173 results found
Creamer A, Lo Fiego A, Agliano A, et al., 2023, Modular synthesis of semiconducting graft co-polymers to achieve ‘clickable’ fluorescent nanoparticles with long circulation and specific cancer targeting, Advanced Materials, Pages: 1-14, ISSN: 0935-9648
Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9′-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically “clicked” onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.
Najer A, Blight J, Ducker CB, et al., 2022, Potent virustatic polymer-lipid nanomimics block viral entry and inhibit malaria parasites in vivo, ACS Central Science, Vol: 8, Pages: 1238-1257, ISSN: 2374-7943
Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer–lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition.
Hogset H, Horgan C, Bergholt M, et al., 2020, In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy, Nature Communications, Vol: 11, Pages: 1-12, ISSN: 2041-1723
Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivotemporal monitoring of the wound response in living zebrafish embryos.Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis in fully intact and living zebrafish embryos.
Bottiglione F, Dee CT, Lea R, et al., 2020, Zebrafish IL-4-like cytokines and IL-10 suppress inflammation but only IL-10 is essential for gill homeostasis, Journal of Immunology, Vol: 205, Pages: 994-1008, ISSN: 0022-1767
Mucosal surfaces such as fish gills interface between the organism and the external environment and as such are major sites of foreign Ag encounter. In the gills, the balance between inflammatory responses to waterborne pathogens and regulatory responses toward commensal microbes is critical for effective barrier function and overall fish health. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune responses to pathogens and suppressing inflammation. Although considerable progress has been made in the field of fish immunology in recent years, whether the fish counterparts of these key mammalian cytokines perform similar roles is still an open question. In this study, we have generated IL-4/13A and IL-4/13B mutant zebrafish (Danio rerio) and, together with an existing IL-10 mutant line, characterized the consequences of loss of function of these cytokines. We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4–like cytokines and is essential for gill homeostasis. Thus, both IL-4/13 and IL-10 paralogs in zebrafish exhibit aspects of conserved function with their mammalian counterparts.
Wenz R, Conibear E, Bugeon L, et al., 2020, Fast, easy and early (larval) identification of transparent mutant zebrafish using standard fluorescence microscopy [version 1; peer review: 2 approved], F1000Research, Vol: 9, ISSN: 2046-1402
The availability of transparent zebrafish mutants (either TraNac: tra b6/b6; nac w2/w2 or casper: roy a9/a9; nac w2/w2 ) for live imaging studies together with the ease of generating transgenic lines are two of the strengths of the zebrafish model organism. The fact that transparent casper ( roy a9/a9;nac w2/w2) and silver nacre ( nac w2/w2) mutants are indistinguishable by eye at early stages (1-5 days post-fertilization; dpf) means many fish must be raised and later culled if they are not transparent. To identify translucent mutants early and easily at the early larval stage (≤5 dpf) before they are classified as protected animals, we developed a simple screening method using standard fluorescence microscopy. We estimate that this procedure could annually save 60,000 animals worldwide.
Moroz-Omori EV, Satyapertiwi D, Ramel MC, et al., 2020, Photoswitchable gRNAs for spatiotemporally controlled CRISPR-Cas-based genomic regulation, ACS Central Science, Vol: 6, Pages: 695-703, ISSN: 2374-7943
The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.
Progatzky F, Jha A, Wane M, et al., 2019, Induction of innate cytokine responses by respiratory mucosal challenge with R848 in zebrafish, mice and humans, Journal of Allergy and Clinical Immunology, Vol: 144, Pages: 342-345.e7, ISSN: 0091-6749
We compared live zebrafish, mouse and human nasal challenge responses to the TLR7/8 agonist resiquimod (R848). We found remarkably similar induction of mediators in the three species, offering novel mucosal models of innate anti-viral immunity.
Vervoort SJ, de Jong OG, Roukens MG, et al., 2018, Global transcriptional analysis identifies a novel role for SOX4 in 2 tumor-induced angiogenesis, eLife, Vol: 7, ISSN: 2050-084X
The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.
