Zebrafish embryos and larvae: a new generation of disease models and drug screens

Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.     

Organization of the CTX prophage in environmental isolates of Vibrio mimicus

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment.     

Microbial transformation of 17alpha-ethynyl- and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products

The microbial transformation of the 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17alpha-ethyl-17beta-hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17alpha-ethynyl-17beta-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17alpha-ethynyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17alpha-ethyl-17beta-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17alpha-ethyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (6) and 17alpha-ethyl-6alpha,17beta-dihydroxy-5alpha-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC(50)=1.72 microM).     

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.     

Neurogenesis and neuronal communication on micropatterned neurochips

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.     

An innovative application of a small-scale motion analysis technique to quantify human skin deformation in vivo

This study highlights a new experimental method developed to measure full-field deformation of human skin in vivo. The technique uses a small-scale Qualisys (Sweden) 3D motion capture system and an array of reflective markers placed on the forearm of five healthy volunteers. A load of up to 1.5 N was applied to induce skin deformation by pulling a fine wire attached to the centre of the marker configuration. Loading and marker displacements were recorded simultaneously. 3D marker trajectory data was generated for three different load directions. Tests were repeated to investigate accuracy and repeatability. Calibration results indicate the accuracy of the motion capture system with an average residual of 0.05 mm. The procedure was found to be repeatable and accurate for five repeated tests of measured displacements with a maximum variance of 5%. Experimental data are presented to demonstrate robustness and the ability to produce significant outputs. For all five subjects, at 1 N load, the mean and standard deviations of skin axial and lateral displacements were found to be 11.7±1.6 mm and 12.3±3.3 mm, respectively. The axial displacements ratio (u₉₀/u₀) ranges from 0.63 to 1.45 with mean±standard deviation of 0.982±0.34 and 0.982±0.32 for left and right arms, respectively. The experiments generated useful and accurate data that can be used to study the viscoelastic, hyperelastic or anisotropic behaviour of human skin. The measured displacements will be analysed further to determine the mechanical properties of skin using inverse Finite Element Analysis and Ogden model.     

Induction of specific T cell immunity in patients with prostate cancer by vaccination with PSA146–154 peptide

T cell immunotherapy of prostate cancer (CaP) offers the potential for less toxic, more effective outcomes. A clinical trial was conducted in 28 patients with locally advanced or metastatic CaP to determine whether an HLA-A2 binding epitope of prostate-specific antigen, PSA146–154 (PSA-peptide), can induce specific T cell immunity. Patients were vaccinated either by intradermal injection of PSA-peptide and GM-CSF or by intravenous administration of autologous dendritic cells pulsed with PSA-peptide at weeks 1, 4 and 10. Delayed-type hypersensitivity (DTH) skin testing was performed at weeks 4, 14, 26 and 52. Fifty percent of the patients developed positive DTH responses to PSA-peptide. The size of the DTH induration progressively increased over time in the majority of responding patients. Skin biopsies from seven DTH-positive patients were available and T cells that developed in situ were also characterized. The phenotype of recovered T cells demonstrated variable proportions of CD4⁺CD8⁻, CD4⁻CD8⁺ and CD4⁺CD8⁺ T cell populations. Cytokine analysis of PSA-peptide stimulated T cells per bead array assay exhibited specific IFN-γ and TNF-α response in six of seven patients. Specific IL-4 response was observed in five patients, while IL-10 response was detected in one patient. Purified CD4⁻CD8⁺ T cells isolated from four patients demonstrated specific cytolytic activity per chromium release assay. In conclusion, immunization with PSA-peptide induced specific T cell immunity in one-half of the patients with locally advanced and hormone-sensitive, metastatic CaP. DTH-derived T cells exhibited PSA-peptide-specific cytolytic activity and predominantly expressed a type-1 cytokine profile.