Since obtaining his Science degree with Honours in Pharmacology from Monash University in 1996, Dr Cranfield completed his PhD in Biophysics at Swinburne University of Technology in 2002 where he investigated the effects of mobile phone radiation on calcium levels in T lymphocytes. His first post-doctoral position saw him travel to Keele University in England under the mentorship of Prof Jon Dobson to investigate how biogenic magnetic nanoparticles might interact with mobile phone radiation. Dr Cranfield then returned to his native Melbourne where, under the auspices of Prof Min Gu at the Centre for Micro-Photonics at Swinburne University of Technology, he assisted in the development of the world’s first non-linear endoscope. Dr Cranfield was then the beneficiary of 2 Marie Curie Transfer of Knowledge Fellowships. The first was at the Tyndall National Institute in Cork, Ireland, to assist in the development of fluorescence based point-of care diagnostic devices. The second was to work with biomolecular photonics expert Prof Christoph Biskup at University Hospital Jena, Germany, where he helped develop novel fluorescent ion nanosensors. From 2010 - 2013 he then worked in the laboratory of Prof Boris Martinac at the Victor Chang Cardiac Research Institute researching the lipid protein interactions of mechanosensitive ion channels. In 2014 he was appointed as Senior Lecturer at University of Technology Sydney and in 2015 he was appointed Team Leader of the Molecular Biosciences Team within the newly formed School of Life Sciences. In 2018 he became Deputy Leader of the Biology and Biotechnology Discipline.
- Member of the Australian Society for Biophysics
Can supervise: YES
Dr Cranfield's current research interests are in using the tethered lipid bilayer membranes (tBLMs) in conjunction with electrical impedance spectroscopy to investigate the insertion and function of biological metabolites and antimicrobial peptides.
His other research interests Dr Cranfield has published in include the study of:
- mechanosensitive ion channels
- advanced fluorescence imaging techniques,
- fluorescent ion nanosensors,
- the possible effects of mobile phone radiation on immune cells
Research Group (UTS Membrane Biophysics)
- Dr Alvaro Garcia (Chancellor's Postdoctroal Fellow)
- Dr Evelyne Deplazes (Chancellor's Postdoctoral Fellow)
- Adj Prof Bruce Cornell
- Dr Stephen Holt (Visiting Scholar, ANSTO)
- Dr Krishanthi Jayasundera (Postdoctoral Research Associate- IDEAL HUB)
- Amani Alghayalini (PhD Student - IDEAL HUB)
- Thomas Berry (PhD Student)
- Lissy Hartmann (Honours 2019)
- Samantha Sandelowsky (Intern student 2019)
- Surithi Yogalingham (Intern student 2019)
- Jacqueline White (Intern student 2018)
- Christopher Murphy (Intern Student 2018)
- Metabolic Biochemistry
- Physiological Systems
Facey, JA, Steele, JR, Violi, JP, Mitrovic, SM & Cranfield, C 2019, 'An examination of microcystin-LR accumulation and toxicity using tethered bilayer lipid membranes (tBLMs).', Toxicon, vol. 158, pp. 51-56.View/Download from: Publisher's site
Microcystin-LR (MC-LR) is a potent cyanobacterial toxin responsible for animal and human poisonings worldwide. MC-LR is found in organisms throughout the foodweb, however there is conjecture regarding whether it biomagnifies. Few studies have investigated how MC-LR interacts with lipid membranes, a determinant of biomagnification potential. We tested whether 1M MC-LR irreversibly associates with lipid bilayers or causes the creation of pore defects upon short and long-term exposure. Using tethered bilayer lipid membranes (tBLMs), we observed an increase in membrane conduction in tBLMs, representing an interaction of microcystin-LR with the lipid bilayer and a change in membrane packing properties. However, there were minimal changes in membrane capacitance upon short and long-term exposure, and MC-LR exhibited a rapid off-rate. Upon 24h exposure to the toxin, no lipophilic multimeric complexes were detected capable of altering the toxin's off-rate. There was no evidence of the creation of new pores. This study demonstrates that MC-LR does not irreversibly imbed itself into lipids membranes after short or long-term exposure and suggests MC-LR does not biomagnify through the food web via lipid storage.
