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Higher order structural characterisation of 2’-O-methyl phosphorothioate linkage modification of RNA oligonucleotides
Tabassum Khair Barbhuiya(1,2), Mark Fisher(2,3), Serene El-Kamand(4), Roland Gamsjaeger(4), Sam Beard(2,3), Laura Croft(2,3), Derek Richard(2,3) & Neha S. Gandhi(1,2)
Centre for Genomics and Personalised Health, School of Chemistry and Physics, Faculty of Science, Queensland University of Technology, Brisbane, QLD 4000, Australia
Cancer and Ageing Research Program, Translational Research Institute (TRI), Woolloongabba, QLD 4102, Australia
Centre for Genomics and Personalised Health, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, QLD, Australia
School of Science, Western Sydney University, Locked bag 1797, Penrith, NSW 2751, Australia
The RNA oligonucleotides-based therapeutics have rapidly evolved as one of the alternate treatment modalities to small molecules for a wide range of diseases. Their capabilities to modulate several biological targets have been widely explored as personalised therapeutic regime for cancer treatments. However, the instability of the native oligonucleotides owing to degradation in biological systems, have led to incorporation of a variety of chemical modifications within the backbone and of the bases, in order to improve stability and pharmacokinetic properties [1, 2]. We performed a cytotoxicity screen in cancer cells using an RNA oligonucleotide library of 300 phosphorothioate (PS) and 2’-O-Methyl (2’-O-Me) modified oligonucleotides and identified a subset of oligonucleotides with cytotoxic properties. Using circular dichroism (CD) spectropolarimeter and thermal melting analysis we investigated the effect of these modifications on the solution structure of the lead cytotoxic oligonucleotide and compared to the unmodified as well as to a non-cytotoxic sequence. The CD spectra of these RNA oligonucleotides identified the formation of hairpin confirmation in unmodified, cytotoxic sequence. The 2’-O-Me and PS modified oligonucleotide retained the hairpin characteristic, however, it also incurred an intermediate state between hairpin and B-form RNA structure. The thermal analyses showed a reduction in stability of the 2’-O-Me- and PS-modified oligonucleotide, compared to unmodified sequence. Overall, these results suggest that 2’-O-Me- and PS- modifications lead to the formation of less stable hairpin structure while also forming other structures. These structural changes may have some biological implications, in particular structure dependent binding to the target protein, human single-stranded DNA binding protein 1 (hSSB1).
1. MacLeod AR, Crooke ST: RNA therapeutics in oncology: advances, challenges, and future directions. The Journal of Clinical Pharmacology 2017, 57:S43-S59.
2. Corey DR: Chemical modification: the key to clinical application of RNA interference? The Journal of clinical investigation 2007, 117(12):3615-3622.
DNA Sequencing using functionalised graphene nanopore
Mohammad M. Mohammadi(1), Omid Bavi(1) & Yousef Jamali(2)
1. Department of Mechanical and Aerospace Engineering, Shiraz University of Technology, Shiraz, 71557-13876, Iran.
2. Biomathematics Laboratory, Department of Applied Mathematics, School of Mathematical Science, Tarbiat Modares University, Tehran, Iran
Solid-state nanopores have been given tremendous attention for DNA sequencing [1, 2]. Among various materials used as a solid-state nanopore, graphene is a well-studied one. . In this study, with the aid of constant velocity steered molecular dynamic, a single stranded DNA (ssDNA) is pulled across a nanopore in graphene. The carbon atoms of the rim are functionalized using hydrogen and hydroxyl groups. Pulling force and base orientation during translocation are tracked to verify whether a distinction between bases is possible or not. For the hydrogenated pore, guanine has the maximum pulling force, while for the hydroxylated pore thymine has the maximum pulling force. Studying the base orientation during translocation reveals that thymine rotates its base plane about 110 degrees which is the most probable among other bases and in the hydroxylated pore the most probable orientation belongs to cytosine that rotates its base plane about 80 degrees. It could be concluded that guanine, cytosine, and thymine could be distinguished, but adenine remains indistinguishable.
1. Mohammadi, M.M. and O. Bavi, DNA Sequencing: An overview of solid-state and biological nanopore-based methods Biophysical Reviews, 2021. 14: p. 1-5.
2. Zeng, X., et al., Nanopore Technology for the Application of Protein Detection. Nanomaterials, 2021. 11(8): p. 1942.
3. Heerema, S.J. and C. Dekker, Graphene nanodevices for DNA sequencing. Nature nanotechnology, 2016. 11(2): p. 127-136.
Interactive analysis of biomolecular motions using dynamics network analysis
Po-chia Chen(1), Miro Astore(1) & Serdar Kuyucak(1)
1. School of Physics, University of Sydney, NSW, Australia 2006
Molecular dynamics (MD) trajectories contain essential information about the structure-function relationships of biomolecules. The main process of interpreting MD data involves reducing high dimensional atomistic data into human-intelligible metrics, which can then be used to pose explanations for relevant biological questions, such as mechanisms underlying Cystic Fibrosis pathogenesis.
Graph theory is one such means of interpreting biomolecular motion. By coarse-graining trajectory data at the residue and domain levels, a simplified network of nodes and neighbourhood information can be constructed. Metrics based on this network thus report on relationships between functional subunits, mappable to biophysical notions such as allostery and fold stability. For example, notions of betweenness between high-correlated nodes suggest likely allosteric pathways, whereas clustering coefficients represent tightness of local packing.
This technically-inclined presentation discusses applications of basic graph theory to protein networks, based on generalised correlation coefficients and the Dynamic Network Analysis workflow by Melo et al. (2020). The above notions will be illustrated in model systems such as periplasmic ligand-binding proteins and the biologically-relevant Cystic Fibrosis Transmembrane-conductance Regulator.
