induction-micro-ct-imaging-cerebral-cavernous-malformations-mouse

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

University of Sydney

The North American Symptomatic Carotid Endarterectomy Trial (NASCET) confirmed that carotid endarterectomy (CEA) can significantly cut the risk of stroke in patients with moderate and severe blockage. The standard today is that patients who have internal carotid artery stenosis > 70% with associated symptoms and who are appropriate surgical candidates should be offered CEA. Aneurysmal disease, a growing public health concern, poses the threat of death from rupture, and lower extremity arterial occlusive disease poses a significant risk of critical leg ischemia and limb loss. Both conditions involve surgical options. In treating their patients, primary care physicians should become familiar with the benefits and risks of vascular surgery to manage the various aspects of peripheral arterial disease.

Vascular surgery for peripheral arterial disease.

Clinical assessment of the patient with a suspected pulmonary embolism.

Vertically aligned ZnO nanowires were synthesized on a sapphire ([Formula: see text]) substrate by vapour deposition and their light-emitting properties were characterized using photoluminescence and cathodoluminescence spectroscopies. Besides the nanowires, there exists a mosaic ZnO film on the substrate surface. Comparison of the luminescent properties of the as-grown ensemble and the nanowires extracted from it shows that the mosaic film is the major source of the defect-related green emission while the nanowires possess highly crystalline quality with virtually no defects. Photoemission spectroscopy shows that the valence band states associated with O 2p from the as-grown sample are diminished compared with those from the extracted nanowires. These findings suggest that the green emission partly arises from oxygen vacancies located on the surface of the mosaic film.

Luminescent properties of ZnO nanowires and as-grown ensembles.

Ionic liquid-facilitated mobilization and reorganization of biopolymers in natural fibrous materials is visualized by confocal fluorescent spectromicroscopy. Ionic liquid-based processes controllably fuse adjacent fibres while simultaneously leaving selected amounts of biopolymers in their native states. These processes generate congealed materials with extended intermolecular hydrogen bonding networks and enhanced properties.

Fluorescence monitoring of ionic liquid-facilitated biopolymer mobilization and reorganization.

Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive.

Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.

Evolution in fast forward: a potential role for mutators in accelerating Staphylococcus aureus pathoadaptation.

Reducing emergency department utilization: is this the answer?

To demonstrate the novel application of nano X-ray computed tomography (NanoXCT) for visualizing and quantifying the internal structures of pharmaceutical particles.

NanoXCT: A Novel Technique to Probe the Internal Architecture of Pharmaceutical Particles.

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane-associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut.

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale.

The Cretaceous Sanagasta neosauropod nesting site (La Rioja, Argentina) was the first confirmed instance of extinct dinosaurs using geothermal-generated heat to incubate their eggs. The nesting strategy and hydrothermal activities at this site led to the conclusion that the surprisingly 7 mm thick-shelled eggs were adapted to harsh hydrothermal microenvironments. We used micro-CT scans in this study to obtain the first three-dimensional microcharacterization of these eggshells. Micro-CT-based analyses provide a robust assessment of gas conductance in fossil dinosaur eggshells with complex pore canal systems, allowing calculation, for the first time, of the shell conductance through its thickness. This novel approach suggests that the shell conductance could have risen during incubation to seven times more than previously estimated as the eggshell erodes. In addition, micro-CT observations reveal that the constant widening and branching of pore canals form a complex funnel-like pore canal system. Furthermore, the high density of pore canals and the presence of a lateral canal network in the shell reduce the risks of pore obstruction during the extended incubation of these eggs in a relatively highly humid and muddy nesting environment.

Micro-CT scan reveals an unexpected high-volume and interconnected pore network in a Cretaceous Sanagasta dinosaur eggshell.

Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause strokes and seizures in younger individuals. CCMs arise from endothelial cell loss of KRIT1, CCM2 or PDCD10, non-homologous proteins that form an adaptor complex. How disruption of the CCM complex results in disease remains controversial, with numerous signalling pathways (including Rho, SMAD and Wnt/β-catenin) and processes such as endothelial-mesenchymal transition (EndMT) proposed to have causal roles. CCM2 binds to MEKK3 (refs 7, 8, 9, 10, 11), and we have recently shown that CCM complex regulation of MEKK3 is essential during vertebrate heart development. Here we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we show that expression of the MEKK3 target genes Klf2 and Klf4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. By contrast, we find no evidence of EndMT or increased SMAD or Wnt signalling during early CCM formation. Endothelial-specific loss of Map3k3 (also known as Mekk3), Klf2 or Klf4 markedly prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we show that endothelial expression of KLF2 and KLF4 is increased in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates the MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signalling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-uptake system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex.

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes.

Corrigendum: Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

Fungal growth in indoor environments is associated with many negative health effects. Many studies focus on brown- and white-rot fungi and their effect on wood, but there is none that reveals the influence of soft-rot fungi, such as Stachybotrys spp. and Chaetomium spp., on the structure of building materials such as plywood and gypsum wallboard. This study focuses on using micro-computed tomography (microCT) to investigate changes of the structure of plywood and gypsum wallboard during fungal degradation by S. chartarum and C. globosum. Changes in the materials as a result of dampness and fungal growth were determined by measuring porosity and pore shape via microCT. The results show that the composition of the building material influenced the level of penetration by fungi as shown by scanning electron microscopy (SEM). Plywood appeared to be the most affected, with the penetration of moisture and fungi throughout the whole thickness of the sample. Conversely, fungi grew only on the top cardboard in the gypsum wallboard and they did not have significant influence on the gypsum wallboard structure. The majority of the observed changes in gypsum wallboard occurred due to moisture. This paper suggests that the mycelium distribution within building materials and the structural changes, caused by dampness and fungal growth, depend on the type of the material.

Visualization of the structural changes in plywood and gypsum board during the growth of Chaetomium globosum and Stachybotrys chartarum.

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice.

We sought to: (1) explore the feasibility of using email for seasonal influenza vaccination reminders to parents of adolescents and (2) assess influenza vaccination rates among adolescents whose parents were randomized to either receive or not receive email reminders.

Article	Year
Vascular surgery for peripheral arterial disease. Clinical cornerstone\| PubMed ID: 12425183	2002
Clinical assessment of the patient with a suspected pulmonary embolism. Annals of emergency medicine\| PubMed ID: 18584772	2008
Luminescent properties of ZnO nanowires and as-grown ensembles. Nanotechnology\| PubMed ID: 21832651	2008
Fluorescence monitoring of ionic liquid-facilitated biopolymer mobilization and reorganization. Chemical communications (Cambridge, England)\| PubMed ID: 22614442	2012
Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation. Current biology : CB\| PubMed ID: 22704991	2012
Evolution in fast forward: a potential role for mutators in accelerating Staphylococcus aureus pathoadaptation. Journal of bacteriology\| PubMed ID: 23204459	2013
Reducing emergency department utilization: is this the answer? Academic emergency medicine : official journal of the Society for Academic Emergency Medicine\| PubMed ID: 24127711	2013
NanoXCT: A Novel Technique to Probe the Internal Architecture of Pharmaceutical Particles. Pharmaceutical research\| PubMed ID: 24867423	2014
Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale. Molecular microbiology\| PubMed ID: 25388295	2015
Micro-CT scan reveals an unexpected high-volume and interconnected pore network in a Cretaceous Sanagasta dinosaur eggshell. Journal of the Royal Society, Interface\| PubMed ID: 27009182	2016
Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling. Nature\| PubMed ID: 27027284	2016
The Sus operon: a model system for starch uptake by the human gut Bacteroidetes. Cellular and molecular life sciences : CMLS\| PubMed ID: 27137179	2016
Corrigendum: Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling. Nature\| PubMed ID: 27281211	2016
Visualization of the structural changes in plywood and gypsum board during the growth of Chaetomium globosum and Stachybotrys chartarum. Journal of microbiological methods\| PubMed ID: 27476483	2016
Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice. PloS one\| PubMed ID: 27513872	2016
The impacts of email reminder/recall on adolescent influenza vaccination. Vaccine\| PubMed ID: 28455173	2017

Matthew Foley

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