Alghalayini, A, Garcia, A, Berry, T & Cranfield, CG 2019, 'The Use of Tethered Bilayer Lipid Membranes to Identify the Mechanisms of Antimicrobial Peptide Interactions with Lipid Bilayers.', Antibiotics (Basel, Switzerland), vol. 8, no. 1.View/Download from: Publisher's site
This review identifies the ways in which tethered bilayer lipid membranes (tBLMs) can be used for the identification of the actions of antimicrobials against lipid bilayers. Much of the new research in this area has originated, or included researchers from, the southern hemisphere, Australia and New Zealand in particular. More and more, tBLMs are replacing liposome release assays, black lipid membranes and patch-clamp electrophysiological techniques because they use fewer reagents, are able to obtain results far more quickly and can provide a uniformity of responses with fewer artefacts. In this work, we describe how tBLM technology can and has been used to identify the actions of numerous antimicrobial agents.
Kuppusamy, R, Yasir, M, Berry, T, Cranfield, CG, Nizalapur, S, Yee, E, Kimyon, O, Taunk, A, Ho, KKK, Cornell, B, Manefield, M, Willcox, M, Black, DS & Kumar, N 2018, 'Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents.', European journal of medicinal chemistry, vol. 143, pp. 1702-1722.View/Download from: UTS OPUS or Publisher's site
Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alternatives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many microbes. AMPs are part of the natural defenses of most living organisms and they also have a unique mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics were synthesized by incorporating the 3'-amino-[1,1'-biphenyl]-3-carboxylic acid backbone to mimic the essential properties of natural AMPs. By altering hydrophobicity and charge, we identified the most potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC = 15.6 M) and Gram-negative Escherichia coli (MIC = 7.8 M) bacteria. Cytoplasmic permeability assay results revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using tethered bilayer lipid membranes (tBLMs) and their activity against established biofilms of S. aureus and E. coli.
Deplazes, E, Poger, D, Cornell, B & Cranfield, CG 2018, 'The effect of hydronium ions on the structure of phospholipid membranes.', Physical Chemistry Chemical Physics, vol. 20, no. 1, pp. 357-366.View/Download from: UTS OPUS or Publisher's site
This work seeks to identify the mechanisms by which hydronium ions (H3O+) modulate the structure of phospholipid bilayers by studying the interactions of H3O+ with phospholipids at the molecular level. For this, we carried out multiple microsecond-long unrestrained molecular dynamics (MD) simulations of a POPC bilayer at different H3O+ concentrations. The results show that H3O+ accumulates at the membrane surface where it displaces water and forms strong and long-lived hydrogen bonds with the phosphate and carbonyl oxygens in phospholipids. This results in a concentration-dependent reduction of the area per lipid and an increase in bilayer thickness. This study provides an important molecular-level insight into the mechanism of how H3O+ modulates the structure of biological membranes and is a critical step towards a better understanding of the effect of low pH on mammalian and bacterial membranes.
