Bio21 - Research Publications

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 52
  • Item
    Thumbnail Image
    Development of Matrix-Embedded Bovine Tracheal Organoids to Study the Innate Immune Response against Bovine Respiratory Disease
    Quah, PS ; Tran, BM ; Corbin, VDA ; Chang, JJ-Y ; Wong, CY ; Diaz-Méndez, A ; Hartley, CA ; Zeng, W ; Hanssen, E ; Trifunovic, Z ; Reading, PC ; Jackson, DC ; Vincan, E ; Coin, LJM ; Deliyannis, G (MDPI, 2023)
    Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle. Bovine herpesvirus-1 (BHV-1) is one of the main culprits of BRD; however, research on BHV-1 is hampered by the lack of suitable models for infection and drug testing. In this study, we established a novel bovine tracheal organoid culture grown in a basement membrane extract type 2 (BME2) matrix and compared it with the air–liquid interface (ALI) culture system. After differentiation, the matrix-embedded organoids developed beating cilia and demonstrated a transcriptomic profile similar to the ALI culture system. The matrix-embedded organoids were also highly susceptible to BHV-1 infection and immune stimulation by Pam2Cys, an immunomodulator, which resulted in robust cytokine production and tracheal antimicrobial peptide mRNA upregulation. However, treatment of bovine tracheal organoid cultures with Pam2Cys was not sufficient to inhibit viral infection or replication, suggesting a role of the non-epithelial cellular microenvironment in vivo.
  • Item
    Thumbnail Image
    Characterising the influence of milk fat towards an application for extrusion-based 3D-printing of casein-whey protein suspensions via the pH-temperature-route
    Daffner, K ; Ong, L ; Hanssen, E ; Gras, S ; Mills, T (ELSEVIER SCI LTD, 2021-09)
    This study presents the design and characterisation of casein−whey protein suspensions (8.0/10.0% (w/w) casein and 2.0/2.5% (w/w) whey protein) mixed with dairy fat (1.0, 2.5 and 5.0% (w/w) total fat) processed via the pH−temperature-route in preparation for 3D-printing. Mechanical treatment was applied to significantly decrease the particle size of the milk fat globules and increase surface area, creating small fat globules (<1 μm) covered with proteins, which could act as pseudo protein particles during gelation. Different proteins covered the fat globule surface after mechanical treatment, as a result of differences in the pH adjusted just prior to heating (6.55, 6.9 or 7.1). The protein-fat suspensions appeared similar by transmission electron cryogenic microscopy and the zeta-potential of all particles was unchanged by the heating pH, with a similar charge to the solution (~−20 mV) occurring after acidification (pH 4.8/5.0) at low temperatures (2 °C). A low heating pH (6.55) resulted in increased sol−gel transition temperatures (G՛ = 1 Pa) and a decreased rate of aggregation for protein−fat suspensions. A higher heating pH (6.9 and 7.1) caused an increased rate of aggregation (aggregation rate ≥ 250 Pa/10 K), resulting in materials more promising for application in extrusion-based printing. 3D-printing of formulations into small rectangles, inclusive of a sol−gel transition in a heated nozzle, was conducted to relate the aggregation rate towards printability.
  • Item
    Thumbnail Image
    Effects of altered cellular ultrastructure on energy metabolism in diabetic cardiomyopathy: an in silico study.
    Ghosh, S ; Guglielmi, G ; Orfanidis, I ; Spill, F ; Hickey, A ; Hanssen, E ; Rajagopal, V (The Royal Society, 2022-11-21)
    Diabetic cardiomyopathy is a leading cause of heart failure in diabetes. At the cellular level, diabetic cardiomyopathy leads to altered mitochondrial energy metabolism and cardiomyocyte ultrastructure. We combined electron microscopy (EM) and computational modelling to understand the impact of diabetes-induced ultrastructural changes on cardiac bioenergetics. We collected transverse micrographs of multiple control and type I diabetic rat cardiomyocytes using EM. Micrographs were converted to finite-element meshes, and bioenergetics was simulated over them using a biophysical model. The simulations also incorporated depressed mitochondrial capacity for oxidative phosphorylation (OXPHOS) and creatine kinase (CK) reactions to simulate diabetes-induced mitochondrial dysfunction. Analysis of micrographs revealed a 14% decline in mitochondrial area fraction in diabetic cardiomyocytes, and an irregular arrangement of mitochondria and myofibrils. Simulations predicted that this irregular arrangement, coupled with the depressed activity of mitochondrial CK enzymes, leads to large spatial variation in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio profile of diabetic cardiomyocytes. However, when spatially averaged, myofibrillar ADP/ATP ratios of a cardiomyocyte do not change with diabetes. Instead, average concentration of inorganic phosphate rises by 40% owing to lower mitochondrial area fraction and dysfunction in OXPHOS. These simulations indicate that a disorganized cellular ultrastructure negatively impacts metabolite transport in diabetic cardiomyopathy. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.
  • Item
    Thumbnail Image
    CardioVinci: building blocks for virtual cardiac cells using deep learning
    Khadangi, A ; Boudier, T ; Hanssen, E ; Rajagopal, V (ROYAL SOC, 2022-11-21)
    Advances in electron microscopy (EM) such as electron tomography and focused ion-beam scanning electron microscopy provide unprecedented, three-dimensional views of cardiac ultrastructures within sample volumes ranging from hundreds of nanometres to hundreds of micrometres. The datasets from these samples are typically large, with file sizes ranging from gigabytes to terabytes and the number of image slices within the three-dimensional stack in the hundreds. A significant bottleneck with these large datasets is the time taken to extract and statistically analyse three-dimensional changes in cardiac ultrastructures. This is because of the inherently low contrast and the significant amount of structural detail that is present in EM images. These datasets often require manual annotation, which needs substantial person-hours and may result in only partial segmentation that makes quantitative analysis of the three-dimensional volumes infeasible. We present CardioVinci, a deep learning workflow to automatically segment and statistically quantify the morphologies and spatial assembly of mitochondria, myofibrils and Z-discs with minimal manual annotation. The workflow encodes a probabilistic model of the three-dimensional cardiomyocyte using a generative adversarial network. This generative model can be used to create new models of cardiomyocyte architecture that reflect variations in morphologies and cell architecture found in EM datasets. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.
