Investigation of blood-brain barrier maturation using fluorescent dyes.

<p>Ai-iv) One hour after pericardial injection of Evans blue into 8 d.p.f. Tg(fli1a:EGFP)^y1 larvae, strong Evan’s blue fluorescence could be observed in the dorsal aorta (arrows) and vena cava (arrowheads) and weak fluorescence could be observed in individual segmental vessels of the trunk. Bi-iv) 4 hours after injection of Evan’s blue, strong fluorescence is still observed in dorsal aorta and vena cava and additionally in segmental vessels. In addition, fluorescence is also observed in trunk muscles between the segmental vessels and in the fin mesenchyme demonstrating that the dye has penetrated into surrounding tissue. Ci) Parasagittal section of a 3 d.p.f. zebrafish larva 3 hours after pericardial injection with saline control. Cii) High magnification fluorescent imaging of the region of marked in Ci). Di) Parasagittal section of a 3 d.p.f. zebrafish larva 3 hours after pericardial injection with Evan’s blue. Dii) High magnification fluorescent imaging of the region of marked in Di). Ei) Parasagittal section of a 5 d.p.f. zebrafish larva 3 hours after pericardial injection with Evan’s Blue. Cii) High magnification fluorescent imaging of the region of marked in Ci). Cii – Eii) The fluorescent intensity of dye within the brain was quantified using image thesholding (pseudo-coloured green) and area over threshold was measured using AnalySis software. F & G) The fluorescence intensity of injected dyes was measured in the brain of zebrafish following peripheral injection at various time points of zebrafish development. Graphs show mean fluorescent intensity (± std dev.) for each treatment. F) Evans blue, (961 Da) a large molecule known to form multimers with serum proteins, is excluded from the brain from day 5. G) Sodium fluorescein (376 Da) permeates into the brain until 8 d.p.f. but is excluded at 10 d.p.f. Scale bar represents 250 µm in A and B and 50 µm in C - E.</p

Rhodamine 123 distribution is altered in the presence of verapamil.

<p>A) Dye distribution and quantification experiments using rhodamine 123 (R123) were performed as described for Figure 1. Graph shows mean fluorescent intensity (± std dev.) for each treatment. Rhodamine 123 (grey bars), a substrate for mammalian ABCB1, ABCB4 and ABCB5, was excluded from the brain by 8 d.p.f. which, coincides with the onset of zebrafish Abcb/4/5 staining in the vasculature of the CNS. When 8 d.p.f. and 10 d.p.f. larvae were incubated with verapamil (white bars), an inhibitor of ABCB1, ABCB4 and ABCB5 in mammals, R123 failed to be excluded, consistent with blocked Abcb1/4/5 function. B and C) Representative images of parasagittal section of 5 d.p.f. larvae following pericardial injection of saline (B) or Rhodamine 123 (C). Fluorescence in the brain is observed following injection of Rhodamine 123. Scale bar represents 50 µm. </p

Sequence comparison and expression of zebrafish ABCB1/4/5 homologues.

<p><b>A</b>) <b>Sequence alignment of human and mouse ABCB1 protein sequences with zebrafish Abcb4 and Abcb5</b>. <b>The zebrafish proteins show a high level of identity with the mouse and human ABCB1, ABCB4 and ABCB5 proteins</b>. <b>An antibody recognising the peptide sequences VQAALD (yellow) and VQEALD (blue) (Covance) was selected for use in zebrafish as the similarity of these peptides was conserved</b>. <b>B (low magnification) and C) (high magnification) 3D projections of optically sectioned wholemount Tg(fli1a:EGFP)^y1 larvae stained with the anti-VQAALD antibody</b>. <b>Positive </b><b>staining </b><b>is </b><b>observed </b><b>in </b><b>the </b><b>vascular </b><b>endothelium </b><b>of </b><b>the </b><b>CNS </b><b>at 8 </b><b>d.p.f , but </b><b>not </b><b>at </b><b>earlier </b><b>timepoints </b>(<b>see</b> Figure S2). <b>High magnification images (Ci-iii) demonstrate the co-localisation of ABCB1/4/5 antibody staining (red) on cerebral vessels (green)</b>. <b>Bi and Ci – GFP channel – maximum intensity projection of the cerebral vasculature of Tg(fli1a:EGFP)^y1 transgenic larvae; Bii and Cii – Alexa 568 labelled antibody staining with ABCB1/4/5 antibody; Biii and Ciii – overlay</b>. </p

Phylogenetic analysis of mammalian and zebrafish <i>ABCB</i> genes.

<p>Phylogenetic analysis of the <i>ABCB</i> gene family in human, mouse and zebrafish. The zebrafish genome does not contain an annotated orthologue of mammalian <i>ABCB1</i>. Zebrafish <i>abcb4</i> and <i>abcb5</i> are identified as the closest homologues to mammalian <i>ABCB1, 4</i> and <i>5</i>. </p

Transmission electron microscopy analysis of blood-brain barrier maturation.