Nguyen, T, Li, GE, Chen, H, Cranfield, CG, McGrath, KC & Gorrie, CA 2018, 'Maternal E-Cigarette Exposure Results in Cognitive and Epigenetic Alterations in Offspring in a Mouse Model.', Chemical research in toxicology, vol. 31, no. 7, pp. 601-611.View/Download from: UTS OPUS or Publisher's site
Electronic cigarette (e-cigarette) use is on the rise worldwide and is particularly attractive to young people and as a smoking substitute by pregnant woman. There is a perception in pregnant women and women of child-bearing age that the use of e-cigarettes (vaping) is safer than smoking tobacco cigarettes during pregnancy. However, there is little evidence to support this perception. Here, we examined the offspring from mouse dams that had been exposed during and after pregnancy to ambient air (sham) ( n = 8), e-cigarette aerosols with nicotine ( n = 8), or e-cigarette aerosols without nicotine ( n = 8). Offspring underwent cognitive testing at 12 weeks of age and epigenetic testing of brain tissues at 1 day, 20 days, and 13 weeks after birth. The findings showed deficits in short-term memory, reduced anxiety, and hyperactivity in offspring following maternal e-cigarette exposure using the novel object recognition and elevated plus maze tests. In addition, global DNA methylation was increased in the brains of offspring soon after birth. Using a quantitative-PCR array specific to chromatin modification enzymes on genomic DNA and histones,13 key genes were identified to be significantly altered in the offspring brains from the e-cigarette groups compared to the nonexposed groups. The changes to genes Aurka, Aurkb, Aurkc, Kdm5c, Kdm6b, Dnmt3a, Dnmt3b, and Atf2, all associated with modulating neurological activity, were validated using RT-qPCR. In conclusion, in a mouse model, maternal exposure to e-cigarette aerosols resulted in both cognitive and epigenetic changes in offspring. This suggests that the use of e-cigarettes during pregnancy may have hitherto undetected neurological consequences on newborns.
Berry, T, Dutta, D, Chen, R, Leong, A, Wang, H, Donald, WA, Parviz, M, Cornell, B, Willcox, M, Kumar, N & Cranfield, CG 2018, 'Lipid Membrane Interactions of the Cationic Antimicrobial Peptide Chimeras Melimine and Cys-Melimine.', Langmuir : the ACS journal of surfaces and colloids, vol. 34, no. 38, pp. 11586-11592.View/Download from: UTS OPUS or Publisher's site
Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilized melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using ac electrical impedance spectroscopy. The addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. The results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the noncysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability, which is then reduced as the melimines aggregate and phase-separate within the membrane. The actions of these antimicrobials are modeled in terms of altering the critical packing parameter (CPP) of the membranes. The variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. The results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for the Gram-positive and Gram-negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy- sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studied here and their modulation of membrane conductivity.
Deplazes, E, Poger, D, Cornell, B & Cranfield, CG 2018, 'The effect of H3O+ on the membrane morphology and hydrogen bonding of a phospholipid bilayer.', Biophysical reviews, vol. 10, no. 5, pp. 1371-1376.View/Download from: Publisher's site
At the 2017 meeting of the Australian Society for Biophysics, we presented the combined results from two recent studies showing how hydronium ions (H3O+) modulate the structure and ion permeability of phospholipid bilayers. In the first study, the impact of H3O+ on lipid packing had been identified using tethered bilayer lipid membranes in conjunction with electrical impedance spectroscopy and neutron reflectometry. The increased presence of H3O+ (i.e. lower pH) led to a significant reduction in membrane conductivity and increased membrane thickness. A first-order explanation for the effect was assigned to alterations in the steric packing of the membrane lipids. Changes in packing were described by a critical packing parameter (CPP) related to the interfacial area and volume and shape of the membrane lipids. We proposed that increasing the concentraton of H3O+ resulted in stronger hydrogen bonding between the phosphate oxygens at the water-lipid interface leading to a reduced area per lipid and slightly increased membrane thickness. At the meeting, a molecular model for these pH effects based on the result of our second study was presented. Multiple s-long, unrestrained molecular dynamic (MD) simulations of a phosphatidylcholine lipid bilayer were carried out and showed a concentration dependent reduction in the area per lipid and an increase in bilayer thickness, in agreement with experimental data. Further, H3O+ preferentially accumulated at the water-lipid interface, suggesting the localised pH at the membrane surface is much lower than the bulk bathing solution. Another significant finding was that the hydrogen bonds formed by H3O+ ions with lipid headgroup oxygens are, on average, shorter in length and longer-lived than the ones formed in bulk water. In addition, the H3O+ ions resided for longer periods in association with the carbonyl oxygens than with either phosphate oxygen in lipids. In summary, the MD simulations support a model where the hydrogen bon...