Melo, M. C. R.; Bernardi, R. C.; de la Fuente-Nunez, C.; Luthey-Schulten, Z. Generalized Correlation-Based Dynamical Network Analysis: A New High-Performance Approach for Identifying Allosteric Communications in Molecular Dynamics Trajectories. J. Chem. Phys. 2020, 153 (13), 134104. https://doi.org/10.1063/5.0018980.
The electrostatic switch mechanism of peripheral and integral membrane protein regulation
Molly Carter(1), Matthew Fakhoury(1), Nicole Lin(1), Rachel Luo(1), Lachlan J. E. Robertson(1), Julia M. Spiker(1), Chi Him Wong(1), Sariena Ye(1), Shelley Young(1), Zack Zuccolotto(1) & Ron J. Clarke(1)
1. School of Chemistry, The University of Sydney, Sydney NSW 2006
In recent years it has become increasingly clear that the lipid membrane surrounding membrane proteins does not merely serve a structural role, providing the support to which proteins are either attached to or embedded in, but instead plays an active role in determining membrane protein activity. As the words membrane protein imply, it is impossible to consider the function of the protein without the membrane. One clear example of how the protein and the membrane are intertwined is the electrostatic switch mechanism. This mechanism involves an electrostatic interaction between clusters of positively charged basic residues (lysine or arginine), usually at the protein’s N- or C-terminus, and negatively charged lipid headgroups (predominantly phosphatidylserine) on the cytoplasmic surface of the plasma membrane. This interaction, which plays an important role in the trafficking of peripheral membrane proteins and potentially in the regulation of integral membrane proteins (Clarke et al, 2020), can be switched off in two possible ways. One is by a decrease in positive charge on the protein segment involved in the interaction, which can occur via phosphorylation of conserved serine residues by protein kinase C. The other way is by a reduction in the negative surface charge density of the membrane, which would occur via the transport of phosphatidylserine to the extracellular leaflet of the membrane by a membrane-bound scramblase. The second way is particularly relevant when a cell is in the process of undergoing apoptosis.
The purpose of this talk is to describe in detail the electrostatic switch mechanism and to demonstrate examples amongst different peripheral and integral membrane proteins where it is operative.
Clarke, R. J., K. R. Hossain and K. Cao, Physiological roles of transverse lipid asymmetry of animal membranes, Biochim. Biophys. Acta – Biomembr. 1862 (2020) 183382.
Jasleen Kaur Daljit Singh
Lipid-interacting switchable DNA origami nanostructures: Minimising aggregation and maximising binding
Jasleen Kaur Daljit Singh(1,2,3), Es Darley(4) , Pietro Ridone(4) , James P Gaston(4) , Ali Abbas(2,3), Matthew AB Baker(4,6) & Shelley FJ Wickham(1,3,5)
1. School of Chemistry, The University of Sydney
2. School of Chemical and Biomolecular Engineering, The University of Sydney
3. The University of Sydney Nano Institute
4. School of Biotechnology and Biomolecular Sciences, University of New South Wales
5. School of Physics, The University of Sydney 6 CSIRO Synthetic Biology Future Science Platform
Lipid-integrated dynamic DNA nanotechnology(1) has been implemented to achieve diverse functions, such as biosensing, cell surface engineering, membrane shaping, and as transmembrane nanopore. Modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, the optimal conditions to facilitate stable, high-yield DNA-lipid binding while allowing for controlled switching are not known. At the same time, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and inhibition of membrane-interaction(2).
Here, a systematic characterisation of the number, position and geometry of cholesterol attachment sites on a DNA nanostructure is conducted to improve its lipid-binding efficiency(3). It is shown that 4 – 8 cholesterol modifications are optimal, while edge positions and longer spacers increase the yield of lipid-binding. Furthermore, DNA strand displacement is shown to achieve controlled removal of DNA nanostructures from membranes, but is inhibited by overhang domains, which are used to prevent cholesterol aggregation. Cholesterol-induced aggregation of DNA origami nanostructures was shown to be minimised by reducing the number of cholesterols from 6 to 4, optimising the cholesterol configuration, decreasing spacer length, and using protective singlestranded DNA 10T overhangs.
These findings provide fundamental guidelines to minimising cholesterol-induced aggregation, improving yield of well-formed structures, while at the same time maximising DNA-lipid binding. This paves the way for achieving dynamic control of complex membrane-interacting DNA nanostructures with potential applications in nanomedicine and biophysics.
1. Darley, E.; Singh, J. K. D.; Surace, N. A.; Wickham, S. F. J.; Baker, M. A. B. The Fusion of Lipid and DNA Nanotechnology. Genes 2019, 10 (12), 1001. https://doi.org/10.3390/genes10121001.
2. Chidchob, P.; Offenbartl-Stiegert, D.; McCarthy, D.; Luo, X.; Li, J.; Howorka, S.; Sleiman, H. F. Spatial Presentation of Cholesterol Units on a DNA Cube as a Determinant of Membrane Protein-Mimicking Functions. J. Am. Chem. Soc. 2019, 141 (2), 1100–1108. https://doi.org/10.1021/jacs.8b11898.
3. Singh, J. K. D.; Darley, E.; Ridone, P.; Gaston, J. P.; Abbas, A.; Wickham, S. F. J.; Baker, M. A. B. Binding of DNA Origami to Lipids: Maximizing Yield and Switching via Strand Displacement. Nucleic Acids Res. 2021, No. gkab888. https://doi.org/10.1093/nar/gkab888.
