Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.     

An Individual-Based Model of Zebrafish Population Dynamics Accounting for Energy Dynamics

Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch’s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.     

Predictive Modeling of Neuroblastoma Growth Dynamics in Xenograft Model After Bevacizumab Anti-VEGF Therapy

Neuroblastoma is the leading cause of cancer death in young children. Although treatment for neuroblastoma has improved, the 5-year survival rate of patients still remains less than half. Recent studies have indicated that bevacizumab, an anti-VEGF drug used in treatment of several other cancer types, may be effective for treating neuroblastoma as well. However, its effect on neuroblastoma has not been well characterized. While traditional experiments are costly and time-consuming, mathematical models are capable of simulating complex systems quickly and inexpensively. In this study, we present a model of vascular tumor growth of neuroblastoma IMR-32 that is complex enough to replicate experimental data across a range of tumor cell properties measured in a suite of in vitro and in vivo experiments. The model provides quantitative insight into tumor vasculature, predicting a linear relationship between vasculature and tumor volume. The tumor growth model was coupled with known pharmacokinetics and pharmacodynamics of the VEGF blocker bevacizumab to study its effect on neuroblastoma growth dynamics. The results of our model suggest that total administered bevacizumab concentration per week, as opposed to dosage regimen, is the major determining factor in tumor suppression. Our model also establishes an exponentially decreasing relationship between administered bevacizumab concentration and tumor growth rate.     

Axitinib Improves Radiotherapy in Murine Xenograft Lung Tumors

A third of patients with non-small cell lung cancer (NSCLC) present with un-resectable stage III locally advanced disease and are currently treated by chemo-radiotherapy but the median survival is only about 21 months. Using an orthotopic xenograft model of lung carcinoma, we have investigated the combination of radiotherapy with the anti-angiogenic drug axitinib (AG-013736, Pfizer), which is a small molecule receptor tyrosine kinase inhibitor that selectively targets the signal transduction induced by VEGF binding to VEGFR receptors. We have tested the combination of axitinib with radiotherapy in nude mice bearing human NSCLC A549 lung tumors. The therapy effect was quantitatively evaluated in lung tumor nodules. The modulation of radiation-induced pneumonitis, vascular damage and fibrosis by axitinib was assessed in lung tissue. Lung irradiation combined with long-term axitinib treatment was safe resulting in minimal weight loss and no vascular injury in heart, liver and kidney tissues. A significant decrease in the size of lung tumor nodules was observed with either axitinib or radiation, associated with a decrease in Ki-67 staining and a heavy infiltration of inflammatory cells in tumor nodules. The lungs of mice treated with radiation and axitinib showed a complete response with no detectable residual tumor nodules. A decrease in pneumonitis, vascular damage and fibrosis were observed in lung tissues from mice treated with radiation and axitinib. Our studies suggest that axitinib is a potent and safe drug to use in conjunction with radiotherapy for lung cancer that could also act as a radioprotector for lung tissue by reducing pneumonitis and fibrosis.     

