Development and application of platform technologies for the study of lipid phase behaviour and biomembrane mechanics

This thesis presents the design, development and application of a number of platform technologies for the study of lipid-phase behaviour and bio-membrane mechanics. This includes an automated laboratory based X-ray beamline with a multi-capillary sample chamber capable of undertaking small angle X-ray scattering measurements on a maximum of 104 samples at a time as a function of temperature between 5 and 85 C. The modular format of the system enables the user to simultaneously equilibrate samples at eight different temperatures with an accuracy of +/-0.005 C. This system couples a rotating anode generator and 2-D optoelectronic detector with Franks X-ray optics, leading to typical exposure times of less than ve minutes for lyotropic liquid crystalline samples. Beamline control including sample exchange and data acquisition has been fully automated via a custom designed LabVIEW framework. In addition this thesis presents an overview of the development of a suite of tools for undertaking fluctuation analysis measurements of lipid vesicles under a variety of conditions including as a function of hydrostatic pressure. These and other biophysical techniques have been used to study a variety of binary lipidic systems determining key parameters ranging from spontaneous curvature and bending rigidity through to p-T dependent phase behaviour.Open Acces

Dependence of norfloxacin diffusion across bilayers on lipid composition.

Antibiotic resistance is a growing concern in medicine and raises the need to develop and design new drug molecules that can efficiently inhibit bacterial replication. Spurring the passive uptake of the drug molecules is an obvious solution. However our limited understanding of drug-membrane interactions due to the presence of an overwhelming variety of lipids constituting cellular membranes and the lack of facile tools to probe the bio-physical interactions between drugs and lipids imposes a major challenge towards developing new drug molecules that can enter the cell via passive diffusion. Here, we used a label-free micro-fluidic platform combined with giant unilamellar lipid vesicles to investigate the permeability of membranes containing mixtures of DOPE and DOPG in DOPC, leading to a label-free measurement of passive membrane-permeability of autofluorescent antibiotics. A fluoroquinolone drug, norfloxacin was used as a case study. Our results indicate that the diffusion of norfloxacin is strongly dependent on the lipid composition which is not expected from the traditional octanol-lipid partition co-efficient assay. The anionic lipid, DOPG, slows the diffusion process whereas the diffusion across liposomes containing DOPE increases with higher DOPE concentration. Our findings emphasise the need to investigate drug-membrane interactions with focus on the specificity of drugs to lipids for efficient drug delivery, drug encapsulation and targeted drug-delivery.SP and UFK acknowledge funding from an ERC starting grant, Passmembrane 261101 and an EPSRC grant GRAPHTED, EP/ K016636/1, and JC acknowledges the support from an Internal Graduate Studentship, Trinity College, Cambridge and a Research Studentship from the Cambridge Philosophical Society.This is the final version of the article. It first appeared from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C5SM02371

Permeability and Line-Tension-Dependent Response of Polyunsaturated Membranes to Osmotic Stresses

The lipidome of plant plasma membranes-enriched in cellular phospholipids containing at least one polyunsaturated fatty acid tail and a variety of phytosterols and phytosphingolipids-is adapted to significant abiotic stresses. But how mesoscale membrane properties of these membranes such as permeability and deformability, which arise from their unique molecular compositions and corresponding lateral organization, facilitate response to global mechanical stresses is largely unknown. Here, using giant vesicles reconstituting mixtures of polyunsaturated lipids (soy phosphatidylcholine), glucosylceramide, and sitosterol common to plant membranes, we find that the membranes adopt "janus-like" domain morphologies and display anomalous solute permeabilities. The former textures the membrane with a single sterol-glucosylceramide-enriched, liquid-ordered domain separated from a liquid-disordered phase consisting primarily of soy phosphatidylcholine. When subject to osmotic downshifts, the giant unilamellar vesicles (GUVs) respond by transiently producing well-known swell-burst cycles. In each cycle, the influx of water swells the GUV, rendering the membrane tense. Subsequent rupture of the membrane through transient poration, which localizes in the liquid-disordered phase or at the domain boundaries, reduces the osmotic stress by expelling some of the excess osmolytes (and solvent) before sealing. When subject to abrupt hypertonic stress, they deform by nucleating buds at the domain phase boundaries. Remarkably, this incipient vesiculation is reversed in a statistically significant fraction of GUVs because of the interplay with solute permeation timescales, which render osmotic stresses short-lived. This, then, suggests a novel control mechanism in which an interplay of permeability and deformability regulates osmotically induced membrane deformation and limits vesiculation-induced loss of membrane material. Interestingly, recapitulation of such dynamic morphological reconfigurability-switching between budded and nonbudded morphologies-due to the interplay of membrane permeability, which temporally reverses the osmotic gradient, and domain boundaries, which select modes of deformations, might prove valuable in endowing synthetic cells with novel morphological responsiveness

