Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism

Noncanonical Wnt signaling regulates midline convergence of organ primordia during zebrafish development

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. Here we show that the key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. We also show that three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, our results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development

Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase activation in Jurkat T-cell leukemia tumor cells

7 pages, 6 figures, 2 tables.-- PMID: 12351627 [PubMed].The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC(50) concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G(1) peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodide- and annexin V-stained cells indicated that the growth-inhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased (13)C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC(50) (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.This work was supported by Grants PPQ 2000-0688-CO5-03 and PPQ 2000-0688-C05-04 from the Spanish government, by NATO Collaborative Grant LST.CLG.976283, by Grant PHS M01-RR00425 from the General Clinical Research Unit, and by Grant P01-CA42710 of the UCLA Clinical Nutrition Research Unit Stable Isotope Core.Peer reviewe

Dose-effect analysis of QT and non-QT prolongation inducers.

<p>Four QT prolongation inducers (Terfenadine, Cisapride, Astemizole and Haloperidiol) and 2 non-QT prolongation inducers (Digitoxin and Nitrendipine) were tested on 48 hpf zebrafish embryos. Predominant observed effects are indicated: normal heart rate (N), bradycardia (B), 2∶1 arrhythmia (A), ventricle failure (VF), heart failure (HF).</p

Image analysis of the heart rate.

<p>The figure shows the analysis of the heart rate of a representative control (A), arrhythmic heart (B) and cardiac arrest (C). OF and ED algorithms are applied for the analysis of each video. Only when the OF is above a specific threshold FFT is applied to ED. Control and arrhythmic hearts are classified based on the number of the significant frequencies detected in the FFT, one for control (A) two or more for arrhythmic (B). (C) OF representative of a dead embryo. In FFTs, Periodogram is shown in blue, Original Fourier Transform of the ED signal in black and Heartbeat is shown in red.</p

Two different phenotypes induced by angiogenesis inhibitors.

<p>24 hpf zebrafish embryos were treated with different inhibitors and checked for angiogenesis defects at 48 hpf (A) or at 72 hpf (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036690#s4" target="_blank">material and methods</a> section. (A) Representative fluorescent images of embryos treated with inhibitors that alter the development of ISVs at the level of sprouting and extension (superior panel) or that perturb later stages of maturation and/or DLAV formation (lower panel). Compared with control ones, treated embryos present a lower number of incomplete ISVs in the case of Sorafenib and AG1478 while AG1296 and PD173074 inhibit proper development of the DLAV and the ISVs looks thinner. (B) Representative fluorescent images of embryos treated with the indicated inhibitors showing the presence of thinner ISVs, some of them disrupted (arrows) and interrupted DLAV formation (asterisks).</p

HTS platform layout.

<p>The figure shows all the components of the platform: master PC, embryo sorter, two liquid handlings (LH1 and LH2), plate hotel, incubator and reader. The robotic arm in the central part transports the plate to all the devices.</p

Repeatability assessment.

<p>Calculation of the Coefficient of Variation (C.V) for ten independent experiments for the compounds and concentration indicated.</p

Coefficient of Variation of the angiogenesis assay.

<p>Calculation of the Coefficient of Variation of the indicated parameters calculated from data obtained after 3 independent experiments carried out with the positive compounds shown at one dose close to the IC50 or the effective concentration.</p

Images analysis for ISVs formation.

<p>The figure shows the different steps in the process of image analysis for the determination of the anti-angiogenesis effect. Control embryo (A, B, C and D); embryo treated with KRN633 (A’, B’, C’ and D’). (A) and (A’) are the original images acquired by the microscope; (B) and (B’) are images showing the segmentation between the head and the tail of the embryo to separate the ROI and measure the length; (C) and (C’) are images corresponding to an intermediate step in the filtering process where all the vessels in the tail can be identified; (D) and (D’) are images presenting the area enclosed between ISVs and DLAVs (in white) that are the ones to be quantified.</p