Looking beyond fluorescence intensity: Lifetime estimations to study aquatic microbial interactions
Pranali Deore(1), Elizabeth Hinde(2), Madeleine van Oppen(1,3) & Linda Blackall(1)
1. School of BioSciences, The University of Melbourne, Parkville, VIC 3010
2. School of Physics, The University of Melbourne, Parkville, VIC 3010
3. Australian Institute of Marine Science, Townsville, QLD 4810, Australia
Diboflagellate microalgae in the family Symbiodiniaceae (5-8 µm) are important microbial symbionts of corals. They provide fixed carbon to their host in exchange of ammonia-nitrogen and other essential nutrients. Cultured free-living Symbiodinaceae are reported to harbour bacteria (~1 µm) such as Labrenzia and Marinobacter sp. in intra- and immediate extra-cellular spaces(1). The interaction among Symbiodiniaceae and bacteria is poorly studied, yet these symbioses are an important piece of the puzzle in understanding climate change impacts on coral reefs. Nucleic acid labelling techniques with fluorophores such as fluorescence in situ hybridization (FISH) are widely explored to study spatial and temporal dynamics of microbial interactions. However, traditional fluorescent intensity mapping of FISH labels is particularly difficult in microbes containing multiple intrinsic fluorescent pigments such as chlorophyll a, c2, pheophytin a. These pigments absorb photo-energy in the visible and far-red region of the light spectrum which is re-emitted as fluorescence commonly referred to as autofluorescence.
Symbiodiniaceae widely absorb energy in the visible spectrum (400–580 and 550–700 nm)(2) which leads to high background autofluorescence. The spectral overlap due to wide absorption and emission wavelengths limit the choice of FISH labels to study the spatial dynamics of the symbiotic microbes. Therefore, we exploit the lifetime property of fluorescence, i.e. the length of time a molecule remains in an excited state prior to returning to the ground state, to visualise and resolve the presence of bacteria in intra- and extra-cellular spaces of Symbiodiniaceae. For example, the chlorophyll a autofluorescence (λex/em = 675/697 nm) spectrally overlaps with ATTO 647N dye (λex/em = 646/664 nm). However, their lifetimes (τ) are distinct, 0.25 (3) and 1.8 ns (4), respectively, which will improve resolution and spatial tracking of Symbiodiniaceae and FISH-labelled bacteria.
1. Maire, J., et al., Intracellular bacteria are common and taxonomically diverse in cultured and in hospite algal endosymbionts of coral reefs. The ISME journal, 2021. 15: p. 2028–2042.
2. Wangpraseurt, D., et al., Spectral effects on Symbiodinium photobiology studied with a programmable light engine. PloS One, 2014. 9(11): p. e112809.
3. Kristoffersen, A.S., et al., Chlorophyll a and NADPH fluorescence lifetimes in the microalgae Haematococcus pluvialis (chlorophyceae) under normal and astaxanthin-accumulating conditions. Applied Spectroscopy, 2012. 66(10): p. 1216-1225.
4. Bückers, J., et al., Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses. Optics Express, 2011. 19(4): p. 3130-3143.
Optimising co-localisation analysis for fluorescent imaging of liposomes and DNA nanotechnology
James P. Gaston(1), Pietro Ridone(1), Jasleen Kaur Daljit Singh(2, 3, 4), Shelley F.J. Wickham(2, 4, 5), & Matthew A.B. Baker(1, 6)
1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
2. School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia
3. School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, New South Wales, Australia
4. The University of Sydney Nano Institute, The University of Sydney, Sydney, New South Wales, Australia
5. School of Physics, The University of Sydney, Sydney, New South Wales, Australia
6. CSIRO Synthetic Biology Future Science Platform, Brisbane, Australia
Membrane bilayer structures are vital for controlling the compartmentalisation of cells and their contents within biological systems. Modified liposome structures capable of recognising and binding to surface ligands can be designed by incorporating membrane-bound DNA nanostructures (Singh et al., 2021) (Darley et al., 2019). These DNA-liposomes have the potential to build interconnected synthetic ex vivo systems that can demonstrate directed membrane fusion, act as a model for cellular communication and tissue build-up, and can build responsive vesicle networks that dynamically interact with their environment.
To quantify DNA-liposome systems, we optimised the measurement of colocalisation between differently labelled fluorescent DNA-liposomes to determine the best method for quantifying broad-scale spatial interactions. We used fluorescence microscopy to distinguish purposefully colocalised and non-colocalised scenarios. We then show an optimised object-based method of selecting and measuring liposome colocalisation, with this method being compared to other established pixel-intensity based modes of analysis. We found that our method was comparable to existing methods such as the Pearson’s correlation coefficient (Manders et al., 1992), Mander’s overlap coefficient (Manders, Verbeek and Aten, 1993), and the Costes autocorrelation method (Costes et al., 2004). Furthermore, our method was better at specifically selecting liposomes and measuring spatial overlap. We found that our set of control samples behaved predictably, establishing a good baseline for future work measuring complementary binding interactions driven by DNA.
Costes, S.V. et al. (2004) ‘Automatic and quantitative measurement of protein-protein colocalization in live cells’, Biophysical Journal, 86(6), pp. 3993–4003. doi:10.1529/biophysj.103.038422.
Darley, E. et al. (2019) ‘The Fusion of Lipid and DNA Nanotechnology’, Genes, 10(12), p. 1001. doi:10.3390/genes10121001.
Manders, E. et al. (1992) ‘Dynamics of three dimensional replication patterns during the S-phase, analyzed by double labeling of DNA and confocal microscopy’, Journal of cell science, 103 ( Pt 3), pp. 857–62. doi:10.1242/jcs.103.3.857.
Manders, E.M.M., Verbeek, F.J. and Aten, J.A. (1993) ‘Measurement of co-localization of objects in dual-colour confocal images’, Journal of Microscopy, 169(3), pp. 375–382. doi:10.1111/j.1365-2818.1993.tb03313.x.
