In vitro Recapitulation of the Polymicrobial Communities Associated with Cystic Fibrosis Airway Infections

The airways of persons with cystic fibrosis (CF) are prone to lifelong colonisation with dense microbial ecosystems comprised of a diverse combination of bacteria and fungi. The role of interspecies interactions in modulating changes in gene expression and the metabolism of individual members of a polymicrobial community, compared with growth as an axenic population, is becoming increasingly apparent within the field of medical microbiology. In part, these poorly understood interactions explain why, regardless of intense research efforts, therapeutic treatments designed to clear such chronic airway infections often fail within the clinical setting. The paucity of existing in vitro and in vivo models used to study CF-associated infections greatly hinders research into the behaviours of polymicrobial communities. Species that coexist within the same in situ environment often outcompete one another when grown as a mixed population using traditional culture methods. The work presented in this dissertation redresses these problems. I describe the development and characterisation of a novel in vitro continuous-flow culture model. This experimental system permits the stable co-cultivation of distinctly different microbial species commonly-associated with CF airway infections; species that ordinarily readily outcompete one another in existing models. I demonstrate that the metabolic state and mutation rates of species cultured within the model remain stable for extended periods of time. Furthermore, I show that antimicrobial compounds display decreased efficacy against their target organism when grown as part of a polymicrobial community. I also show that different combinations of microbial species display different biofilm-forming potential. This work provides the basis of future research efforts aimed at the in vitro recapitulation of an entire polymicrobial community directly derived from persons with CF. Ultimately, this will help to bridge the bench-to-bedside gap for the development of more efficacious airway infection management regimens.NC3Rs studentship NC/P001564/1 and further consumables support from the UK Cystic Fibrosis Trust (Venture and Innovation Award) and the British Lung Foundation

Universal Properties of Galactic Rotation Curves and a First Principles Derivation of the Tully-Fisher Relation

In a recent paper McGaugh, Lelli, and Schombert showed that in an empirical plot of the observed centripetal accelerations in spiral galaxies against those predicted by the Newtonian gravity of the luminous matter in those galaxies the data points occupied a remarkably narrow band. While one could summarize the mean properties of the band by drawing a single mean curve through it, by fitting the band with the illustrative conformal gravity theory with fits that fill out the width of the band we show here that the width of the band is just as physically significant. We show that at very low luminous Newtonian accelerations the plot can become independent of the luminous Newtonian contribution altogether, but still be non-trivial due to the contribution of matter outside of the galaxies (viz. the rest of the visible universe). We present a new empirical plot of the difference between the observed centripetal accelerations and the luminous Newtonian expectations as a function of distance from the centers of galaxies, and show that at distances greater than 10 kpc the plot also occupies a remarkably narrow band, one even close to constant. Using the conformal gravity theory we provide a first principles derivation of the empirical Tully-Fisher relation.Comment: 6 pages, 15 figures. The paper is a comment on S. S. McGaugh, F. Lelli, and J. M. Schombert, Phys. Rev. Lett. 117, 201101 (2016). Updated to include a first principles derivation of the Tully-Fisher relation using the conformal gravity theory. Submitted to Physics Letters

A Continuous-Flow Model for in vitro Cultivation of Mixed Microbial Populations Associated With Cystic Fibrosis Airway Infections.

The airways of people with cystic fibrosis (CF) provide a nutrient-rich environment which favours colonisation by a variety of bacteria and fungi. Although the dominant pathogen associated with CF airway infections is Pseudomonas aeruginosa, it is becoming increasingly clear that inter-species interactions between P. aeruginosa and other colonists in the airways may have a large impact on microbial physiology and virulence. However, there are currently no suitable experimental models that permit long-term co-culture of P. aeruginosa with other CF-associated pathogens. Here, we redress this problem by describing a "3R's-compliant" continuous-flow in vitro culture model which enables long-term co-culture of three representative CF-associated microbes: P. aeruginosa, Staphylococcus aureus and Candida albicans. Although these species rapidly out-compete one another when grown together or in pairs in batch culture, we show that in a continuously-fed setup, they can be maintained in a very stable, steady-state community. We use our system to show that even numerically (0.1%) minor species can have a major impact on intercellular signalling by P. aeruginosa. Importantly, we also show that co-culturing does not appear to influence species mutation rates, further reinforcing the notion that the system favours stability rather than divergence. The model is experimentally tractable and offers an inexpensive yet robust means of investigating inter-species interactions between CF pathogens