Nizalapur, S, Kimyon, O, Yee, E, Ho, K, Berry, T, Manefield, M, Cranfield, CG, Willcox, M, Black, DS & Kumar, N 2017, 'Amphipathic guanidine-embedded glyoxamide-based peptidomimetics as novel antibacterial agents and biofilm disruptors.', Organic and Biomolecular Chemistry, vol. 15, no. 9, pp. 2033-2051.View/Download from: UTS OPUS or Publisher's site
Antimicrobial resistance in bacteria is becoming increasingly prevalent, posing a critical challenge to global health. Bacterial biofilm formation is a common resistance mechanism that reduces the effectiveness of antibiotics. Thus, the development of compounds that can disrupt bacterial biofilms is a potential strategy to combat antimicrobial resistance. We report herein the synthesis of amphipathic guanidine-embedded glyoxamide-based peptidomimetics via ring-opening reactions of N-naphthoylisatins with amines and amino acids. These compounds were investigated for their antibacterial activity by the determination of minimum inhibitory concentration (MIC) against S. aureus and E. coli. Compounds 35, 36, and 66 exhibited MIC values of 6, 8 and 10 g mL(-1) against S. aureus, respectively, while compounds 55 and 56 showed MIC values of 17 and 19 g mL(-1) against E. coli, respectively. Biofilm disruption and inhibition activities were also evaluated against various Gram-positive and Gram-negative bacteria. The most active compound 65 exhibited the greatest disruption of established biofilms by 65% in S. aureus, 61% in P. aeruginosa, and 60% in S. marcescens respectively, at 250 M concentration, while compound 52 inhibited the formation of biofilms by 72% in S. marcescens at 250 M. We also report here the in vitro toxicity against MRC-5 human lung fibroblast cells. Finally, the pore forming capability of the three most potent compounds were tested using tethered bilayer lipid membrane (tBLM) technology.
Tsz, TU, Nizalapur, S, Ho, KKK, Yee, E, Berry, T, Cranfield, CG, Willcox, M, Black, DS & Kumar, N 2017, 'Design, Synthesis and Biological Evaluation of N-Sulfonylphenyl glyoxamide-Based Antimicrobial Peptide Mimics as Novel Antimicrobial Agents', Chemistry Select, vol. 2, no. 12, pp. 3452-3461.View/Download from: UTS OPUS or Publisher's site
Antibiotic resistance is a major global health concern. There is an urgent need for the development of novel antimicrobials. Recently, phenylglyoxamide-based small molecular antimicrobial peptide mimics have been identified as potential new leads to treat bacterial infections. Here, we describe the synthesis of novel phenylglyoxamide derivatives via the ring-opening reaction of N-sulfonylisatins with primary amines, followed by conversion into hydrochloride, quaternary ammonium iodide or gunidinium salts. The antibacterial activity of the compounds against Staphylococcus aureus was evaluated by invitro assays. Structure-activity relationship studies revealed that 5-bromo-substituent at the phenyl ring, octyl group appended to the ortho sulfonamide group or guanidine hydrochloride salt as the terminal group significantly contributed to potency. The most potent compound, the gunidinium salt 35d, exhibited a minimum inhibitory concentration value of 12 M and a therapeutic index of 15. It also demonstrated its potential to act as antimicrobial pore-forming agent. Overall, the results identified 35d as a new lead antimicrobial compound.
Cranfield, CG, Henriques, ST, Martinac, B, Duckworth, P, Craik, DJ & Cornell, B 2017, 'Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes.', Langmuir: the ACS journal of surfaces and colloids, vol. 33, no. 26, pp. 6630-6637.View/Download from: UTS OPUS or Publisher's site
Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.
