An automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos.

The knowledge on environmentally relevant chemicals that may interfere with thyroid signaling is scarce. Here, we present a method for the screening of goitrogens, compounds that disrupt the thyroid gland function, based on the automatic orientation of zebrafish in a glass capillary and a subsequent imaging of reporter gene fluorescence in the thyroid gland of embryos of the transgenic zebrafish line tg(tg:mCherry). The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to a negative control compound (3,4-dichloroaniline), or a concentration series of known goitrogenic compounds (resorcinol, methimazole, potassium perchlorate, 6-propyl-2-thiouracil, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on mortality and/or solubility. Exposure to 3,4-dichloroaniline decreased the fluorescence signal. All goitrogenic compounds exhibited clear concentration-dependent inductions of reporter fluorescence 1.4 to 2.6 fold above control levels. Concentration-response modelling was used to calculate goitrogenic potencies based on EC50 values. The new automated method offers an efficient screening approach for goitrogenic activity.

A comparison of fate and toxicity of selenite, biogenically, and chemically synthesized selenium nanoparticles to zebrafish (Danio rerio) embryogenesis.

Microbial reduction of selenium (Se) oxyanions to elemental Se is a promising technology for bioremediation and treatment of Se wastewaters. But a fraction of biogenic nano-Selenium (nano-Seb) formed in bioreactors remains suspended in the treated waters, thus entering the aquatic environment. The present study investigated the toxicity of nano-Seb formed by anaerobic granular sludge biofilms on zebrafish embryos in comparison with selenite and chemogenic nano-Se (nano-Sec). The nano-Seb formed by granular sludge biofilms showed a LC50 value of 1.77 mg/L, which was 3.2-fold less toxic to zebrafish embryos than selenite (LC50 = 0.55 mg/L) and 10-fold less toxic than bovine serum albumin stabilized nano-Sec (LC50 = 0.16 mg/L). Smaller (nano-Secs; particle diameter range: 25-80 nm) and larger (nano-Secl; particle diameter range: 50-250 nm) sized chemically synthesized nano-Sec particles showed comparable toxicity on zebrafish embryos. The lower toxicity of nano-Seb in comparison with nano-Sec was analyzed in terms of the stabilizing organic layer. The results confirmed that the organic layer extracted from the nano-Seb consisted of components of the extracellular polymeric substances (EPS) matrix, which govern the physiochemical stability and surface properties like ζ-potential of nano-Seb. Based on the data, it is contented that the presence of humic acid like substances of EPS on the surface of nano-Seb plays a major role in lowering the bioavailability (uptake) and toxicity of nano-Seb by decreasing the interactions between nanoparticles and embryos.

Establishment and optimization of a high throughput setup to study Staphylococcus epidermidis and Mycobacterium marinum infection as a model for drug discovery.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

A high-throughput screen for tuberculosis progression.

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.