Singh, J.K.D. et al. (2021) ‘Binding of DNA origami to lipids: maximizing yield and switching via strand displacement’, Nucleic Acids Research [Preprint], (gkab888). doi:10.1093/nar/gkab888.
A mathematical model for axonal cargo transport
Nizhum Rahman & Dietmar Oelz
School of Mathematics and Physics, The University of Queensland
Axonal transport is the process by which cargo is delivered by motor proteins through the axon of neuron cells. Actin plays a crucial role in this phenomenon through perpendicular actomyosin rings which are wrapped around the circumference of axons. Some cargo vesicles are larger in diameter than the axon so the axon and with it actomyosin rings are locally dilate as the cargo vesicle moves. We construct a mathematical model to describe the resulting deformation of the axon. The model is reminiscent of the classical obstacle problem. It allows to relate the cargo velocity to the mechanical properties of the actomyosin rings and to explain the observation that the speed of axonal cargoes is inversely correlated with their size.
In-cell DNP NMR reveals multiple targeting effect of the antimicrobial peptide maculatin 1.1
Frances Separovic(1), Vinzenz Hofferek(2), Anthony Duff(3), Malcom J. McConville(2) & Marc-Antoine Sani(1)
1. School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC 3010, Australia
2. Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Melbourne, VIC 3010, Australia
3. National Deuteration Facility, Australian Nuclear Science and Technology Organisation, Kirrawee DC, NSW 2232, Australia
Using dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (ssNMR), we report the impact of an antimicrobial peptide (AMP), on lipid membrane and macromolecular components (i.e., proteins and nucleic acids) of Escherichia coli. Global scanning of the cellular components was achieved by monitoring the nitrogen (15N) signals by cross polarization (CP) magic angle spinning (MAS) NMR of whole bacteria grown in isotopically enriched media (15N, 13C and 2H 98% enriched isotopes). The different 15N chemical shifts of cellular components served as an atomic marker for monitoring the action of the AMP maculatin 1.1 on E. coli (Fig. 1). The enhanced 15N ssNMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on bacterial cells.
Figure 1 DNP-enhanced 15N CPMAS spectra of untreated E. coli cells (black line) and in the presence of Mac1 at 15:1 w/w ratio (red line). The left panel is scaled 4-fold compared to the right panel to increase visibility. The DNA bases, amino acids with nitrogen containing sidechains and the phospholipid palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) structures are displayed in the inserts with nitrogen (blue), oxygen (red), phosphorous (orange), carbon (grey) and hydrogen (white) atoms.
Fluorescent micropipette aspiration assay to examine calcium mobilization of red blood cell mechanosensing
Peyman Obeidy(1), Haoqing Wang(1,2) & Lining Arnold Ju(1,2,3)
1. School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, NSW 2006, Australia.
2. Charles Perkins Centre, The University of Sydney, NSW 2006, Australia
3. Heart Research Institute, Camperdown, NSW 2006, Australia
Introduction: Red blood cells (RBCs) experience significant mechanical forces while recirculating, such as pressure, stretch and touch, which play a critical role in regulating their physiological functions. In RBCs calcium is an important and universal signaling entity (1). Merging studies have demonstrated the existence of mechanogating ion channels such as Piezo1, which regulate the calcium influx as well as the volume and deformability of RBCs in response to mechanical forces (2). However, the molecular mechanism of how RBCs sense the mechanical stimuli and trigger the calcium signaling process has not yet been fully investigated.
Method: Here, we developed a new system combining a fluorescent confocal microscope with micropipette aspiration, image thresholding segmentation, and probe quantification to examine the mechanosignaling of calcium in RBCs at the cellular level. This method provides a pathway to quantitatively study the impact of controlled mechanical stimuli on cell behavior.
Result: A remarkable calcium mobilization is observed when a single RBC is being aspirated, but the increased intensity of calcium signal slowly decays when aspiration reaches the steady state. Results indicate that the increased membrane tension opens mechanosensitive ion channels and consequentially causes an influx of calcium. We conclude that our newly developed fluorescent micropipette assay is a robust technique to characterize the relationship between external stimuli and intracellular mechanosignaling in live cells.
1. A. Bogdanova, A. Makhro, J. Wang, P. Lipp, L. Kaestner, Int J Mol Sci 2013, 14 (5), 9848, https://doi.org/10.3390/ijms14059848.
2. S. M. Cahalan, V. Lukacs, S. S. Ranade, S. Chien, M. Bandell, A. Patapoutian, Elife 2015, 4, https://doi.org/10.7554/eLife.07370.
Examination of calcium mobilization in RBCs under forces using micropipette aspiration, image thresholding segmentation, and probe quantification.
Rapid measurement of the effects of antimicrobial drug candidates on bacterial motility
Monerh AL-Shahrani(1,2) & Gary Bryant(1)
1. Physics, School of Science, RMIT University, Melbourne, Australia
2. Department of Physics, College of Science, University of Bisha, Saudi Arabia
Antimicrobial drug resistance is a growing health crisis, and there is an urgent need for new antimicrobial agents that work differently than existing drugs and are thus more likely to be successful (1). There are 12 types of dangerous bacteria that threaten human health and have developed resistance to antibiotics that are used to treat a number of common diseases. Antibiotic-resistant bacteria are estimated to cause the death of about one million people annually (2). The ability of bacteria to investigate their environment and spread, known as motility, is of great importance, as this is how bacteria propagate and proliferate (3). Because standard approaches to monitor bacterial movement are limited, antimicrobial medicines that inhibit or reduce motility have received little attention despite their potential.
We will use the innovative technique of Differential Dynamic Microscopy (DDM) in combination with Dynamic Light Scattering (DLS) to evaluate the efficacy of possible candidate antibacterial compounds and understand how they affect motility.
