Biology subjects

Cama, J.

Publications and source records attributed to Cama, J..

7 recordsLinked to original sources

bioRxiv2021

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions - their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptides physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

microbiology↗

bioRxiv2021

Conformable and robust force sensors to enable precision joint replacement surgery

Balancing forces within weight-bearing joints such as the hip during joint replacement surgeries is essential for implant longevity. Minimising implant failure is vital to improve patient wellbeing and alleviate pressure on healthcare systems. With improvements in surgery, hip replacement patients are now often younger and more active than in previous generations, and their implants correspondingly need to survive higher stresses. However, force balancing currently depends entirely on surgical skill: no sensors can provide quantitative force feedback within the hip joints small, complex geometry. Here, we solve this unmet clinical need by presenting a thin and conformable microfluidic force sensor, which is compatible with the standard surgical process. We optimised the design using finite element modelling, then incorporated and calibrated our sensor in a model hip implant. Using a bespoke testing rig, we demonstrated high sensitivity at typical forces experienced during hip replacements. We anticipate that these sensors will aid implant positioning, increasing the lifetime of hip replacements, and represent a powerful new surgical tool for a range of orthopaedic procedures where force balancing is crucial.

bioengineering↗

bioRxiv2021

Measuring thousands of single vesicle leakage events reveals the mode of action of antimicrobial peptides

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, existing characterization assays lack the resolution necessary to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations, and related the observed modes with the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner, and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

biophysics↗

bioRxiv2019

Biophysical characterization reveals the similarities of liposomes produced using microfluidics and electroformation

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 m2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

biophysics↗

bioRxiv2019

Label-free single-cell microfluidics reveals antibiotic transport kinetics in Gram-negative bacteria

The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to the complexity of the problem and a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to quantify antibiotic uptake label-free in hundreds of individual Escherichia coli cells. By manipulating the microenvironment, we showed that drug (ofloxacin) accumulation is higher in growing versus non-growing cells. Using genetic knockouts, we provide the first direct evidence that growth phase is more important for drug accumulation than the presence or absence of individual transport pathways. We use our experimental results to inform a mathematical model that predicts drug accumulation kinetics in subcellular compartments. These novel experimental and theoretical results pave the way for the rational design of new Gram-negative antibiotics.

biophysics↗

bioRxiv2019

An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological salt and pH levels, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin across biomimetic liposomes.

biophysics↗

bioRxiv2018

Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner

Bacterial cells are critically dependent upon pH regulation. Most proteins function over a limited pH range and the pH gradient across the bacterial cell membrane is central to energy production and transduction1. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of E. coli. Indole is an aromatic molecule with diverse signalling roles that in bacteria is produced from tryptophan by the enzyme tryptophanase (TnaA)2. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry3,4. We show that E. coli cells growing under conditions where no indole is produced maintain their cytoplasmic pH at 7.8 {+/-} 0.2. In contrast, under conditions permitting indole production, pH is maintained at 7.2 {+/-} 0.2. Experiments where indole was added experimentally to non-producing cultures showed that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, the application of an artificial pulse of either of two non-biological proton ionophores (DNP or CCCP) caused a similar effect, suggesting that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane5. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a \"memory\" to maintain the correct cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.

microbiology↗