  • Item
    Thumbnail Image
    Biochemical Characterization of Caenorhabditis elegans Ferritins
    Mubarak, SSM ; Malcolm, TR ; Brown, HG ; Hanssen, E ; Maher, MJ ; McColl, G ; Jameson, GNL (AMER CHEMICAL SOC, 2023-05-02)
    The nematode Caenorhabditis elegans contains genes for two types of ferritin (ftn-1 and ftn-2) that express FTN-1 and FTN-2. We have expressed and purified both proteins and characterized them by X-ray crystallography, cryo-electron microscopy, transmission electron microscopy, dynamic light scattering, and kinetically by oxygen electrode and UV-vis spectroscopy. Both show ferroxidase activity, but although they have identical ferroxidase active sites, FTN-2 is shown to react approximately 10 times faster than FTN-1, with L-type ferritin character over longer time periods. We hypothesize that the large variation in rate may be due to differences in the three- and four-fold channels into the interior of the protein 24-mer. FTN-2 is shown to have a wider entrance into the three-fold channel than FTN-1. Additionally, the charge gradient through the channel of FTN-2 is more pronounced, with Asn and Gln residues in FTN-1 replaced by Asp and Glu residues in FTN-2. Both FTN-1 and FTN-2 have an Asn residue near the ferroxidase active site that is a Val in most other species, including human H ferritin. This Asn residue has been observed before in ferritin from the marine pennate diatom Pseudo-mitzchia multiseries. By replacing this Asn residue with a Val in FTN-2, we show that the reactivity decreases over long time scales. We therefore propose that Asn106 is involved in iron transport from the ferroxidase active site to the central cavity of the protein.
  • Item
    Thumbnail Image
    Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte
    Dasgupta, S ; Auth, T ; Gov, NS ; Satchwell, TJ ; Hanssen, E ; Zuccala, ES ; Riglar, DT ; Toye, AM ; Betz, T ; Baum, J ; Gompper, G (CELL PRESS, 2014-07-01)
    The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.
  • Item
    Thumbnail Image
    Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes
    Li, J ; Shami, GJ ; Cho, E ; Liu, B ; Hanssen, E ; Dixon, MWA ; Tilley, L (NATURE PORTFOLIO, 2022-08-27)
    The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and -4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.
  • Item
    Thumbnail Image
    MeasureIce: accessible on-the-fly measurement of ice thickness in cryo-electron microscopy
    Brown, HG ; Hanssen, E (NATURE PORTFOLIO, 2022-08-15)
    Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo-electron microscopy (cryo-EM). The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some additional noise in cryo-EM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables, also usable in SerialEM and Leginon, using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map.
  • Item
    No Preview Available
    Antifungal versus antibacterial defence of insect wings
    Ivanova, EP ; Linklater, DP ; Aburto-Medina, A ; Phuc, L ; Baulin, VA ; Huu, KDN ; Curtain, R ; Hanssen, E ; Gervinskas, G ; Ng, SH ; Vi, KT ; Luque, P ; Ramm, G ; Wosten, HAB ; Crawford, RJ ; Juodkazis, S ; Maclaughlin, S (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2021-12)
    HYPOTHESIS: The ability exhibited by insect wings to resist microbial infestation is a unique feature developed over 400 million years of evolution in response to lifestyle and environmental pressures. The self-cleaning and antimicrobial properties of insect wings may be attributed to the unique combination of nanoscale structures found on the wing surface. EXPERIMENTS: In this study, we characterised the wetting characteristics of superhydrophobic damselfly Calopteryx haemorrhoidalis wings. We revealed the details of air entrapment at the micro- and nano scales on damselfly wing surfaces using a combination of spectroscopic and electron microscopic techniques. Cryo-focused-ion-beam scanning electron microscopy was used to directly observe fungal spores and conidia that were unable to cross the air-liquid interface. By contrast, bacterial cells were able to cross the air-water interface to be ruptured upon attachment to the nanopillar surface. The robustness of the air entrapment, and thus the wing antifungal behaviour, was demonstrated after 1-week of water immersion. A newly developed wetting model confirmed the strict Cassie-Baxter wetting regime when damselfly wings are immersed in water. FINDINGS: We provide evidence that the surface nanopillar topography serves to resist both fungal and bacterial attachment via a dual action: repulsion of fungal conidia while simultaneously killing bacterial cells upon direct contact. These findings will play an important role in guiding the fabrication of biomimetic, anti-fouling surfaces that exhibit both bactericidal and anti-fungal properties.
  • Item
    Thumbnail Image
    Extracellular Vesicles Secreted by Glioma Stem Cells Are Involved in Radiation Resistance and Glioma Progression
    Ma, C ; Nguyen, HPT ; Jones, JJ ; Stylli, SS ; Whitehead, CA ; Paradiso, L ; Luwor, RB ; Areeb, Z ; Hanssen, E ; Cho, E ; Putz, U ; Kaye, AH ; Morokoff, AP (MDPI, 2022-03)
    Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs' specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.