Differential Dynamic Microscopy has been used to study bacterial motility previously and has been shown to be a viable technique for Escherichia coli (4-5), the bacteria responsible for maladies such as gastroenteritis, meningitis in newborns, pneumonia, and urinary tract infections. We will build on earlier research by investigating bacterial motility in a variety of organisms and evaluate the effects of both existing and novel drugs on bacterial motility, with the aim of informing the development of a new class of antibacterial.
1. J. Carlet et al., "Society's failure to protect a precious resource: antibiotics," The Lancet, vol. 378, no. 9788, pp. 369-371, 2011.
2. D. J. Wilson, "Insights from genomics into bacterial pathogen populations," p. e1002874, 2012.
3. H. C. Berg, "The rotary motor of bacterial flagella," Annual review of biochemistry, vol. 72, no. 1, pp. 19-54, 2003.
4. Y. Hu et al., "Photomodulation of bacterial growth and biofilm formation using carbohydrate-based surfactants," Chemical science, vol. 7, no. 11, pp. 6628-6634, 2016.
5. L. G. Wilson et al., "Differential dynamic microscopy of bacterial motility," Physical review letters, vol. 106, no. 1, p. 018101, 2011.
Diverse simulation techniques to understand cystic fibrosis pathogenesis
Miro Astore, Po-chia Chen & Serdar Kuyucak
1. School of Physics, University of Sydney, NSW, Australia 2006
Cystic Fibrosis (CF) is the most common fatal hereditary disease in Caucasians. It is caused by loss-of-function mutations to a chloride channel, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). To date, more than 300 disease causing mutations have been identified. Two classes of drug known as CFTR modulators have been designed: Correctors which rescue misfolded CFTR; and potentiators which increase the stability of the channel’s conducting state. However, certain drugs are only clinically effective when treating the defects introduced by certain mutations.
A significant component of the presented work was using computational techniques to augment the 2018 ATP-bound human CFTR (PDB ID: 6MSM), where the pore is not sufficiently dilated for ion conduction. Part of this involved adding a section of CFTRs Regulatory domain into our model. This enabled us to discover the pathogenic interactions between the Regulatory domain and the novel mutation I37R, which functionally inhibits channel opening. Simulations of other mutants reveal a network of stable water molecules which are disrupted by mutations. For example, G551D and S945L interrupt contacts with water molecules important for ATP catalysis and protein structure, respectively.
Surprisingly, despite the independent modes of pathogenesis these mutations are all rescued by CFTR potentiators. To increase our understanding of CFTR’s function, we used conformational flooding to discover a conducting conformation of CFTR. This will help us resolve the pathogenesis of more mutations and elucidate the mechanisms by which so many defects are treated by CFTR modulators.
Determining membrane permeability to optimise cryopreservation
Saffron J. Bryant(1), Aaron Elbourne(1), Tamar L. Greaves(1) & Gary Bryant(1)
1. School of Science, College of STEM, RMIT University, Melbourne, Australia
Cryopreservation has facilitated numerous breakthroughs including in assisted reproductive technology, stem cell therapies, and species preservation. Successful cryopreservation requires the addition of cryoprotective agents to protect against freezing damage and dehydration. For decades, cryopreservation has largely relied on the same two primary agents: dimethylsulfoxide and glycerol. However, both of these are toxic which limits their use for cells destined for clinical applications. Furthermore, these two agents are ineffective for hundreds of cell types, and organ and tissue preservation has not been achieved.
The activity of these cryoprotectants relies on them getting inside of the cell to provide intracellular protection. However, the rate of influx into a cell varies widely depending on both the solute and the cell type. This rate must be taken into account when designing cryopreservation protocols to ensure that there is sufficient uptake of the cryoprotectant, without excess toxicity.
We have used ‘shrink-swell’ experiments to monitor volume changes of two different cell types on exposure to different cryoprotectants. The results have been modelled using the two parameter transport formalism(1-3), which was implemented in Python. The code is freely available(4).
The results demonstrated significantly different permeability behaviour for different cryoprotectants and different cells and this information was then used to tailor the cryopreservation protocols for different agents to maximise viability. Thus, biophysical measurements of permeability can be used to optimise cryopreservation procedures in a specific and directed way.
1. Kleinhans, F. W., Membrane Permeability Modeling: Kedem–Katchalsky vs a Two-Parameter Formalism. Cryobiology 1998, 37 (4), 271-289.
2. Jacobs, M. H., The simultaneous measurement of cell permeability to water and to dissolved substances. Journal of Cellular and Comparative Physiology 1933, 2 (4), 427-444.
3. Kedem, O.; Katchalsky, A., Thermodynamic analysis of the permeability of biological membranes to non-electrolytes. Biochimica et Biophysica Acta 1958, 27, 229-246.
4. Martin, A. V. shrinkSwell. https://github.com/amartinrmit/shrinkSwell (accessed 11/03/2021).
Energy based modelling of bacterial signalling systems
School of Mathematics and Statistics, University of Melbourne
A key challenge in systems biology is creating mathematical models that can be easily and accurately combined with other models. Such models will need to share a consistent modelling framework and be easily reusable by systems biologists.
One solution to this challenge is to use a physics-based approach to modelling. Bond graphs are an energy-based modelling framework that describe the rate of energy flow (power) moving through system components. By construction, bond graphs models enforce physical and thermodynamic constraints, making model components physically consistent with one another. Bond graphs also provide a graphical representation of the model and allow for easy hierarchical modelling.