Constantine, M, Liew, CK, Lo, V, Macmillan, A, Cranfield, CG, Sunde, M, Whan, R, Graham, RM & Martinac, B 2016, 'Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer', SCIENTIFIC REPORTS, vol. 6.View/Download from: UTS OPUS or Publisher's site
Nizalapur, S, Ho, KKK, Kimyon, O, Yee, E, Berry, T, Manefield, M, Cranfield, CG, Willcox, M, Black, DS & Kumar, N 2016, 'Synthesis and biological evaluation of N-naphthoyl-phenylglyoxamide-based small molecular antimicrobial peptide mimics as novel antimicrobial agents and biofilm inhibitors', ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 14, no. 14, pp. 3623-3637.View/Download from: UTS OPUS or Publisher's site
Cranfield, CG, Berry, T, Holt, SA, Hossain, KR, Le Brun, AP, Carne, S, Al Khamici, H, Coster, H, Valenzuela, SM & Cornell, B 2016, 'Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology', LANGMUIR, vol. 32, no. 41, pp. 10725-10734.View/Download from: UTS OPUS or Publisher's site
Hoiles, W, Gupta, R, Cornell, B, Cranfield, C & Krishnamurthy, V 2016, 'The Effect of Tethers on Artificial Cell Membranes: A Coarse-Grained Molecular Dynamics Study', PLOS ONE, vol. 11, no. 10.View/Download from: UTS OPUS or Publisher's site
Cranfield, CG, Bettler, T & Cornell, B 2015, 'Nanoscale Ion Sequestration To Determine the Polarity Selectivity of Ion Conductance in Carriers and Channels', LANGMUIR, vol. 31, no. 1, pp. 292-298.View/Download from: UTS OPUS or Publisher's site
Hoiles, W, Krishnamurthy, V, Cranfield, CG & Cornell, B 2014, 'An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface', BIOPHYSICAL JOURNAL, vol. 107, no. 6, pp. 1339-1351.View/Download from: UTS OPUS or Publisher's site
Cranfield, CG, Cornell, BA, Grage, SL, Duckworth, P, Carne, S, Ulrich, AS & Martinac, B 2014, 'Transient potential gradients and impedance measures of tethered bilayer lipid membranes: pore-forming peptide insertion and the effect of electroporation.', Biophysical Journal, vol. 106, no. 1, pp. 182-189.View/Download from: UTS OPUS or Publisher's site
In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >~150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.
Martinac, B, Nomura, T, Chi, G, Petrov, E, Rohde, PR, Battle, AR, Foo, A, Constantine, M, Rothnagel, R, Carne, S, Deplazes, E, Cornell, B, Cranfield, CG, Hankamer, B & Landsberg, MJ 2014, 'Bacterial mechanosensitive channels: models for studying mechanosensory transduction.', Antioxidants and Redox Signaling, vol. 20, no. 6, pp. 952-969.View/Download from: UTS OPUS or Publisher's site
SIGNIFICANCE: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.
Martinac, B. & Cranfield, C.G. 2012, 'Shining a light on the structural dynamics of ion channels using Förster resonance energy transfer (FRET).', IPSI BgD Transactions on Advanced Research, vol. 8, pp. 19-24.
Nomura, T, Cranfield, CG, Deplazes, E, Owen, DM, Macmillan, A, Battle, AR, Constantine, M, Sokabe, M & Martinac, B 2012, 'Differential effects of lipids and lyso-lipids on the mechanosensitivity of MscL and MscS', Proceedings of The National Academy of Sciences of the United States of America, vol. 109, no. 22, pp. 8770-8775.View/Download from: UTS OPUS or Publisher's site
Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study,we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.
