umbilical vein endothelial cells (HUVEC) were treated with cinnamon extract at a concentration of 25 µg/mL for 1, 3, or 6 h followed by treatment with phorbol ester (TPA) at a concentration of 10 nmol/L to induce mitogen-activated protein kinase (MAPK) expression. Results show that cinnamon extract inhibited TPA-induced phosphorylation of MAPK and AKT in a dose-dependent manner. Gene expression results in HUVEC showed that cinnamon extract treatment inhibited TPA induction of protein kinase C, PKCα and PKCη messenger RNA (mRNA) expression in a dose-dependent manner along with suppression of vascular endothelial growth factor receptor 1 (VEGFR1/Flt1) and vascular endothelial growth factor receptor 2 (VEGFR2/KDR/Flk1) mRNA expression. Cinnamon extract was administered to zebrafish embryos during gastrulation at 6-8 h post fertilization (hpf). The embryos were observed for changes in morphology, toxicity, and blood vessel development. The intersegmental vessels in the zebrafish embryos were attenuated and underdeveloped at an effective cinnamon extract dose of 250 µg/mL compared with the DMSO-treated control. Exposure to cinnamon extract for 36 h resulted in gross morphological deformities. The results suggest the effect of cinnamon extract on angiogenesis is mediated by PKC-dependent phosphorylation of MAPK.

Structural identification of theaflavin trigallate and tetragallate from black tea using liquid chromatography/electrospray ionization tandem mass spectrometry.

Black tea contains two major pigments, theaflavins and thearubigins. These polyphenols have been associated with certain health benefits including prevention of heart disease and cancer. Elucidating and characterizing the structural aspects of thearubigins, the most abundant pigment in black tea, has been a challenge for many years. Therefore further studies of black tea polyphenols must be conducted in effort to solve this thearubigin dispute. In the present study, black tea extract was found to possess theaflavin trigallate and tetragallate by means of liquid chromatography/electrospray ionization mass spectrometry. These structures were confirmed by analysis of the MS(n) (n = 1-4) spectra and comparison of the MS/MS spectra of the product ions to the MS/MS spectra of authentic (-)-epigallocatechin-3-gallate, (-)-epicatechin-3-gallate and theaflavin-3,3'-digallate. To our knowledge, this is the first report to confirm the presence of theaflavin trigallate and tetragallate in black tea.

6-gingerdiols as the major metabolites of 6-gingerol in cancer cells and in mice and their cytotoxic effects on human cancer cells.

6-Gingerol, a major pungent component of ginger (Zingiber officinale Roscoe, Zingiberaceae), has been reported to have antitumor activities. However, the metabolic fate of 6-gingerol and the contribution of its metabolites to the observed activities are still unclear. In the present study, we investigated the biotransformation of 6-gingerol in different cancer cells and in mice, purified and identified the major metabolites from human lung cancer cells, and determined the effects of the major metabolites on the proliferation of human cancer cells. Our results show that 6-gingerol is extensively metabolized in H-1299 human lung cancer cells, CL-13 mouse lung cancer cells, HCT-116 and HT-29 human colon cancer cells, and in mice. The two major metabolites in H-1299 cells were purified and identified as (3R,5S)-6-gingerdiol (M1) and (3S,5S)-6-gingerdiol (M2) based on the analysis of their 1D and 2D NMR data. Both metabolites induced cytotoxicity in cancer cells after 24 h, with M1 having a comparable effect to 6-gingerol in H-1299 cells.

Ginger stimulates hematopoiesis via Bmp pathway in zebrafish.

BACKGROUND: Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1. CONCLUSIONS/SIGNIFICANCE: Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.

Discriminating different cancer cells using a zebrafish in vivo assay.

Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase) inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell metastasis. This assay represents not only the useful test for patient diagnosis, but also has the potential for evaluating anti-cancer drugs treatment.

Zebrafish model: worth considering in defining tumor angiogenesis.

The process of angiogenesis is essential for tumor progression and metastasis; however, antiangiogenesis therapy-induced hypoxia and inflammation are perhaps the driving force for tumor escape and metastasis formation, thereby compromising its efficacy. This warrants the complete understanding of the molecular and cellular basis of antiangiogenesis therapy and necessitates the identification of potential signaling events in the host microenvironment, which are involved in tumor angiogenesis and metastasis, to improve the treatment of cancer. In this context, the zebrafish/tumor xenograft model represents an emerging vertebrate system to study the correlation between tumor angiogenesis, inflammation, and metastasis and to better understand the modification of tumor microenvironment by antiangiogenesis therapy. This review article describes the necessity to study the microenvironment of host-tumor interface by introducing basic concepts of angiogenesis, emerging paradigms, and challenges of antiangiogenesis therapy and provides an update on the importance of the zebrafish/tumor xenograft model to address these issues.

The orphan G protein-coupled receptor 161 is required for left-right patterning.

Gpr161 (also known as RE2) is an orphan G protein-coupled receptor (GPCR) that is expressed during embryonic development in zebrafish. Determining its biological function has proven difficult due to lack of knowledge regarding its natural or synthetic ligands. Here, we show that targeted knockdown of gpr161 disrupts asymmetric gene expression in the lateral plate mesoderm, resulting in aberrant looping of the heart tube. This is associated with elevated Ca(2+) levels in cells lining the Kupffer's vesicle and normalization of Ca(2+) levels, by over-expression of ncx1 or pmca-RNA, is able to partially rescue the cardiac looping defect in gpr161 knockdown embryos. Taken together, these data support a model in which gpr161 plays an essential role in left-right (L-R) patterning by modulating Ca(2+) levels in the cells surrounding the Kupffer's vesicle.

Expression of the G protein gammaT1 subunit during zebrafish development.

Here, we report the identification and expression analysis of the zebrafish G protein gammaT1 subunit gene (gngT1) during development. Similar to its human and mouse homologs, we confirm zebrafish gngT1 is expressed in the developing retina, where its transcription overlaps with the photoreceptor cell-specific marker, rhodopsin (rho). Surprisingly, we also show zebrafish gngT1 is expressed in the dorsal diencephalon, where its transcription overlaps with the pineal specific markers, arylalkylamine N-acetyltransferase-2 (annat-2) and extra-ocular rhodopsin (exorh). Analysis of the proximal promoter sequence of the zebrafish gngT1 gene identifies several conserved binding sites for the cone-rod homeobox/orthodenticle (Crx/Otx) homeodomain family of transcription factors. Using a morpholino anti-sense approach in zebrafish, we show that targeted knockdown of otx5 potently suppresses gngT1 expression in the pineal gland, whereas knockdown of crx markedly reduces gngT1 expression in the retina. Taken together, these data indicate that pineal- and retinal-specific expression of the gngT1 gene are controlled by different transcription factors and exogenous signals.