To demonstrate bond graph modelling applied to biological systems, we have applied this framework to Two Component Systems (TCS). TCS are a signalling mechanism found in many common bacteria such as E. coli and B. subtilis. By modelling the explicit energy dependence of TCS using bond graphs, we find new insights into the behaviour of the system in different energy contexts. A modular framework also means we can combine models together to investigate coupling dynamics of TCS. In future, we argue that such an approach could lead towards the development of a systems-wide, physically plausible whole-cell model.
Flow-fields generated by rotation of rod-shaped, tethered bacterial cell body in the surrounding medium
Jyoti P Gurung(1), Moein Navvab Kashani(2,3), Charitha de Silva(4) & Matthew AB Baker(1,5).
School of Biotechnology and Biomolecular Science, UNSW Sydney
Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095
Australian National Fabrication Facility – South Australia Node, Mawson Lakes, SA 5095
School of Mechanical and Manufacturing Engineering, UNSW Sydney
CSIRO Future Science Platform for Synthetic Biology
Many motile bacteria are propelled by the rotation of flagellar filaments, driven by a transmembrane nano-machine, known as the bacterial flagellar motor. The high rotational speed of the ion-powered bacterial flagellar motor shows promising potential for various applications such as mixing, actuation, and biosensing.
Here, we focus mainly on the mixing applications of motile bacteria. Two approaches can be employed to utilize bacterial motility for mixing, either by the flagellar bundle rotation or the bacterial cell body rotation. One of the approaches, i.e., rotation of rod-shaped bacterial cell body, was attained by adhering genetically engineered bacteria via truncated sticky filaments onto the solid surface. This rotation of the bacterial cell body is analogous to the rotation of a micron-sized magnetic stir bar.
To determine the mixing efficiency of bacterial flagellar rotation at these length scales, we measure velocity flow-fields generated by the rotation of a single cell. We investigated the flow-fields generated by the bacterial cell body using micro-particle image velocimetry (µPIV). The flow field revealed that a rotating bacterial cell body generated micro-vortices in the surrounding medium. These results confirmed that directional mixing could be observed using the two types of bacterial strain, one which rotates clockwise (FliG-∆PAA (1)) and the other which rotates in an anti-clockwise direction (∆cheY (2)). Our observations matched our numerical simulations for mixing driven by a tethered cell body. We further investigated the effect of parameters such as length of the cell body and axis of rotation in the generation of micro-vortices.
1. F. Togashi, S. Yamaguchi, M. Kihara, S. I. Aizawa, and R. M. Macnab, “An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB,” J Bacteriol, vol. 179, no. 9, pp. 2994–3003, May 1997, doi: 10.1128/jb.179.9.2994-3003.1997.
2. M. I. Islam et al., “Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types,” J Bacteriol, p. JB0036721, Sep. 2021, doi: 10.1128/JB.00367-21.
Md. Sirajul Islam
Assembling 3D DNA snub-cube origamis in 2D arrays
Md. Sirajul Islam(1,2), Gerrit David Wilkens(2), Karol Wolski(3) , Szczepan Zapotoczny(3) & Jonathan Gardiner Heddle(2)
1. School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW 2052, Australia.
2. Malopolska Centre of Biotechnology, Jagiellonian University, Krakow 30-387, Poland. 3Faculty of Chemistry, Jagiellonian University, Krakow 30-387, Poland.
The DNA origami technique allows the facile design and production of three-dimensional shapes from single template strands of DNA (1). These can act as functional devices with multiple potential applications including biosensing, drug delivery, nanophotonics and plasmonics, nanoelectronics and biomimetics. Multi-functionality can be achieved by connecting together distinct DNA origami modules in a higher order structure. Arraying of nonidentical, three-dimensional DNA origamis in an ordered manner is challenging including the necessity for single-stranded DNA scaffolds with custom sequence and length to avoid large unstructured sequences which would interfere with origami-origami interactions and in producing ordered arrays of 3D structures when individual structures are not identical – leading to poor tiling ability between individual units. Here we show that we can design and build ordered wireframe DNA structures (2) using nonidentical 3D building blocks by using DNA origami snub-cubes in left-handed (SnL) and righthanded (SnR) forms. Taking a step-by-step approach, we assembled single SnL and SnR separately (Figure 1A). Next, SnL and SnR were constructed with external ssDNA strands with the strands on the R-form being complementary to those on the L-form, with the expectation that these would anneal to form heterodimers (Figure 1B), 1D chain (Figure 1C) and 2D lattices (Figure 1D). This approach is particularly useful for formation of convex polyhedra DNA origami structures having potential as cargo carrying nanostructures and even as artificial vaccines if their exterior is decorated with antigens. This work on 3D DNA origami is from my recent postdoctoral research in Poland (3). We plan to extend this work in the Baker Lab at UNSW using 3D DNA origamis to control transmembrane communication.
1. Rothemund, P.W.K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297–302.
2. Veneziano, R.; Ratanalert, S.; Zhang, K.; Zhang, F.; Yan, H.; Chiu, W.; Bathe, M. Designer Nanoscale DNA Assemblies Programmed from the Top Down. Science 2016, 352, 1534–1534.
3. Islam, M.S.; Wilkens, G.D.; Wolski, K.; Zapotoczny, S.; Heddle, J.G. Chiral 3D DNA Origami Structures for Ordered Heterologous Arrays. Nanoscale Adv. 2021, 3, 4685–4691.
Figure 1: Wireframe DNA origami structure of snub-cube is designed both in the left-handed (SnL) and right-handed (SnR) forms (A). Taking a step-by-step approach, SnL and SnR monomeric units were assembled into heterodimers (B); 1D chain (C); and 2D lattices (D).
Md. Imtiazul Islam
Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types
MI Islam(1), JH Bae(1), T Ishida(2), P Ridone(1), J Lin(1), MJ Kelso(3,4), Y Sowa(2,5), BJ Buckley(3,4) & MAB Baker(1,6).