Lam EW, Yao S, Dallman M, et al., 2017, p62/SQSTM1 interacts with vimentin to enhance breast cancer metastasis, Carcinogenesis, Vol: 38, Pages: 1092-1103, ISSN: 1460-2180
The signalling adaptor p62 is frequently overexpressed in numerous cancer types. Here, we found that p62 expression was elevated in metastatic breast cancer and its overexpression correlated with reduced metastasis-free and relapse-free survival times. Analysis of p62 expression in breast cancer cell lines demonstrated that high p62 expression was associated with the invasive phenotypes of breast cancer. Indeed, silencing p62 expression attenuated the invasive phenotypes of highly metastatic cells, whereas overexpressing p62 promoted the invasion of non-metastatic cells in in vitro microfluidic model. Moreover, MDA-MB-231 cells with p62 depletion which were grown in a three-dimensional culture system exhibited a loss of invasive protrusions. Consistently, genetic ablation of p62 suppressed breast cancer metastasis in both zebrafish embryo and immunodeficient mouse models, as well as decreased tumorigenicity in vivo. To explore the molecular mechanism by which p62 promotes breast cancer invasion, we performed a co-immunoprecipitation (co-IP)-MS analysis and revealed that p62 interacted with vimentin, which mediated the function of p62 in promoting breast cancer invasion. Vimentin protein expression was downregulated upon p62 suppression and upregulated with p62 overexpression in breast cancer cells. Linear regression analysis of clinical breast cancer specimens showed a positive correlation between p62 and vimentin protein expression. Together, our findings provide strong evidence that p62 functions as a tumour metastasis promoter by binding vimentin and promoting its expression. This finding might help to develop novel molecular therapeutic strategies for breast cancer metastasis treatment.
Watson, Andrews N, Davis S, et al., 2017, OPTiM: optical projection tomography integrated microscope using open-source hardware and software, PLOS One, Vol: 12, ISSN: 1932-6203
We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 μm). All three methods use the microscope’s existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations’ viability.
Andrews N, Davis S, Hay C, et al., 2017, Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography, Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, Publisher: Society of Photo-Optical Instrumentation Engineers (SPIE), ISSN: 0277-786X
Jha A, Progatzky F, Wane M, et al., 2016, Human nasal mucosal responses to TLR agonists are mirrored by the zebrafish gill, British Association of Lung Research Summer Congress
Introduction: There are few reliable ways to study respiratory mucosal immune responses to viruses, viral-type toll-like receptor (TLR) agonists and vaccines. To investigate innate immune responses to TLR agonists (TLR3: poly IC/ poly ICLC; TLR7/8: resiquimod), we compared the effects on human nasal mucosa and zebrafish gills in vivo. Methods: Nasal challenge of adult volunteers was performed with saline, poly IC (n=4), poly ICLC (n=4) or resiquimod (n=8; 5 non-atopic, 3 atopic). Nasal mucosal lining fluid (MLF) was obtained by nasosorption at regular intervals up to 24 hours after challenge; nasal obstruction was monitored by peak nasal inspiratory flow (PNIF) and total nasal symptom scores (TNSS). Cytokines and interferons were measured in MLF using electrochemiluminescence on the Meso Scale Discovery (MSD) platform. Adult zebrafish gills were exposed to the same TLR agonists and gene expression was quantified in gill tissue at similar time-points. Results: Nasal challenge with TLR3 agonists failed to elicit any significant responses when compared to saline. In contrast resiquimod (10μg/100μl per nostril) caused a potent induction of cytokines with an early release (1-3 hours) of IFN-α2a, TNF-α and IL-1β and a later release (after 4 hours) of IFN-γ. The 3 volunteers with the highest levels of IFN-α2a were atopic. Six volunteers were asymptomatic and two volunteers had flu-like symptoms. There were no significant changes in clinical correlates of nasal obstruction. After resiquimod administration, but not TLR3 agonists, zebrafish gills showed an immune profile remarkably analogous to human nasal responses. Conclusion: The TLR7/8 agonist resiquimod is a potent mucosal inducer of IFN-α2a, IFN-γ and proinflammatory cytokines, whilst TLR3 agonists failed to stimulate mucosal innate immune responses. Zebrafish gills accurately mimic human nasal mucosal responses following exposure to TLR agonists, offering translational app
Kumar S, Lockward N, Ramel M-C, et al., 2016, Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish, Oncotarget, Vol: 7, Pages: 43939-43948, ISSN: 1949-2553
We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.
