Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

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.

Potent Virustatic Polymer-Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo.

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.

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy.

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 vivo temporal monitoring of the wound response in living zebrafish embryos.

Fast, easy and early (larval) identification of transparent mutant zebrafish using standard fluorescence microscopy.

The availability of transparent zebrafish mutants (either : or ) 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 ( and silver ( 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.

Zebrafish IL-4-like Cytokines and IL-10 Suppress Inflammation but Only IL-10 Is Essential for Gill Homeostasis.

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.

Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation.

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.

p62/SQSTM1 interacts with vimentin to enhance breast cancer metastasis.

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- 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.

OPTiM: Optical projection tomography integrated microscope using open-source hardware and software.

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.

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish.

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.

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

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.

Mucosal inflammation at the respiratory interface: a zebrafish model.

Inflammatory diseases of the respiratory system such as asthma and chronic obstructive pulmonary disease are increasing globally and remain poorly understood conditions. Although attention has long focused on the activation of type 1 and type 2 helper T cells of the adaptive immune system in these diseases, it is becoming increasingly apparent that there is also a need to understand the contributions and interactions between innate immune cells and the epithelial lining of the respiratory system. Cigarette smoke predisposes the respiratory tissue to a higher incidence of inflammatory disease, and here we have used zebrafish gills as a model to study the effect of cigarette smoke on the respiratory epithelium.

Inference of random walk models to describe leukocyte migration.

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.

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish.

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.

Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation.

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.

P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish.

The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation.

The CD46-Jagged1 interaction is critical for human TH1 immunity.

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (T(H)1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46.

Noncanonical Notch signaling modulates cytokine responses of dendritic cells to inflammatory stimuli.

Dendritic cell (DC)-derived cytokines play a key role in specifying adaptive immune responses tailored to the type of pathogen encountered and the local tissue environment. However, little is known about how DCs perceive the local environment. We investigated whether endogenous Notch signaling could affect DC responses to pathogenic stimuli.

The role of Nfil3 in zebrafish hematopoiesis.

Nfil3, a transcription factor that has an array of functions in immune cells, has been described as key regulator of CD8α(+) dendritic cell and natural killer cell development in mice. In this report we show that Nfil3 is enriched in the myeloid compartment of adult zebrafish including eosinophils. Knockdown of Nfil3 in pu.

Incorporation of an experimentally determined MTF for spatial frequency filtering and deconvolution during optical projection tomography reconstruction.

We demonstrate two techniques to improve the quality of reconstructed optical projection tomography (OPT) images using the modulation transfer function (MTF) as a function of defocus experimentally determined from tilted knife-edge measurements. The first employs a 2-D binary filter based on the MTF frequency cut-off as an additional filter during back-projection reconstruction that restricts the high frequency information to the region around the focal plane and progressively decreases the spatial frequency bandwidth with defocus. This helps to suppress "streak" artifacts in OPT data acquired at reduced angular sampling, thereby facilitating faster OPT acquisitions.

Calibrating spatio-temporal models of leukocyte dynamics against in vivo live-imaging data using approximate Bayesian computation.

In vivo studies allow us to investigate biological processes at the level of the organism. But not all aspects of in vivo systems are amenable to direct experimental measurements. In order to make the most of such data we therefore require statistical tools that allow us to obtain reliable estimates for e.

The NOTCH pathway contributes to cell fate decision in myelopoiesis.

Background: Controversy persists regarding the role of Notch signaling in myelopoiesis. We have used genetic approaches, employing two Notch zebrafish mutants deadly seven (DES) and beamter (BEA) with disrupted function of notch1a and deltaC, respectively, and Notch1a morphants to analyze the development of leukocyte populations in embryonic and mature fish.

Design And Methods: Myelomonocytes were quantified in early embryos by in situ hybridization using a myeloper-oxidase (mpx) probe.

In vivo fluorescence lifetime optical projection tomography.

We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.

Regulation of IL-17 in chronic inflammation in the human lung.

The regulation of human Th17 cell effector function by Treg cells (regulatory T-cells) is poorly understood. In the present study, we report that human Treg (CD4(+)CD25(+)) cells inhibit the proliferative response of Th17 cells but not their capacity to secrete IL (interleukin)-17. However, they could inhibit proliferation and cytokine production by Th1 and Th2 cells as determined by IFN-γ (interferon-γ) and IL-5 biosynthesis.

Podocin inactivation in mature kidneys causes focal segmental glomerulosclerosis and nephrotic syndrome.

Podocin is a critical component of the glomerular slit diaphragm, and genetic mutations lead to both familial and sporadic forms of steroid-resistant nephrotic syndrome. In mice, constitutive absence of podocin leads to rapidly progressive renal disease characterized by mesangiolysis and/or mesangial sclerosis and nephrotic syndrome. Using established Cre-loxP technology, we inactivated podocin in the adult mouse kidney in a podocyte-specific manner.