Measurements of the effect of membrane asymmetry on the mechanical properties of lipid bilayers

We detail an approach for constructing asymmetric membranes and characterising their mechanical properties, leading to the first measurement of the effect of asymmetry on lipid bilayer mechanics.</p

Recurrent dynamics of rupture transitions of giant lipid vesicles at solid surfaces

Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a "heart"-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition

Negative Emissions at Negative Cost- An Opportunity for a Scalable Niche

In the face of the rapidly dwindling carbon budgets, negative emission technologies are widely suggested as required to stabilize the earth’s climate. However, finding cost-effective, socially acceptable, and politically achievable means to enable such technologies remains a challenge. We propose solutions based on negative emission technologies to facilitate wealth creation for the stakeholders while helping to mitigate climate change. This paper presents a coffee and jackfruit agroforestry-based case study with an array of technical interventions, having a special focus on bioenergy and biochar, potentially leading to “negative emissions at negative cost.” The strategies for integrating food production with soil and water management, fuel production, adoption of renewable energy systems and timber management are outlined. The emphasis is on combining biological and engineering sciences to devise practically viable niche that is easy to adopt, adapt and scale up for the communities and regions to achieve net negative emissions. The concerns expressed in the recent literature on the implementation of emission reduction and negative emission technologies are briefly presented. The novel opportunities to alleviate these concerns arising from our proposed interventions are then pointed out. Finally, the global outlook for an easily adoptable nature-based approach is presented, suggesting an opportunity to implement revenue-generating negative emission technologies at the gigatonne scale. We anticipate that our approach presented in the paper results in increased attention to the development of practically viable science and technology-based interventions in order to support the speeding up of climate change mitigation efforts

Negative emissions at negative cost-an opportunity for a scalable niche

In the face of the rapidly dwindling carbon budgets, negative emission technologies are widely suggested as required to stabilize the Earth’s climate. However, finding cost-effective, socially acceptable, and politically achievable means to enable such technologies remains a challenge. We propose solutions based on negative emission technologies to facilitate wealth creation for the stakeholders while helping to mitigate climate change. This paper comes up with suggestions and guidelines on significantly increasing carbon sequestration in coffee farms. A coffee and jackfruit agroforestry-based case study is presented along with an array of technical interventions, having a special focus on bioenergy and biochar, potentially leading to “negative emissions at negative cost.” The strategies for integrating food production with soil and water management, fuel production, adoption of renewable energy systems and timber management are outlined. The emphasis is on combining biological and engineering sciences to devise a practically viable niche that is easy to adopt, adapt and scale up for the communities and regions to achieve net negative emissions. The concerns expressed in the recent literature on the implementation of emission reduction and negative emission technologies are briefly presented. The novel opportunities to alleviate these concerns arising from our proposed interventions are then pointed out. Our analysis indicates that 1 ha coffee jackfruit-based agroforestry can additionally sequester around 10 tonnes of CO2-eq and lead to an income enhancement of up to 3,000–4,000 Euros in comparison to unshaded coffee. Finally, the global outlook for an easily adoptable nature-based approach is presented, suggesting an opportunity to implement revenue-generating negative emission technologies on a gigatonne scale. We anticipate that our approach presented in the paper results in increased attention to the development of practically viable science and technology-based interventions in order to support the speeding up of climate change mitigation efforts