Andrews N, Ramel M-C, Kumar S, et al., 2016, Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time, Journal of Biophotonics, Vol: 9, Pages: 414-424, ISSN: 1864-0648
Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.
Watson TJ, Andrews N, Harry E, et al., 2016, Remote focal scanning and sub-volume optical projection tomography
We present a platform for sub-volume optical projection tomography utilising an electrically tunable lens and tracking technology. Applied to 3D fluorescent bead phantoms and zebrafish embryos, we demonstrate an improvement in resolution and light collection efficiency with respect to conventional optical projection tomography.
Andrews N, Ramel MC, Kumar S, et al., 2016, Fluorescence lifetime optical projection tomography and FRET applied to visualizing apoptosis in live zebrafish larvae
We present the application of FLIM-OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed cleavable FRET biosensor for Caspase-3 as an indicator of gamma radiation induced apoptosis.
Progatzky F, Cook HT, Lamb JR, et al., 2016, Mucosal inflammation at the respiratory interface: a zebrafish model, AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, Vol: 310, Pages: L551-L561, ISSN: 1040-0605
Kumar S, Lockwood N, Ramel MC, et al., 2016, In vivo multiplexed OPT and FLIM OPT of an adult zebrafish cancer disease model
We report angular multiplexed OPT and FLIM OPT applied to in vivo imaging of cancer and FRET biosensors in adult zebrafish. Multiple-spectral 3-D datasets of entire adult zebrafish can be acquired in 3 minutes.
Jones PJM, Sim A, Taylor HB, et al., 2015, Inference of random walk models to describe leukocyte migration, Physical Biology, Vol: 12, ISSN: 1478-3975
While the majority of cells in an organism are static and remain relatively immobile in their tissue, migrating cells occur commonly during developmental processes and are crucial for a functioning immune response. The mode of migration has been described in terms of various types of random walks. To understand the details of the migratory behaviour we rely on mathematical models and their calibration to experimental data. Here we propose an approximate Bayesian inference scheme to calibrate a class of random walk models characterized by a specific, parametric particle re-orientation mechanism to observed trajectory data. We elaborate the concept of transition matrices (TMs) to detect random walk patterns and determine a statistic to quantify these TM to make them applicable for inference schemes. We apply the developed pipeline to in vivo trajectory data of macrophages and neutrophils, extracted from zebrafish that had undergone tail transection. We find that macrophage and neutrophils exhibit very distinct biased persistent random walk patterns, where the strengths of the persistence and bias are spatio-temporally regulated. Furthermore, the movement of macrophages is far less persistent than that of neutrophils in response to wounding.
Correia T, Lockwood N, Kumar S, et al., 2015, Accelerated optical projection tomography applied to in vivo imaging of zebrafish, PLOS One, Vol: 10, ISSN: 1932-6203
Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections—achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.
Broncel M, Serwa RA, Ciepla P, et al., 2015, Myristoylation profiling in human cells and zebrafish., Data in Brief, Vol: 4, Pages: 379-383, ISSN: 2352-3409
Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.
Broncel M, Serwa RA, Ciepla P, et al., 2015, Multifunctional Reagents for Quantitative Proteome-Wide Analysis of Protein Modification in Human Cells and Dynamic Profiling of Protein Lipidation During Vertebrate Development, Angewandte Chemie-International Edition, Vol: 54, Pages: 5948-5951, ISSN: 1521-3773
Novel multifunctional reagents were applied incombination with a lipid probe for affinity enrichment ofmyristoylated proteins and direct detection of lipid-modifiedtryptic peptides by mass spectrometry. This method enableshigh-confidence identification of the myristoylated proteomeon an unprecedented scale in cell culture, and allowed the firstquantitative analysis of dynamic changes in protein lipidationduring vertebrate embryonic development.