A Novel Mitochondrial Sphingomyelinase in Zebrafish Cells

Sphingolipids are important signaling molecules in many biological processes, but little is known regarding their physiological roles in the mitochondrion. We focused on the biochemical characters of a novel sphingomyelinase (SMase) and its function in mitochondrial cer a mide generation in zebrafish embryonic cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61,300) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain. The mature endogenous enzyme was predicted to have a molecular weight of 57,000, and matrix-assisted laser de sorp tion ionization time-of-flight mass spectrometry analysis indicated that the N-terminal amino acid residue of the mature enzyme was Ala-36. The purified enzyme optimally hydrolyzed [¹⁴C]sphingomyelin in the presence of 10 mm Mg²⁺ at pH 7.5. In HEK293 cells that overexpressed SMase cDNA, the enzyme was localized to the mitochondrial fraction, whereas mutant proteins lacking MLS or both the MLS and the transmembrane domain were absent from the mitochondrial fraction. Endogenous SMase protein co-localized with a mitochondrial cytostaining marker. Using a protease protection assay, we found that SMase was distributed throughout the intermembrane space and/or the inner membrane of the mitochondrion. Furthermore, the overexpression of SMase in HEK293 cells induced cer a mide generation and sphingomyelin hydrolysis in the mitochondrial fraction. Antisense phosphorothioate oligonucleotide-induced knockdown repressed cer a mide generation and sphingomyelin hydrolysis in the mitochondrial fraction in zebrafish embryonic cells. These observations indicate that SMase catalyzes the hydrolysis of sphingomyelin and generates cer a mide in mitochondria in fish cells.Sphingomyelinase (SMase,² sphingomyelin phosphodiesterase, EC 3.1.4.12) hydrolyzes sphingomyelin and produces ceramide and phosphocholine. Ceramide plays an important role as a signaling molecule in cell proliferation, apoptosis, cell cycle arrest, differentiation, and the stress response in animal cells (1–5). To date, three distinct classes of acid, neutral, and alkaline SMases have been identified according to optimum pH, cation dependence, amino acid sequence, and subcellular localization (3).The Mg²⁺-dependent neutral SMases have emerged as major candidates in the mediation of ceramide-induced cell signaling (6). Recent research has identified at least three distinct neutral SMases in human and mouse, designated as neutral SMase 1, SMase 2, and SMase 3 (7–9). Neutral SMase 1 was the first SMase identified in human and mouse. Although mammalian enzymes exhibited Mg²⁺-dependent neutral SMase activity in vitro (9), no significant biological functions in sphingomyelin and ceramide metabolism were identified in SMase 1-overexpressing cells (10) or neutral SMase 1 knock-out mice (11). In zebrafish embryos, Mg²⁺-dependent neutral SMase 1 produced ceramide and caused thalidomide-induced vascular defects (12). In addition, SMase 1 was found to mediate heat-induced ceramide generation and apoptosis (13).The neutral SMase 2 gene SMPD3, has also been identified based on its similarity to Bacillus cereus SMase DNA sequences (7). This gene encodes a membrane-bound protein expressed in the brain and liver that has two highly hydrophobic segments near the N-terminal region, both of which are thought to function as transmembrane domains. Unlike neutral SMase 1, neutral SMase 2 possesses Mg²⁺-dependent neutral SMase activity in vivo in MCF-7 cells (14). When overexpressed in the confluent phase of MCF-7 cells, mouse neutral SMase 2 was palmitoylated via thioester bonds and localized in the inner leaflet of the plasma membrane (15). In MCF-7 cells stably expressing neutral SMase 2, the enzyme inhibited cell growth and was required for cells to undergo confluence-induced cell cycle arrest (16). Interestingly, neutral SMase 2 was isolated as the confluent 3Y1 cell-associated 1 gene (cca1) in rat 3Y1 cells (17). Neutral SMase 2 has been implicated in signal transduction events in cell growth and the cellular response to cytokines (18, 19), oxidative stress (20), and amyloid β-peptide (21).Stoffel et al. (22) demonstrated that gene-targeted mice deficient for neutral SMase 2 developed a novel form of dwarfism and had delayed puberty as part of a hypothalamus-induced pituitary hormone deficiency. Strikingly, positional cloning of the recessive mutation fragilitas ossium in mice identified a deletion in the gene that encodes neutral SMase 2, leading to the complete loss of neutral SMase activity (23). The mutant fragilitas ossium mice develop severe osteogenesis and dentinogenesis imperfecta, with no collagen defect. Thus, mouse neutral SMase 2 is essential for late embryonic and postnatal development.Mitochondria contain small amounts of a variety of sphingolipids, including ceramide and sphingomyelin (24–26), which may be derived from the endoplasmic reticulum via intimate membrane contacts or produced in response to apoptosis. For mitochondria isolated from HL-60 cells, treatment with ceramide inhibited the mitochondrial respiratory chain complex III (27). Birbes et al. (28) found that the selective hydrolysis of a mitochondrial pool of sphingomyelin induced apoptosis. They transfected MCF-7 cells with B. cereus SMase targeted to various subcellular organelles, but they observed cytochrome c release and apoptosis induction only when the enzyme was targeted to the mitochondria. Ceramide activated the mitochondrial protein phosphatase 2A, which dephosphorylated Bcl-2 and led to apoptosis (29). In MCF-7 cells, mitochondrial ceramide generation in response to tumor necrosis factor-α induced Bax translocation to mitochondria and subsequent cytochrome c release and apoptosis (30). The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane (31). The concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (31). In isolated mitochondria, ceramide can also form membrane channels large enough to release cytochrome c and other small proteins (32). Ceramide-metabolizing enzymes, such as a bovine liver ceramide synthase (33) and human ceramidase (34), are localized to the mitochondrion. These observations suggest the existence of a mitochondrial pool of sphingomyelin and the function of a sphingomyelin-specific metabolic pathway in mitochondria. However, no SMase has been identified in mitochondria.We identified and examined the biochemical properties of a novel SMase localized to the zebrafish mitochondrion. The enzyme was cloned from a cDNA library of embryonic zebrafish cells. It was found to regulate mitochondrial ceramide levels.     

