TULIPs decorate the three-dimensional genome of PFA ependymoma.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs.

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights: The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions.The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs.Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture.The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

Linked-read based analysis of the medulloblastoma genome.

Introduction: Medulloblastoma is the most common type of malignant pediatric brain tumor with group 4 medulloblastomas (G4 MBs) accounting for 40% of cases. However, the molecular mechanisms that underlie this subgroup are still poorly understood. Point mutations are detected in a large number of genes at low incidence per gene while the detection of complex structural variants in recurrently affected genes typically requires the application of long-read technologies. Methods: Here, we applied linked-read sequencing, which combines the long-range genome information of long-read sequencing with the high base pair accuracy of short read sequencing and very low sample input requirements. Results: We demonstrate the detection of complex structural variants and point mutations in these tumors, and, for the first time, the detection of extrachromosomal DNA (ecDNA) with linked-reads. We provide further evidence for the high heterogeneity of somatic mutations in G4 MBs and add new complex events associated with it. Discussion: We detected several enhancer-hijacking events, an ecDNA containing the MYCN gene, and rare structural rearrangements, such a chromothripsis in a G4 medulloblastoma, chromoplexy involving 8 different chromosomes, a TERT gene rearrangement, and a PRDM6 duplication.

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses.

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in situ hybridization probes are used to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof of principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell.

BACKGROUND: Extracellular vesicles (EVs) produced by human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are currently investigated for their clinical effectiveness towards immune-mediated diseases. The large amounts of stem cell-derived EVs required for clinical testing suggest that bioreactor production systems may be a more amenable alternative than conventional EV production methods for manufacturing products for therapeutic use in humans. METHODS: To characterize the potential utility of these systems, EVs from four hBM-MSC donors were produced independently using a hollow-fiber bioreactor system under a cGMP-compliant procedure. EVs were harvested and characterized for size, concentration, immunophenotype, and glycan profile at three separate intervals throughout a 25-day period. RESULTS: Bioreactor-inoculated hBM-MSCs maintained high viability and retained their trilineage mesoderm differentiation capability while still expressing MSC-associated markers upon retrieval. EVs collected from the four hBM-MSC donors showed consistency in size and concentration in addition to presenting a consistent surface glycan profile. EV surface immunophenotypic analyses revealed a consistent low immunogenicity profile in addition to the presence of immuno-regulatory CD40 antigen. EV cargo analysis for biomarkers of immune regulation showed a high abundance of immuno-regulatory and angiogenic factors VEGF-A and IL-8. CONCLUSIONS: Significantly, EVs from hBM-MSCs with immuno-regulatory constituents were generated in a large-scale system over a long production period and could be frequently harvested with the same quality and quantity, which will circumvent the challenge for clinical application.

DNA Based Vaccine Expressing SARS-CoV-2 Spike-CD40L Fusion Protein Confers Protection Against Challenge in a Syrian Hamster Model.

SARS-CoV-2 infections present a tremendous threat to public health. Safe and efficacious vaccines are the most effective means in preventing the infections. A variety of vaccines have demonstrated excellent efficacy and safety around the globe. Yet, development of alternative forms of vaccines remains beneficial, particularly those with simpler production processes, less stringent storage conditions, and the capability of being used in heterologous prime/boost regimens which have shown improved efficacy against many diseases. Here we reported a novel DNA vaccine comprised of the SARS-CoV-2 spike protein fused with CD40 ligand (CD40L) serving as both a targeting ligand and molecular adjuvant. A single intramuscular injection in Syrian hamsters induced significant neutralizing antibodies 3-weeks after vaccination, with a boost substantially improving immune responses. Moreover, the vaccine also reduced weight loss and suppressed viral replication in the lungs and nasal turbinates of challenged animals. Finally, the incorporation of CD40L into the DNA vaccine was shown to reduce lung pathology more effectively than the DNA vaccine devoid of CD40L. These results collectively indicate that this DNA vaccine candidate could be further explored because of its efficacy and known safety profile.

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles.

BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. METHODS: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. RESULTS: Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. CONCLUSION: The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma.

We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.

Intratumoral Genetic and Functional Heterogeneity in Pediatric Glioblastoma.

Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. To understand the mechanisms of tumor evolution in this cancer, we performed whole-genome sequencing with linked reads on longitudinally resected pGBM samples. Our analyses showed that all diagnostic and recurrent samples were collections of genetically diverse subclones. Clonal composition rapidly evolved at recurrence, with less than 8% of nonsynonymous single-nucleotide variants being shared in diagnostic-recurrent pairs. To track the origins of the mutational events observed in pGBM, we generated whole-genome datasets for two patients and their parents. These trios showed that genetic variants could be (i) somatic, (ii) inherited from a healthy parent, or (iii) de novo in the germlines of pGBM patients. Analysis of variant allele frequencies supported a model of tumor growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to nondividing cells. Interestingly, radiation and antimitotic chemotherapeutics did not increase overall tumor burden upon recurrence. These findings support an important role for slow-cycling stem cell populations in contributing to recurrences, because slow-cycling cell populations are expected to be less prone to genotoxic stress induced by these treatments and therefore would accumulate few mutations. Our results highlight the need for new targeted treatments that account for the complex functional hierarchies and genomic heterogeneity of pGBM. SIGNIFICANCE: This work challenges several assumptions regarding the genetic organization of pediatric GBM and highlights mutagenic programs that start during early prenatal development.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2111/F1.large.jpg.

Endoscopic balloon dilatation of Crohn's strictures: a safe method to defer surgery in selective cases.

BACKGROUND: Endoscopic balloon dilatation (EBD) provides a valuable alternative to surgery for strictures in Crohn's disease (CD). Data are lacking regarding the factors that improve the safety and effectiveness of EBD in CD. The aim of this study is to determine the safety and efficacy of EBD and the clinical variables, which are predictive of successful treatment of CD strictures with EBD. METHODS: The records of all patients with CD in whom EBD was attempted between 2008 and 2013 were reviewed. Procedures were conducted at a single tertiary referral centre using a Boston Scientific CRE® TTS balloon. Technical success was defined as the ability to traverse the stricture with the endoscope and clinical success as the resolution of obstructive symptoms at review. RESULTS: Forty-seven patients with a total of 58 strictures (19 primary and 39 anastomotic strictures) were treated with EBD with median follow-up of 37 months. A total of 161 dilatation procedures were performed, with technical success reported in 139/158 (88%) cases and clinical success reported in 105/137 (76.7%) cases with complete data. Complications occurred in 7/161 dilatations (4.3% dilatations, 15% patients), three patients with perforation, one with acute bleeding and three admitted with abdominal pain. Eighteen of the 47 patients required surgery (38%). Strictures of <50 mm (P = 0.04) and those dilated to a diameter of ≥15 mm (P = 0.031) were less likely to require surgical resection. CONCLUSIONS: EBD is safe for both primary and post-surgical strictures. Stricture length and diameter of dilatation are predictive of success. In selected patients, treatment with EBD may reduce or delay the need for surgery.

Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche.

In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. We have found that the phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period.

Effect of intestinal resection on quality of life in Crohn's disease.

INTRODUCTION: Patients with Crohn's disease have poorer health-related quality of life [HRQoL] than healthy individuals, even when in remission. Although HRQoL improves in patients who achieve drug-induced or surgically induced remission, the effects of surgery overall have not been well characterised. METHODS: In a randomised trial, patients undergoing intestinal resection of all macroscopically diseased bowel were treated with postoperative drug therapy to prevent disease recurrence. All patients were followed prospectively for 18 months. C-reactive protein [CRP], Crohn's Disease Activity Index [CDAI], and faecal calprotectin [FC] were measured preoperatively and at 6, 12, and 18 months. HRQoL was assessed with a general [SF36] and disease-specific [IBDQ] questionnaires at the same time points. RESULTS: A total of 174 patients were included. HRQoL was poor preoperatively but improved significantly [p < 0.001] at 6 months postoperatively. This improvement was sustained at 18 months. Females and smokers had a poorer HRQoL when compared with males and non-smokers, respectively. Persistent endoscopic remission, intensification of drug treatment at 6 months, and anti-tumour necrosis factor therapy were not associated with HRQoL outcomes different from those when these factors were not present. There was a significant inverse correlation between CDAI, [but not endoscopic recurrence, CRP, or FC] on HRQoL. CONCLUSION: Intestinal resection of all macroscopic Crohn's disease in patients treated with postoperative prophylactic drug therapy is associated with significant and sustained improvement in HRQoL irrespective of type of drug treatment or endoscopic recurrence. HRQoL is lower in female patients and smokers. A higher CDAI, but not direct measures of active disease or type of drug therapy, is associated with a lower HRQoL.

Measurement of fecal calprotectin improves monitoring and detection of recurrence of Crohn's disease after surgery.