A Test for Characterizing Delamination Migration in Carbon/Epoxy Tape Laminates

A new test method is presented for the purpose of investigating migration of a delamination between neighboring ply interfaces in fiber-reinforced, polymer matrix tape laminates. The test is a single cantilever beam configuration consisting of a cross-ply laminate with a polytetrafluoroethylene insert implanted at the mid-plane and spanning part way along the length of the specimen. The insert is located between a 0- degree ply (specimen length direction) and a stack of four 90-degree plies (specimen width direction). The specimen is clamped at both ends onto a rigid baseplate and is loaded on its upper surface via a piano hinge. Tests were conducted with the load-application point located on the intact portion of the specimen in order to initiate delamination growth onset followed by migration of the delamination to a neighboring 90/0 ply interface by kinking through the 90-degree ply stack. Varying this position was found to affect the distance relative to the load-application point at which migration initiated. In each specimen, migration initiated by a gradual transition of the delamination at the 0/90 interface into the 90-degree ply stack. In contrast, transition of the kinked crack into the 90/0 interface was sudden. Fractography of the specimens indicated that delamination prior to migration was generally mixed mode-I/II. Inspection of the kink surface revealed mode-I fracture. In general, use of this test allows for the observation of the growth of a delamination followed by migration of the delamination to another ply interface, and should thus provide a means for validating analyses aimed at simulating migration

Biomechanical locomotion adaptations on uneven surfaces can be simulated with a randomly deforming shoe midsole

Background: A shoe with unsystematic perturbations, similar to natural uneven terrain, may offer an enhanced training stimulus over current unstable footwear technologies. This study compared the instability of a shoe with unpredictably random midsole deformations, an irregular surface and a control shoe-surface whilst treadmill walking and running. Methods: Three-dimensional kinematics and electromyography were recorded of the lower limb in 18 active males. Gait cycle characteristics, joint angles at initial ground contact and maximum values during stance, and muscle activations prior to initial contact and during loading were analysed. Perceived stability, injury-risk and energy consumption were evaluated. Instability was assessed by movement variability, muscular activations and subjective ratings. Results: Posture alterations at initial contact revealed active adaptations in the irregular midsole and irregular surface to maintain stability whilst walking and running. Variability of the gait cycle and lower limb kinematics increased on the irregular surface compared to the control across locomotion types. Similarly increased variability (coefficient of variation) were found in the irregular midsole compared to the control for frontal ankle motion (walk: 31.1 and 14.9, run: 28.1 and 11.6), maximum sagittal knee angle (walk: 7.6 and 4.8, run: 2.8 and 2.4), and global gait characteristics during walking only (2.1 ± 0.5 and 1.6 ± 0.3). Tibialis anterior pre-activation reduced and gastrocnemius activation increased in the irregular midsole compared to the control across locomotion types. During running, peroneus longus activation increased in the irregular midsole and irregular surface. Conclusions: Results indicate random shoe midsole deformations enhanced instability relative to the control and simulated certain locomotion adaptations of the irregular surface, although less pronounced. Thus, a shoe with unpredictable instability revealed potential as a novel instability-training device

From Centroided to Profile Mode: Machine Learning for Prediction of Peak Width in HRMS Data

Centroiding is one of the major approaches used for size reduction of the data generated by high-resolution mass spectrometry. During centroiding, performed either during acquisition or as a pre-processing step, the mass profiles are represented by a single value (i.e., the centroid). While being effective in reducing the data size, centroiding also reduces the level of information density present in the mass peak profile. Moreover, each step of the centroiding process and their consequences on the final results may not be completely clear. Here, we present Cent2Prof, a package containing two algorithms that enables the conversion of the centroided data to mass peak profile data and vice versa. The centroiding algorithm uses the resolution-based mass peak width parameter as the first guess and self-adjusts to fit the data. In addition to the m/z values, the centroiding algorithm also generates the measured mass peak widths at half-height, which can be used during the feature detection and identification. The mass peak profile prediction algorithm employs a random-forest model for the prediction of mass peak widths, which is consequently used for mass profile reconstruction. The centroiding results were compared to the outputs of the MZmine-implemented centroiding algorithm. Our algorithm resulted in rates of false detection ≤5% while the MZmine algorithm resulted in 30% rate of false positive and 3% rate of false negative. The error in profile prediction was ≤56% independent of the mass, ionization mode, and intensity, which was 6 times more accurate than the resolution-based estimated values.publishedVersio