Development of confocal X-ray fluorescence techniques for biological and archaeological applications

X-ray fluorescence imaging (XFI) is a versatile tool for determining spatial distributions of elements or elemental species within a variety of samples. A newer XFI variant, confocal X-ray fluorescence imaging (CXFI), shows significant promise since confocal detection facilitates important advantages such as three-dimensionally resolved elemental investigation, preservation of sample integrity and higher sensitivity. Additionally, a readily available extension of CXFI, confocal X-ray absorption spectroscopy (CXAS), allows determination of elemental speciation within a specific localized volume element. To enable direct visualization of three-dimensional elemental distribution and speciation in intact biological samples without staining or thinning, this thesis aimed at developing CXFI and CXAS for biological and archaeological applications. CXFI and CXAS technique development included installing and refining the polycapillary based setup at beamline 20ID-B, Advanced Photon Source (APS), USA, and verifying the feasibility of different configurations through select measurements. In addition, a novel X-ray detection focusing optic, the spoked channel array (SCA) recently developed at the Cornell High Energy Synchrotron Source (CHESS), USA, was implemented to provide superior spatial resolution. CXFI and CXAS were used to study the following scientific problems: a) Embryonic exposure to elevated selenium in zebrafish: The localization and chemical form of maternally transferred selenium were studied in an early developmental stage of life to understand the effect of exposure. b) Highly localized Fe speciation within intact corrosion layer in antique stained glass: SCA derived CXAS was employed to determine iron speciation in an antique stained glass sample from 13th century Paderborn Cathedral, Germany to aid the study of cleaning methods used in restoration. c) Differentiating diagenesis from biogenicity: Archaeological bone samples from the Royal Naval Hospital Cemetery (c. A.D. 1793-1822), Antigua were studied in an attempt to distinguish between diagenetic and biogenic lead uptake. Systematic comparisons of conventional XFI with polycapillary and SCA-based CXFI showed that lateral spatial resolution was remarkably improved with SCA optics. This research facilitated incorporating CXFI and CXAS at beamline 20ID-B, APS and establishing SCA optics as a valuable addition to the confocal X-ray detection toolkit. These capabilities will be implemented at the BioXAS imaging beamline at the Canadian Light Source

Novel application assigned to toluquinol: inhibition of lymphangiogenesis by interfering with VEGF-C/VEGFR-3 signalling pathway

BACKGROUND AND PURPOSE Lymphangiogenesis is an important biological process associated with the pathogenesis of several diseases, including metastatic dissemination, graft rejection, lymphoedema and other inflammatory disorders. The development of new drugs that block lymphangiogenesis has become a promising therapeutic strategy. In this study, we investigated the ability of toluquinol, a 2-methyl-hydroquinone isolated from the culture broth of the marine fungus Penicillium sp. HL-85-ALS5-R004, to inhibit lymphangiogenesis in vitro, ex vivo and in vivo. EXPERIMENTAL APPROACH We used human lymphatic endothelial cells (LECs) to analyse the effect of toluquinol in 2D and 3D in vitro cultures and in the ex vivo mouse lymphatic ring assay. For in vivo approaches, the transgenic Fli1:eGFPy1 zebrafish, mouse ear sponges and cornea models were used. Western blotting and apoptosis analyses were carried out to search for drug targets. KEY RESULTS Toluquinol inhibited LEC proliferation,migration, tubulogenesis and sprouting of new lymphatic vessels. Furthermore, toluquinol induced apoptosis of LECs after 14 h of treatment in vitro, blocked the development of the thoracic duct in zebrafish and reduced the VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice. Mechanistically, we demonstrated that this drug attenuates VEGF-C-induced VEGFR-3 phosphorylation in a dose-dependentmanner and suppresses the phosphorylation of Akt and ERK1/2. CONCLUSIONS AND IMPLICATIONS Based on these findings, we propose toluquinol as a new candidate with pharmacological potential for the treatment of lymphangiogenesis-related pathologies. Notably, its ability to suppress corneal neovascularization paves the way for applications in vascular ocular pathologies.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work has been supported by personal funding by FP7-PEOPLE-2013-IEF Marie Curie Postdoctoral Fellowship (MGC). Acknowledged are the supporting grants from the Action de Recherche Concertée (ARC) (Université de Liège), the Fonds de la Recherche Scientifique-FNRS (F.R.S.-FNRS), the Foundation Against Cancer (foundation of public interest), the Centre Anticancéreux près l’Université de Liège, the Fonds Léon Fredericq (University of Liège), the Interuniversity Attraction Poles Programme-Belgian Science Policy (all from Belgium) and the Plan National Cancer (« Service Public Federal » from Belgium). Research in the lab of A.R.Q. and M.A.M. was supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER)

Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection.

Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish (Danio rerio) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine

In-vivo Optical Tomography of Small Scattering Specimens: time-lapse 3D imaging of the head eversion process in Drosophila melanogaster

5 fig.Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window - thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously.This work was supported in part by Project ‘‘THALES – BSRC ‘Alexander Fleming’ – Development and employment of Minos-based genetic and functional genomic technologies in model organisms (MINOS)’’ – MIS: 376898, the Fellowship for Young International Scientist of the Chinese Academy of Sciences Grant No. 2010Y2GA03 and the NSFC-NIH Biomedical collaborative research program 81261120414. A. Arranz acknowledges support from Marie Curie Intra-European Fellowship Program FP7-PEOPLE-2010-IEF. J. Ripoll acknowledges support from EC FP7 CIG grant HIGH-THROUGHPUT TOMO, and Spanish MINECO grant MESO-IMAGING FIS2013-41802-R. The authors would like to thank Dr. S. Oehler for the help with the GFP-expressing flies, and G. Livadaras and G. Zacharakis for help with the Drosophila stocks

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale

ToScA North America (6 – 8 June 2017, The University of Texas, Austin, TX) Program

ToScA North America will address key areas of science, including Multi-modal Imaging, Geosciences, Forensics, Increasing Contrast, Educational Outreach, Data, Materials Science and Medical and Biological Science.University of Texas High-Resolution X-ray CT Facility (UTCT); Jackson School of Geosciences, The University of Texas at Austin; Natural History Museum (London); Royal Microscopical Society (Oxford, UK)Geological Science

What determines growth potential and juvenile quality of farmed fish species?

Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut

The Neutrophil's Eye-View: Inference and Visualisation of the Chemoattractant Field Driving Cell Chemotaxis In Vivo

As we begin to understand the signals that drive chemotaxis in vivo, it is becoming clear that there is a complex interplay of chemotactic factors, which changes over time as the inflammatory response evolves. New animal models such as transgenic lines of zebrafish, which are near transparent and where the neutrophils express a green fluorescent protein, have the potential to greatly increase our understanding of the chemotactic process under conditions of wounding and infection from video microscopy data. Measurement of the chemoattractants over space (and their evolution over time) is a key objective for understanding the signals driving neutrophil chemotaxis. However, it is not possible to measure and visualise the most important contributors to in vivo chemotaxis, and in fact the understanding of the main contributors at any particular time is incomplete. The key insight that we make in this investigation is that the neutrophils themselves are sensing the underlying field that is driving their action and we can use the observations of neutrophil movement to infer the hidden net chemoattractant field by use of a novel computational framework. We apply the methodology to multiple in vivo neutrophil recruitment data sets to demonstrate this new technique and find that the method provides consistent estimates of the chemoattractant field across the majority of experiments. The framework that we derive represents an important new methodology for cell biologists investigating the signalling processes driving cell chemotaxis, which we label the neutrophils eye-view of the chemoattractant field