Optical projection tomography is a 3-D imaging approach applicable to transparent samples and model organisms like zebrafish embryos. We present methods to improve the spatial resolution and realize 3-D cell tracking in OPT.
Chen L, Alexandrov Y, Kumar S, et al., 2015, Mesoscopic <i>in vivo</i> 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography, BIOMEDICAL OPTICS EXPRESS, Vol: 6, Pages: 1253-1261, ISSN: 2156-7085
McGinty J, Chen L, Kumar S, et al., 2015, Techniques to improve the spatial and temporal resolution in optical projection tomography: Remote focal scanning and time-lapse cell tracking
© OSA 2015. Optical projection tomography is a 3-D imaging approach applicable to transparent samples and model organisms like zebrafish embryos. We present methods to improve the spatial resolution and realize 3-D cell tracking in OPT.
Progatzky F, Sangha NJ, Yoshida N, et al., 2014, Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation, Nature Communications, Vol: 5, Pages: 1-14, ISSN: 2041-1723
Prolonged ingestion of a cholesterol- or saturated fatty acid-enriched diet induces chronic, often systemic, auto-inflammatory responses resulting in significant health problems worldwide. In vivo information regarding the local and direct inflammatory effect of these dietary components in the intestine and, in particular, on the intestinal epithelium is lacking. Here we report that both mice and zebrafish exposed to high-fat (HFDs) or high-cholesterol (HCDs) diets develop acute innate inflammatory responses within hours, reflected in the localized interleukin-1β-dependent accumulation of myeloid cells in the intestine. Acute HCD-induced intestinal inflammation is dependent on cholesterol uptake via Niemann-Pick C1-like 1 and inflammasome activation involving apoptosis-associated Speck-like protein containing a caspase recruitment domain, which leads to Caspase-1 activity in intestinal epithelial cells. Extended exposure to HCD results in localized, inflammation-dependent, functional dysregulation as well as systemic pathologies. Our model suggests that dietary cholesterol initiates intestinal inflammation in epithelial cells.
Chen L, Kumar S, Kelly D, et al., 2014, Remote focal scanning optical projection tomography with an electrically tunable lens, Biomedical Optics Express, Vol: 5, Pages: 3367-3375, ISSN: 2156-7085
We describe a remote focal scanning technique for optical projection tomography (OPT) implemented with an electrically tunable lens (ETL) that removes the need to scan the specimen or objective lens. Using a 4× objective lens the average spatial resolution is improved by ∼46% and the light collection efficiency by a factor of ∼6.76, thereby enabling increased acquisition speed and reduced light dose. This convenient implementation is particularly appropriate for lower magnifications and larger sample diameters where axial objective scanning would encounter problems with speed and stability.
Progatzky F, Sangha NJ, Yoshida N, et al., 2014, Intestinal inflammation induced by dietary cholesterol in Zebrafish, 9th European-Mucosal-Immunology-Group Meeting, Publisher: WILEY-BLACKWELL, Pages: 27-28, ISSN: 0019-2805
Ciepla P, Konitsiotis AD, Serwa RA, et al., 2014, New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish, Chemical Science, Vol: 5, Pages: 4249-4259, ISSN: 2041-6520
Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in theprogression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requiresattachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidatedShh has been challenging due to the limited array of tools available, and the roles of theseposttranslational modifications are poorly understood. Herein, we describe the development andvalidation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable itsvisualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown tobe an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shhfrom signalling cells, formation of multimeric transport complexes and signalling. We have used thisprobe to determine the size of transport complexes of lipidated Shh in culture medium and expressionlevels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitativechemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy anddetection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probesprovide a set of novel and well-validated tools that can be used to investigate the role of lipidation onactivity of Shh, and potentially other members of the Hedgehog protein family
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