Idzik, KR, Cywinski, PJ, Cranfield, CG, Mohr, GJ & Beckert, R 2011, 'Molecular Recognition of the Antiretroviral Drug Abacavir: Towards the Development of a Novel Carbazole-Based Fluorosensor', Journal of Fluorescence, vol. 21, no. 3, pp. 1195-1204.View/Download from: UTS OPUS or Publisher's site
Due to their optical and electro-conductive attributes, carbazole derivatives are interesting materials for a large range of biosensor applications. In this study, we present the synthesis routes and fluorescence evaluation of newly designed carbazole fluorosensors that, by modification with uracil, have a special affinity for antiretroviral drugs via either WatsonCrick or Hoogsteen base pairing. To an N-octylcarbazole-uracil compound, four different groups were attached, namely thiophene, furane, ethylenedioxythiophene, and another uracil; yielding four different derivatives. Photophysical properties of these newly obtained derivatives are described, as are their interactions with the reverse transcriptase inhibitors such as abacavir, zidovudine, lamivudine and didanosine. The influence of each analyte on biosensor fluorescence was assessed on the basis of the SternVolmer equation and represented by SternVolmer constants. Consequently we have demonstrated that these structures based on carbazole, with a uracil group, may be successfully incorporated into alternative carbazole derivatives to form biosensors for the molecular recognition of antiretroviral drugs.
Stanca, SE, Nietzsche, S, Fritzsche, W, Cranfield, CG & Biskup, C 2010, 'Intracellular ion monitoring using a gold core-polymer shell nanosensor architecture', Nanotechnology, vol. 21, no. 5.View/Download from: UTS OPUS or Publisher's site
In this study, we describe the design of new ratiometric fluorescent nanosensors, whose architecture is based on a gold core surrounded by a poly(vinyl alcohol)polyacetal shell. To the gold core, indicator dyes and reference dyes are attached via a cysteine linker. This nanosensor architecture is flexible with regards to the number and types of fluorophore linkages possible. The robust poly(vinyl alcohol)polyacetal shell protects the fluorophores linked to the core from non-specific interactions with intracellular proteins. The nanosensors developed in this way are biocompatible and can be easily incorporated into mammalian cells without the use of transfection agents. Here, we show the application of these nanosensors for intracellular pH and sodium ion measurements.
Day, D, Cranfield, CG & Gu, M 2006, 'High-speed fluorescence imaging and intensity profiling of femtosecond-induced calcium transients.', International journal of biomedical imaging, vol. 2006, p. 93438.View/Download from: UTS OPUS or Publisher's site
We have demonstrated a combined imaging system, where the physiology of biological specimens can be imaged and profiled at 10-20 frames per second whilst undergoing femtosecond laser irradiation. Individual GH3 cells labeled with the calcium fluorophore Fluo-3 were stimulated using a counter-propagating focused femtosecond beam with respect to the imaging system. As a result of the stimulation, calcium waves can be generated in COS cells, and laser-induced calcium oscillations are initiated in the GH3 cells. Single-photon fluorescence images and intensity profiles of the targeted specimens are sampled in real-time using a modified PerkinElmer UltraView LCI microscope.
Fu, L, Jain, A, Xie, HK, Cranfield, C & Gu, M 2006, 'Nonlinear optical endoscopy based on a double-clad photonic crystal fiber and a MEMS mirror', OPTICS EXPRESS, vol. 14, no. 3, pp. 1027-1032.View/Download from: Publisher's site
Cranfield, C, Bomzon, Z, Day, D, Gu, M & Cartmell, S 2006, 'Mechanical strains induced in osteoblasts by use of point femtosecond laser targeting.', International journal of biomedical imaging, vol. 2006, p. 10427.View/Download from: UTS OPUS or Publisher's site
A study demonstrating how ultrafast laser radiation stimulates osteoblasts is presented. The study employed a custom made optical system that allowed for simultaneous confocal cell imaging and targeted femtosecond pulse laser irradiation. When femtosecond laser light was focused onto a single cell, a rise in intracellular Ca(2+) levels was observed followed by contraction of the targeted cell. This contraction caused deformation of neighbouring cells leading to a heterogeneous strain field throughout the monolayer. Quantification of the strain fields in the monolayer using digital image correlation revealed local strains much higher than threshold values typically reported to stimulate extracellular bone matrix production in vitro. This use of point targeting with femtosecond pulse lasers could provide a new method for stimulating cell activity in orthopaedic tissue engineering.