<p>Parasaggital sections through the brain of zebrafish larvae at 3 d.p.f. and 10 d.p.f. A) At 3 d.p.f., blood vessels (bv) surrounding the brain are simple in structure. B) At high resolution, only a single membrane is observed (arrows) no evidence of double membranes was observed at any location examined. C) At 10 d.p.f., blood vessels surrounding the CNS are more complex in structure. D) At high resolution, a double layer membrane is apparent (arrows), indicative of the presence of tight junctions. E) In some vessels at 10 d.p.f., pericytes (pc) could be observed surrounding endothelial cells (ec). In addition, astrocyte endfeet (ae) were observed around some vessels at 10 d.p.f. but not at earlier ages. Magnification: A & C 5.2K; B, D & E 15.5K. </p

Immunofluorescent staining by confocal microscopy.

<p>Localization of <i>TUSC1</i> in stable transfectants of CHO and 9HTE cell lines. (A) Stably transfected CHO cells were incubated with V5 or <i>TUSC1</i> antibodies. (B–C) Un-transfected 9HTE and SKEMS-1 lung cancer cells were incubated with <i>TUSC1</i> antibody. Subcellular distributions of <i>TUSC1</i> proteins are cytoplasmic and nuclear. V5 antibody was detected with Texas Red-conjugated anti-mouse IgG (Red) and <i>TUSC1</i> antibody was detected with either Texas Red-conjugated anti-rabbit IgG (Red) or by FITC-conjugated anti-rabbit IgG (Green).</p

Overexpression of <i>TUSC1</i> reduces cell growth and tumorigenicity of lung cancer cells.

<p>(A, B) <i>In vitro</i> cell growth of cancer cell lines (Nu6-1 and H290) transfected with <i>TUSC1</i>. (C, D) Tumor volume of subcutaneously injected stably transfected cancer cells with <i>TUSC1</i> in nude mice <i>in vivo</i>. Restoration of <i>TUSC1</i> expression in cell lines with <i>TUSC1</i> homozygous deletion suppressed cell growth and tumor development compared to the parental cell lines transfected with the empty vector (p<0.05 ).</p

Representative immunohistochemical analysis using the TUSC1 antibody in normal and primary lung tissues.

<p>(A) Bronchioepithelium of Normal Lung. The basal layer was not positive for TUSC1 but differentiated cells in the upper layers of epithelium have high TUSC1 expression. Distribution of TUSC1 staining was cytoplasmic, nuclear and nuclear/cytoplasmic in the cells. (B, C) Lung cancer tissue microarray stained with <i>TUSC1</i> antibody and representative tissue cores (1–3) for each level of staining intensity. (D–F) Kaplan-Meier survival plots for the overall patient (D), adenocarcinoma (E) and squamous cell carcinoma (F). Survival status and the associated <i>P</i> value are indicated. Red line represents tumors with higher expression levels of <i>TUSC1</i> (score: 3), green line represents tumor with lower expression levels of <i>TUSC1</i> (score: 0–2).</p

Expression of <i>TUSC1</i> mRNA and protein in cell lines.

<p>(A–B) The tumor cell lines (H290 and Nu6-1) were stably transfected with the <i>TUSC1</i> gene in pcDNA3.1 vector and CHO cells were transfected with <i>TUSC1</i> in pcDNA3.1/V5-His vector. <i>TUSC1</i> mRNA was amplified (RT-PCR) and exogenous protein (Western blot) was detected in all stably transfected cell lines containing <i>TUSC1</i> expression clones but not the parental cell lines transfected with empty vector. (C) Western blot analysis of endogenous TUSC1 proteins in tumor cell lines. Red box: Cell lines with homozygous deletion of <i>TUSC1</i>; Green box: cell lines with reduced TUSC1 expression but without <i>TUSC1</i> deletion. As an internal control for the amount of protein loaded, the same membrane was incubated with Anti-GAPDH antibody.</p

<i>Salmonella</i><i>typhimurium</i> in the Australian egg industry: Multidisciplinary approach to addressing the public health challenge and future directions

<p>In Australia, numerous egg-related human <i>Salmonella</i> <i>typhimurium</i> outbreaks have prompted significant interest among public health authorities and the egg industry to jointly address this human health concern. Nationwide workshops on <i>Salmonella</i> and eggs were conducted in Australia for egg producers and regulatory authorities. State and national regulators represented Primary Production, Communicable Disease Control, Public Health and Food Safety, and Food Standards Australia and New Zealand. All attendees participated in discussions aimed at evaluating current evidence-based information, issues related to quality of egg production, and how to ensure safe eggs in the supply chain, identifying research gaps and practical recommendations. The perceptions from egg producers and regulatory authorities from various states were recorded during the workshops. We presented the issues discussed during the workshops, including <i>Salmonella</i> in the farm environment, <i>Salmonella</i> penetration across eggshell, virulence in humans, food/egg handling in the supply chain, and intervention strategies. We also discussed the perceptions from egg producers and regulators. Recommendations placed emphasis on the future research needs, communication between industry and regulatory authorities, and education of food handlers. Communication between regulators and industry is pivotal to control egg-borne <i>S. typhimurium</i> outbreaks, and collaborative efforts are required to design effective and appropriate control strategies.</p