Novel Formula for Cell Kinetics in Xenograft Model of Hepatocellular Carcinoma Using Histologically Calculable Parameters

The growth rate of tumors should be assessed in terms of both tumor cell proliferation and death. The former is considered to be determined by growth fraction and cell-cycle time, whereas the latter is mainly determined by apoptosis, especially in tumors with a low level of necrosis. While most hepatocellular carcinomas (HCCs) in a relatively early stage contain only a small amount of necrosis, the growth rate supposedly depends mainly on growth fraction, cell-cycle time, and apoptosis. However, their quantitative relationship remains unknown. We have derived a novel theoretical formula for determining this relationship in nonnecrotic HCC, using Ki-67-positive index, apoptotic score, and a correction factor, all calculable by histological assessment without injecting labeling agents. Furthermore, we confirmed the reliability of this formula, using a xenograft model of human HCC with less than 15% necrosis. In this model the values of cell-cycle time calculated from the formula were very close to those estimated by a conventional double-labeling method and showed high correlations. Since our novel formula can clarify the cell kinetics without cumbersome labeling procedures, it is expected to be clinically applicable to HCC with a small portion of necrosis, using the radiographically measured growth rate and the histologically assessed cell kinetic parameters.     

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.     

Lorica Production in Choanoflagellates–A Model System for Investigating the Mechanics, Controls and Dynamics of Secretion

BIOMINERALIZATION is the process by which living organisms assemble structures from naturally occurring inorganic compounds. Mineral deposition is common and widespread amongst Protozoa and in most instances the mineralized structures provide skeletal support and protection for softer organic parts [10]. The 2 most common minerals to be deposited by Protozoa are silica and calcium carbonate. Groups of Protozoa that deposit silica, which we are concerned with here, include the diatoms, chrysophytes, choanoflagellates, Radiolar-ia, Heliozoa and testate amoebae [10]. In the majority of silica-depositing protista, silica is taken up from the medium in the form of monomelic orthosilicic acid Si(OH)₄ (soluble reactive silicate) and deposited as amorphous, polymerised biogenic silica or opal within membrane-bounded vesicles known as silica deposition vesicles (SDV). Often biogenic silica is characteristically patterned and ornamented and for most protozoan groups the morphology of silicified parts is of prime taxonomic importance. By far the most extensively studied group of silica-depositing organisms are the diatoms [1, 12, 13]. To date most of our knowledge of silica metabolism in protists has been based on investigations into this group. Diatoms require silica for the production of their frustules. Uptake and deposition of silica occurs within a closely denned portion of the cell cycle, between nuclear division and cell separation. It occupies about ± of the cell cycle and without an adequate supply of silica diatoms are unable to produce new frustule valves with the result that cell division cannot be completed. Diatoms, therefore, have an obligate requirement for silica and without this nutrient they cease to grow [11]. In contrast to diatoms a number of other silica-depositing protistan groups, such as loricate choanoflagellates and certain chrysophytes, have a facultative requirement for silica. In the past decade the ultras true ture, physiology and ecology of loricate choanoflagellates have been extensively studied by a number of different workers [7] and the significance of these studies to our understanding of the mechanisms, controls and dynamics of silica secretion is summarised and discussed here.     

A Novel Implicit Solvent Model for Simulating the Molecular Dynamics of RNA

Although molecular dynamics simulations can be accelerated by more than an order of magnitude by implicitly describing the influence of the solvent with a continuum model, most currently available implicit solvent simulations cannot robustly simulate the structure and dynamics of nucleic acids. The difficulties become exacerbated especially for RNAs, suggesting the presence of serious physical flaws in the prior continuum models for the influence of the solvent and counter ions on the nucleic acids. We present a novel, to our knowledge, implicit solvent model for simulating nucleic acids by combining the Langevin–Debye model and the Poisson–Boltzmann equation to provide a better estimate of the electrostatic screening of both the water and counter ions. Tests of the model involve comparisons of implicit and explicit solvent simulations for three RNA targets with 20, 29, and 75 nucleotides. The model provides reasonable agreement with explicit solvent simulations, and directions for future improvement are noted.     