1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
2. Department of Frontier Bioscience, Hosei University, Tokyo, Japan.
3. Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia
4. Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.
5. Research Center for Micro-Nano Technology, Hosei University, Tokyo, Japan.
6. CSIRO Synthetic Biology Future Science Platform, Brisbane, Australia.
The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence using ion motive force (most commonly either H+ or Na+). The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. Compounds that inhibit bacterial motility show promise for new anti-virulence agents. Phenamil, a potent and widely used sodium channel inhibitor, can further inhibit the sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We initially assessed the effect of our compounds on motility using semi-solid agar swim plating, single-cell tethered cell rotational measurements, and direct cell-swimming measurements. We then performed high-resolution bead assays using a custom microscopy to examine the mechanism of inhibition at moderate (1 µm polystyrene bead) and low loads (60 nm gold bead). The dark-field gold-bead rotation detection method could measure low-load rotation at speeds of up to 300 Hz to directly assess whether the inhibitors targeted the motor specifically, or interfered with rotation due to surface effects. The high-resolution bead measurements in the presence and absence of stators indeed confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Nevertheless, HM2-16F and BB2-50F showed reversible inhibition of motility across a range of loads, in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains.
Islam, M. I., Bae, J. H., Ishida, T., Ridone, P., Lin, J., Kelso, M. J., et al. (2021). Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types. J. Bacteriol., JB0036721. doi:10.1128/JB.00367-21.
Bead assay (1 μm polystyrene bead) results for phenamil, BB2-611 50F and HM2-16F in different stator types. Each panel shows three X-Y scatter plots (over 1 s) of the position a bead attached to the rotating flagellar filament of a single cell before exposure to drug, after exposure to drug, and after wash to remove drug. All drugs washed in as 50 μL of 10 μM drug, washed out with 200 μL of 85MTB. All lengths in μm.
Monitoring the adsorption of ultra-small gold nanoparticles to model bio-membranes
Rashad Kariuki(1), Saffron Bryant(1), Rebecca Orrell-Trigg(1), Vi Khanh Truong(1), James Chapman(1), Russell J. Crawford(1), Christopher F. McConville(1,2), Gary Bryant(1), Charlotte Conn(1), Kislon Voïtchovsky(3), Andrew Christofferson(1) & Aaron Elbourne(1)
1. School of Science, RMIT University, Melbourne VIC 3001, Australia.
2. Deakin University, Geelong, Australia
3. University of Durham, Physics Department, Durham DH1 3LE, UK.
Nanomaterials - materials with nanoscale dimensions - are widely used in biological applications, including drug delivery, nanomedicines, emerging antimicrobials, disease diagnostics, cellular-imaging, and tumour (cancer) treatment, amongst many others. In general, nanoparticle-based technologies must interact with, and often cross, a cellular membrane to be utilised. However, the precise mechanism by which nanomaterials interact with cell membranes is poorly understood. This work further develops our fundamental knowledge of the physicochemical, nanomechanical, and structural interactions of AuNPs at a model DOPC bio-membrane. Specifically, the adsorptive mechanism of action of 5nm AuNP onto a supported DOPC bilayer – a model biomembrane – was observed in real time. Dual AFM and molecular dynamics simulations were used to interrogate the system. AFM experiments elucidated that the AuNP would spontaneously embed into the model bio-membrane and slowly diffuse through the upper leaflet of the lipid bilayer. Verification of the AuNP-SLB interaction was undertaken via MD simulations and revealed, significant reductions in lipid density upon AuNP introduction, as well as significant structural changes of between the mica surface, the DOPC membrane, and the AuNP. More holistically, it was shown that bilayer self-assembly upon a mica surface was feasible using an atomistic MD model and could characterise its reactions with a AuNP.
Figure 1. Molecular dynamics simulations of 5 nm Au Nanoparticle adsorption to a DOPC lipid bilayer formed at a mica surface. A) Simulation snapshot. B) Radial distribution data.
Engineering transient dynamics of artificial cells by stochastic distribution of enzymes
Alexander F. Mason(1)*, Shidong Song(1), Richard A.J. Post(2), Marco de Corato(3) Rafael Mestre(3) N. Amy Yewdall(1) Shoupeng Cao(1) Remco W. van der Hofstad(2) Samuel Sanchez(3) Loai K.E.A. Abdelmohsen(1) & Jan C.M. van Hest(1)
1. Department of Bio-Organic Chemistry, Eindhoven University of Technology, The Netherlands
2. Department of Mathematics and Computer Science, Eindhoven University of Technology, The Netherlands
3. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Spain
*(current) School of Biotechnology and Biomolecular Science, UNSW, Australia
At the molecular level, the impact of random thermal fluctuations on biological processes is ubiquitous. Nature has evolved an impressive array of mechanisms to control these stochastic events towards useful biological output. These range from the dampening of cellular noise to stabilize stochastic decisions in the development of neuronal cells to the autonomous motion of DNA walkers. However, reproducing this elegant mode of control in synthetic systems represents a significant challenge.
Here we present an artificial cell capable of harnessing molecular fluctuations towards predictable motile behaviors.1 We found that motility-inducing enzymes, when confined to the fluid polymer membrane of a core-shell artificial cell platform, were distributed stochastically in space and time. This transient, asymmetric configuration of motile elements resulted in autonomous motility that could be modulated via tuning relevant parameters. Furthermore, these results were verified by holistic modelling that combines an analytical derivation of an existing motility model with stochastic simulations in silico. Conceptually, this work represents a progression in design philosophy in the construction of bottom-up artificial cells. Random fluctuations will always be present at the molecular level, and instead of ignoring stochasticity or averaging it out, we should view it as an opportunity to engineer more sophisticated biomimetic systems.