BACKGROUND & AIMS: Crohn's disease (CD) usually recurs after intestinal resection; postoperative endoscopic monitoring and tailored treatment can reduce the chance of recurrence. We investigated whether monitoring levels of fecal calprotectin (FC) can substitute for endoscopic analysis of the mucosa. METHODS: We analyzed data collected from 135 participants in a prospective, randomized, controlled trial, performed at 17 hospitals in Australia and 1 hospital in New Zealand, that assessed the ability of endoscopic evaluations and step-up treatment to prevent CD recurrence after surgery. Levels of FC, serum levels of C-reactive protein (CRP), and Crohn's disease activity index (CDAI) scores were measured before surgery and then at 6, 12, and 18 months after resection of all macroscopic Crohn's disease. Ileocolonoscopies were performed at 6 months after surgery in 90 patients and at 18 months after surgery in all patients. RESULTS: Levels of FC were measured in 319 samples from 135 patients. The median FC level decreased from 1347 µg/g before surgery to 166 µg/g at 6 months after surgery, but was higher in patients with disease recurrence (based on endoscopic analysis; Rutgeerts score, ≥i2) than in patients in remission (275 vs 72 µg/g, respectively; P < .001). Combined 6- and 18-month levels of FC correlated with the presence (r = 0.42; P < .001) and severity (r = 0.44; P < .001) of CD recurrence, but the CRP level and CDAI score did not. Levels of FC greater than 100 µg/g indicated endoscopic recurrence with 89% sensitivity and 58% specificity, and a negative predictive value (NPV) of 91%; this means that colonoscopy could have been avoided in 47% of patients. Six months after surgery, FC levels less than 51 µg/g in patients in endoscopic remission predicted maintenance of remission (NPV, 79%). In patients with endoscopic recurrence at 6 months who stepped-up treatment, FC levels decreased from 324 µg/g at 6 months to 180 µg/g at 12 months and 109 µg/g at 18 months. CONCLUSIONS: In this analysis of data from a prospective clinical trial, FC measurement has sufficient sensitivity and NPV values to monitor for CD recurrence after intestinal resection. Its predictive value might be used to identify patients most likely to relapse. After treatment for recurrence, the FC level can be used to monitor response to treatment. It predicts which patients will have disease recurrence with greater accuracy than CRP level or CDAI score.

Crohn's disease management after intestinal resection: a randomised trial.

BACKGROUND: Most patients with Crohn's disease need an intestinal resection, but a majority will subsequently experience disease recurrence and require further surgery. This study aimed to identify the optimal strategy to prevent postoperative disease recurrence. METHODS: In this randomised trial, consecutive patients from 17 centres in Australia and New Zealand undergoing intestinal resection of all macroscopic Crohn's disease, with an endoscopically accessible anastomosis, received 3 months of metronidazole therapy. Patients at high risk of recurrence also received a thiopurine, or adalimumab if they were intolerant to thiopurines. Patients were randomly assigned to parallel groups: colonoscopy at 6 months (active care) or no colonoscopy (standard care). We used computer-generated block randomisation to allocate patients in each centre to active or standard care in a 2:1 ratio. For endoscopic recurrence (Rutgeerts score ≥i2) at 6 months, patients stepped-up to thiopurine, fortnightly adalimumab with thiopurine, or weekly adalimumab. The primary endpoint was endoscopic recurrence at 18 months. Patients and treating physicians were aware of the patient's study group and treatment, but central reading of the endoscopic findings was undertaken blind to the study group and treatment. Analysis included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00989560. FINDINGS: Between Oct 13, 2009, and Sept 28, 2011, 174 (83% high risk across both active and standard care groups) patients were enrolled and received at least one dose of study drug. Of 122 patients in the active care group, 47 (39%) stepped-up treatment. At 18 months, endoscopic recurrence occurred in 60 (49%) patients in the active care group and 35 (67%) patients in the standard care group (p=0.03). Complete mucosal normality was maintained in 27 (22%) of 122 patients in the active care group versus four (8%) in the standard care group (p=0.03). In the active care arm, of those with 6 months recurrence who stepped up treatment, 18 (38%) of 47 patients were in remission 12 months later; conversely, of those in remission at 6 months who did not change therapy recurrence occurred in 31 (41%) of 75 patients 12 months later. Smoking (odds ratio [OR] 2.4, 95% CI 1.2-4.8, p=0.02) and the presence of two or more clinical risk factors including smoking (OR 2.8, 95% CI 1.01-7.7, p=0.05) increased the risk of endoscopic recurrence. The incidence and type of adverse and severe adverse events did not differ significantly between patients in the active care and standard care groups (100 [82%] of 122 vs 45 [87%] of 52; p=0.51) and (33 [27%] of 122 vs 18 [35%] of 52; p=0.36), respectively. INTERPRETATION: Treatment according to clinical risk of recurrence, with early colonoscopy and treatment step-up for recurrence, is better than conventional drug therapy alone for prevention of postoperative Crohn's disease recurrence. Selective immune suppression, adjusted for early recurrence, rather than routine use, leads to disease control in most patients. Clinical risk factors predict recurrence, but patients at low risk also need monitoring. Early remission does not preclude the need for ongoing monitoring. FUNDING: AbbVie, Gutsy Group, Gandel Philanthropy, Angior Foundation, Crohn's Colitis Australia, and the National Health and Medical Research Council.