A Zebrafish Model for Studies on Esophageal Epithelial Biology

Mammalian esophagus exhibits a remarkable change in epithelial structure during the transition from embryo to adult. However, the molecular mechanisms of esophageal epithelial development are not well understood. Zebrafish (Danio rerio), a common model organism for vertebrate development and gene function, has not previously been characterized as a model system for esophageal epithelial development. In this study, we characterized a piece of non-keratinized stratified squamous epithelium similar to human esophageal epithelium in the upper digestive tract of developing zebrafish. Under the microscope, this piece was detectable at 5dpf and became stratified at 7dpf. Expression of esophageal epithelial marker genes (Krt5, P63, Sox2 and Pax9) was detected by immunohistochemistry and in situ hybridization. Knockdown of P63, a gene known to be critical for esophageal epithelium, disrupted the development of this epithelium. With this model system, we found that Pax9 knockdown resulted in loss or disorganization of the squamous epithelium, as well as down-regulation of the differentiation markers Krt4 and Krt5. In summary, we characterized a region of stratified squamous epithelium in the zebrafish upper digestive tract which can be used for functional studies of candidate genes involved in esophageal epithelial biology.     

A Novel Zebrafish Model to Provide Mechanistic Insights into the Inflammatory Events in Carrageenan-Induced Abdominal Edema

A suitable small animal model may help in the screening and evaluation of new drugs, especially those from natural products, which can be administered at lower dosages, fulfilling an urgent worldwide need. In this study, we explore whether zebrafish could be a model organism for carrageenan-induced abdominal edema. The research results showed that intraperitoneal (i.p.) administration of 1.5% λ-carrageenan in a volume of 20 µL significantly increased abdominal edema in adult zebrafish. Levels of the proinflammatory proteins tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were increased in carrageenan-injected adult zebrafish during the development of abdominal edema. An associated enhancement was also observed in the leukocyte marker, myeloperoxidase (MPO). To support these results, we further observed that i.p. methylprednisolone (MP; 1 µg), a positive control, significantly inhibited carrageenan-induced inflammation 24 h after carrageenan administration. Furthermore, i.p. pretreatment with either an anti-TNF-α antibody (1∶5 dilution in a volume of 20 µL) or the iNOS-selective inhibitor aminoguanidine (AG; 1 µg) inhibited carrageenan-induced abdominal edema in adult zebrafish. This new animal model is uncomplicated, easy to develop, and involves a straightforward inducement of inflammatory edema for the evaluation of small volumes of drugs or test compounds.     

The Impact of Tumor Nitric Oxide Production on VEGFA Expression and Tumor Growth in a Zebrafish Rat Glioma Xenograft Model

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2’-7’-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.     

A Zebrafish Model of Germ Cell Aneuploidy

The high frequency of chromosomal nondisjunction in human germ cells impacts society in many ways. Yet, the etiology of chromosome disorders remains unclear. Using a zebrafish strain with a hypomorphic mutation in the kinase Mps1, a genetic association between reduced germ cell mitotic checkpoint activity and aneuploid progeny was recently established. This work highlights the exquisite sensitivity of vertebrate germ cells to disruptions in Mps1 function and mitotic checkpoint activity. In addition, it introduces the zebrafish as a promising tool with which to further investigate the origins of aneuploidy.     

A Novel Detection Scheme for High-Resolution Two-Dimensional Spin-Echo Correlated Spectra in Inhomogeneous Fields

Background

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy is a powerful and non-invasive tool for the analysis of molecular structures, conformations, and dynamics. However, the inhomogeneity of magnetic fields experienced by samples will destroy spectral information and hinder spectral analysis. In this study, a new pulse sequence is proposed based on the modulation of distant dipolar field to recover high-resolution 2D spin-echo correlated spectroscopy (SECSY) from inhomogeneous fields.

Method and Material

By using the new sequence, the correlation information between coupled spins and the J coupled information with straightforward multiplet patterns can be obtained free from inhomogeneous line broadening. In addition, the new sequence is also suitable for non-J coupled spin systems. Although three-dimensional acquisition is needed, the evolution of indirect detection dimensions is carefully designed and the ultrafast acquisition scheme is utilized to improve the acquisition efficiency. A chemical solution of butyl methacrylate (C₈H₁₄O₂) in DMSO (C₂H₆SO) in a deshimmed magnetic field was tested to demonstrate the implementation details of the new sequence. The performance of the new sequence relative to the conventional SECSY sequence was shown by using an aqueous solution of main brain metabolites in a deshimmed magnetic field.