1. Song, S, Mason, AF, et al (2021) ChemRxiv. doi:10.33774/chemrxiv-2021-02c1d
Figure 1: Motility-inducing enzymes catalase or urease, when confined to the membrane of a particle, diffuse stochastically in 2D. This stochastic organisation, or transient asymmetry, of motors enables propulsion in the presence of fuel. Different motile states can be harnessed by controlling different parameters that either dampen or amplify this stochastic, membrane-bound process.
Developing rigid origami as a model for global geometrical constraint
Matthew B. Pinson
Newman College, University of Melbourne
Numerous biological systems are produced through the arrangement of a two-dimensional starting material into a three-dimensional structure. For example, the leg or wing of a holometabolic insect is formed through the folding of an imaginal disc(1). The geometrical constraints involved in embedding a two-dimensional object into three dimensions provide a restriction on what such systems can do and how they can develop. For example, an arbitrary curved surface drawn in three dimensions will have non-zero Gaussian curvature, and hence cannot be constructed by folding a flat sheet without tearing it or producing extra material.
I am interested in when this restriction becomes a benefit: when the geometrical constraint helps stabilise the desired shape or behaviour. Since geometrical constraints are intrinsically global in nature, this stabilisation comes from the overall structure of the system, not through the tuning of particular local interactions. In particular, I look at multistable systems, where the same material is able to adopt either of two or more qualitatively different configurations without getting stuck in a mixture of configurations. Multistability is found, for instance, in memory mechanisms and in achieving cellular differentiation during embryonic development(2).
Another example of such geometrically constrained systems—obviously highly abstracted from a biological context—is rigid origami. A vertex at which four rigid panels meet has a branched, one-dimensional range of motion, and an arbitrary lattice of such vertices is immobile. Foldable lattices can be designed, but to date they have been specific perturbations of highly symmetric patterns. In this talk I present first a method for quantifying how far from being foldable a given pattern is(3) and then an analytic method for designing a generic foldable pattern. Such patterns have engineering applications in areas such as soft robotics(4). By combining patterns and examining when the geometrical protection of the distinct folding modes breaks down and hybrid folding modes appear, they can also be used as a tool for studying the general properties of limitations on geometrically-protected multistability.
1. A. García-Bellido, P. Ripoll, G. Morata, ‘Developmental compartmentalisation of the wing disc of Drosophila’, Nature New Biol. 245, 251-253 (1973).
2. F. Freyer, J.A. Roberts, P. Ritter, M. Breakspear, ‘A canonical model of multistability and scale-invariance in biological systems’, PLOS Comp. Biol. 8, e1002634 (2012).
3. M.B. Pinson, M. Stern, A. Carruthers Ferrero, T.A. Witten, E. Chen, A. Murugan, ‘Self-folding origami at any energy scale’, Nature Comm. 8, 15477 (2017).
4. S. Felton, M. Tolley, E. Demaine, D. Rus, R. Wood, ‘A method for building self-folding machines’, Science 345, 644 (2014).
Figure 1: an example of a rigid origami pattern constructed using the method for quantifying how far from foldable a given pattern is(3).
Ancestral reconstruction and synthesis of the flagellar rotor protein FliG
Janelle Ramos & Matthew Baker
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
The bacterial flagellar motor drives the propulsion of bacteria to more favourable environments. The stator units MotA/B or PomA/B form a transmembrane ion channel that interacts directly with the rotor. The rotor, also known as the C-ring, is composed of FliG, FliM and FliN and can rotate in a clockwise (CW) or counter-clockwise (CCW) direction, and the top-most protein of the rotor, FliG interacts with stators to drive rotation. In this study, we reconstructed eight FliG ancestral sequence reconstructions (ASRs) to examine how switching frequency may have adapted over time and across multiple species. We performed error-prone PCR to rescue the motility of non-functional ASRs. Our FliG-ASRs developed motility after 5 days on a swim plate. Overall, this study demonstrates the use of ASRs to resurrect ancient FliG proteins, characterise them in a contemporary host and better understand the evolution of chemotaxis in bacteria.
Correlation of brightness fluctuations maps protein diffusion as a function of oligomeric state within live cell nuclear architecture
Ashleigh Solano(1), Jieqiong Lou(1), Lorenzo Scipioni(2), Enrico Gratton(2) & Elizabeth Hinde(1)
1. School of Physics, Department of Biochemistry and Molecular Biology, University of
2. Laboratory for Fluorescence Dynamics, University of California, Irvine, USA.
The nucleoplasm is a crowded environment where dynamic rearrangements in local DNA density redefine the space accessible towards nuclear protein diffusion. Transcription factors (TFs) are nuclear proteins that mediate gene expression by scanning the genome and site-specific DNA binding. The extent that nuclear architecture directs the diffusive routes of TFs to their target sequences remains unclear. Protein oligomerisation is known to modulate the exploration volume available to nuclear proteins and is a common feature employed by TFs to guide DNA target search. We explore the role of nuclear protein homo and hetero oligomerisation on DNA target search at a single molecule level within living cells via the correlation of brightness fluctuations. This approach has the capacity to extract the mobility of a fluorescently tagged nuclear protein as a function of self-association states and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture by performing a rapid single channel frame scan acquisition. In addition, we may detect within each pixel, protein homo-oligomer formation and the size dependent obstruction nuclear architecture imparts on this complex’s transport across sub-micron distances. Expanding brightness fluctuations correlation from single to dual channels frame scan acquisitions serves the opportunity to characterize the spatiotemporal dynamics of hetero-oligomer stoichiometries between TF family protein variants. The application of brightness fluctuation correlation techniques to oligomeric transcription factors, demonstrate that homo and hetero oligomer formation differentially regulates chromatin accessibility and interaction with the DNA template.