Cranfield, CG, Dawe, A, Karloukovski, V, Dunin-Borkowski, RE, de Pomerai, D & Dobson, J 2004, 'Biogenic magnetite in the nematode caenorhabditis elegans.', Proceedings. Biological sciences, vol. 271 Suppl 6, pp. S436-S439.View/Download from: Publisher's site
The nematode Caenorhabditis elegans is widely used as a model system in biological research. Recently, examination of the production of heat-shock proteins in this organism in response to mobile phone-type electromagnetic field exposure produced the most robust demonstration to date of a non-thermal, deleterious biological effect. Though these results appear to be a sound demonstration of non-thermal bioeffects, to our knowledge, no mechanism has been proposed to explain them. We show, apparently for the first time, that biogenic magnetite, a ferrimagnetic iron oxide, is present in C. elegans. Its presence may have confounding effects on experiments involving electromagnetic fields as well as implications for the use of this nematode as a model system for iron biomineralization in multi-cellular organisms.
Cranfield, CG, Dawe, A, Karloukovski, V, Dunin-Borkowski, RE, de Pomerai, D & Dobson, J 2004, 'Biogenic magnetite in the nematode Caenorhabditis elegans', PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, vol. 271, pp. S436-S439.View/Download from: Publisher's site
Cranfield, CG, Weiser, HG & Dobson, J 2003, 'Exposure of magnetic bacteria to simulated mobile phone-type RF radiation has no impact on mortality', IEEE TRANSACTIONS ON NANOBIOSCIENCE, vol. 2, no. 3, pp. 146-149.View/Download from: Publisher's site
Cranfield, C, Wieser, HG, Al Madan, J & Dobson, J 2003, 'Preliminary evaluation of nanoscale biogenic magnetite-based ferromagnetic transduction mechanisms for mobile phone bioeffects', IEEE TRANSACTIONS ON NANOBIOSCIENCE, vol. 2, no. 1, pp. 40-43.View/Download from: Publisher's site
Cranfield, CG, Wood, AW, Anderson, V & Menezes, KG 2001, 'Effects of mobile phone type signals on calcium levels within human leukaemic T-cells (Jurkat cells)', INTERNATIONAL JOURNAL OF RADIATION BIOLOGY, vol. 77, no. 12, pp. 1207-1217.View/Download from: Publisher's site
Cranfield, C, Carne, S, Martinac, B & Cornell, B 2015, 'The assembly and use of tethered bilayer lipid membranes (tBLMs).', Humana Press (Springer Imprint), pp. 45-53.View/Download from: UTS OPUS or Publisher's site
Because they are firmly held in place, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cornell et al. Nature 387(6633): 580-583, 1997). Here we describe the procedures required to assemble and test tethered lipid bilayers that can incorporate various lipid species, peptides, and ion channel proteins.
Macmillan, A, Cranfield, CG & Martinac, B 2014, 'Fluorescence methods for monitoring mechanosensitive channels' in Handbook of Imaging in Biological Mechanics, pp. 425-431.View/Download from: Publisher's site
© 2015 by Taylor & Francis Group, LLC. Over the last 10 to 15 years, fluorescence methods have become frequently used for studies of the conformational changes of various membrane proteins including ion channels that function as gated membrane pores in numerous physiological processes essential for the normal function of biological cells. In combination with molecular biological methods that allow introduction of fluorescent labeling in vivo and in vitro, and functional electrophysiological voltage- and patch-clamp recording techniques, fluorescence spectroscopy methods, such as Förster resonance energy transfer (FRET), have made ion channel proteins accessible to studies of protein structure and function relationship. Here we focus on the use of these methods in studies of structural dynamics of bacterial mechanosensitive channels, which to date are among the best studied membrane channels functioning in mechanosensory transduction in living cells.