Conclusion

The results reveal that the new sequence provides an attractive way to eliminate the inhomogeneous spectral line broadening for the spin-echo correlated spectrum and is a promising tool for the study of metabolites in metabonomics, even for the applications on in vivo and in situ high-resolution 2D NMR spectroscopy.     

A Mathematical Model of Lymphangiogenesis in a Zebrafish Embryo

The lymphatic system of a vertebrate is important in health and diseases. We propose a novel mathematical model to elucidate the lymphangiogenic processes in zebrafish embryos. Specifically, we are interested in the period when lymphatic endothelial cells (LECs) exit the posterior cardinal vein and migrate to the horizontal myoseptum of a zebrafish embryo. We wonder whether vascular endothelial growth factor C (VEGFC) is a morphogen and a chemotactic factor for these LECs. The model considers the interstitial flow driving convection, the reactive transport of VEGFC, and the changing dynamics of the extracellular matrix in the embryo. Simulations of the model illustrate that VEGFC behaves very differently in diffusion and convection-dominant scenarios. In the former case, it must bind to the matrix to establish a functional morphogen gradient. In the latter case, the opposite is true and the pressure field is the key determinant of what VEGFC may do to the LECs. Degradation of collagen I, a matrix component, by matrix metallopeptidase 2 controls the spatiotemporal dynamics of VEGFC. It controls whether diffusion or convection is dominant in the embryo; it can create channels of abundant VEGFC and scarce collagen I to facilitate lymphangiogenesis; when collagen I is insufficient, VEGFC cannot influence the LECs at all. We predict that VEGFC is a morphogen for the migrating LECs, but it is not a chemotactic factor for them.     

A Xenograft Mantle Transplantation Technique for Producing a Novel Pearl in an Akoya Oyster Host

The brightness and color of pearls varies among different pearl-producing shellfish and have been a source of human fascination since ancient times. When produced through cultivation, the characteristics and quality of a pearl depend on the kind of shellfish used and also the transplanted mantle graft. This suggests that the Akoya pearl oyster, which is generally used in Japan for pearl culturing, can produce different kinds of pearl through the use of mantles from different species of shellfish. However, a transplanted heterogeneous mantle would be rejected by the immune system of the Akoya oyster. We have therefore developed a new method to suppress the Akoya immune system that archives immune tolerance to other shellfish. It is generally known that small quantities of antigens can be used to produce archived immunological tolerance in a clinical setting. We successfully suppressed the Akoya pearl oyster immune response against a Mabé pearl oyster graft through repeat injections of mantle homogenates. We then transplanted a Mabé pearl oyster mantle graft into the immunologically tolerant Akoya pearl oyster and obtained a Mabé pearl from an Akoya pearl oyster. Our new technique thus makes the production of novel and different pearls in the Akoya possible. We believe that this has significant future potential for the advancement of the pearl industry.     

Podocyte Developmental Defects Caused by Adriamycin in Zebrafish Embryos and Larvae: A Novel Model of Glomerular Damage

The zebrafish pronephros is gaining popularity in the nephrology community, because embryos are easy to cultivate in multiwell plates, allowing large number of experiments to be conducted in an in vivo model. In a few days, glomeruli reach complete development, with a structure that is similar to that of the mammalian counterpart, showing a fenestrated endothelium and a basement membrane covered by the multiple ramifications of mature podocytes. As a further advantage, zebrafish embryos are permeable to low molecular compounds, and this explains their extensive use in drug efficacy and toxicity experiments. Here we show that low concentrations of adriamycin (i.e. 10 and 20 µM), when dissolved in the medium of zebrafish embryos at 9 hours post-fertilization and removed after 48 hours (57 hpf), alter the development of podocytes with subsequent functional impairment, demonstrated by onset of pericardial edema and reduction of expression of the podocyte proteins nephrin and wt1. Podocyte damage is morphologically confirmed by electron microscopy and functionally supported by increased clearance of microinjected 70 kDa fluorescent dextran. Importantly, besides pericardial edema and glomerular damage, which persist and worsen after adriamycin removal from the medium, larvae exposed to adriamycin 10 and 20 µM do not show any myocardiocyte alterations nor vascular changes. The only extra-renal effect is a transient delay of cartilage formation that rapidly recovers once adriamycin is removed. In summary, this low dose adriamycin model can be applied to analyze podocyte developmental defects, such as those observed in congenital nephrotic syndrome, and can be taken in consideration for pharmacological studies of severe early podocyte injury.     

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers Müller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.