Cranfield, CG, Kloda, A, Nomura, T, Petrov, E, Battle, A, Constantine, M & Martinac, B 2013, 'Force from lipids: A multidisciplinary approach to study bacterial mechanosensitive ion channels' in Kamkin & Lozinsky (eds), Mechanically Gated Channels and their Regulation No 6 - Mechanically Gated Channels and their Regula, Springer, USA, pp. 1-34.View/Download from: UTS OPUS
Cranfield, C.G. 2013, 'Techniques for investigating the mechanosensitivity of ion channels' in Encyclopedia of Biophysics, Springer, USA, pp. 1-4.
Martinac, B, Cranfield, CG, Rohde, P & Nomura, T 2013, 'Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.' in Anne Delcour (ed), Methods in Molecular Biology - Bacterial Cell Surfaces, Springer, USA, pp. 367-380.View/Download from: UTS OPUS or Publisher's site
Ion channel studies have been focused on ion channels from animal and human cells over many years. Based on the knowledge acquired, predominantly over the last 20 years, a large diversity of ion channels exists in cellular membranes of prokaryotes as well. Paradoxically, most of what is known about the structure of eukaryotic ion channels is based on the structure of bacterial channels. This is largely due to the suitability of bacterial cells for functional and structural studies of biological macromolecules in a laboratory environment. Development of the "giant spheroplast" preparation from E. coli cells was instrumental for functional studies of ion channels in the bacterial cell membrane. Here we describe detailed protocols used for the preparation of giant spheroplasts as well as protocols used for the patch-clamp recording of native or heterologously expressed ion channels in E. coli spheroplast membrane
Cranfield, CG, Carne, S, Alkhamici, H, Duckworth, P, Lacey, E, Martinac, B & Cornell, B 2014, 'Screening the Insertion of Families of Bioactive Microbial Metabolites into Tethered Bilayer Lipid Membranes (TBLMS)', BIOPHYSICAL JOURNAL, pp. 294A-294A.View/Download from: UTS OPUS or Publisher's site
Cranfield, C.G., Cornell, B., Grage, S.L., Duckworth, P., Carne, S., Ulrich, A.S. & Martinac, B. 2013, 'Characterization of Antimicrobial Peptide Insertion in Tethered Bilayer Lipid Membranes by Pulse Amperometry and Linear Sweep Voltammetry Methods', BIOPHYSICAL JOURNAL, pp. 600A-600A.
Cranfield, C.G., Deplazes, E., MacMillan, A., Owen, D., Nomura, T., Constantine, M., Corry, B. & Martinac, B. 2012, 'Clustering of the Mechanosensitive Ion Channels of Large and Small Conductance MscL and MscS - a FRET-Flim Study', BIOPHYSICAL JOURNAL, pp. 120A-120A.
Dietrich, S, Stanca, SE, Cranfield, CG, Hoffmann, B, Benndorf, K & Biskup, C 2010, 'New strategies to measure intracellular sodium concentrations', MULTIPHOTON MICROSCOPY IN THE BIOMEDICAL SCIENCES X.View/Download from: UTS OPUS or Publisher's site
Fu, L, Jain, A, Xie, H, Cranfield, C & Gu, M 2006, 'Integration of a double-clad photonic crystal fiber, a GRIN lens and a MEMS mirror for nonlinear optical endoscopy', Optics InfoBase Conference Papers.
We report on a prototype of a nonlinear optical endoscope based on a doubleclad photonic crystal fiber and a GRIN lens to improve the detection efficiency and a MEMS mirror to steer the beam. © 2006 Optical Society of America.