Computer-assisted design for scaling up systems based on DNA reaction networks

In the past few years, there have been many exciting advances in the field of molecular programming, reaching a point where implementation of non-trivial systems, such as neural networks or switchable bistable networks, is a reality. Such systems require nonlinearity, be it through signal amplification, digitalization or the generation of autonomous dynamics such as oscillations. The biochemistry of DNA systems provides such mechanisms, but assembling them in a constructive manner is still a difficult and sometimes counterintuitive process. Moreover, realistic prediction of the actual evolution of concentrations over time requires a number of side reactions, such as leaks, cross-talks or competitive interactions, to be taken into account. In this case, the design of a system targeting a given function takes much trial and error before the correct architecture can be found. To speed up this process, we have created DNA Artificial Circuits Computer-Assisted Design (DACCAD), a computer-assisted design software that supports the construction of systems for the DNA toolbox. DACCAD is ultimately aimed to design actual in vitro implementations, which is made possible by building on the experimental knowledge available on the DNA toolbox. We illustrate its effectiveness by designing various systems, from Montagne et al.''s Oligator or Padirac et al.''s bistable system to new and complex networks, including a two-bit counter or a frequency divider as well as an example of very large system encoding the game Mastermind. In the process, we highlight a variety of behaviours, such as enzymatic saturation and load effect, which would be hard to handle or even predict with a simpler model. We also show that those mechanisms, while generally seen as detrimental, can be used in a positive way, as functional part of a design. Additionally, the number of parameters included in these simulations can be large, especially in the case of complex systems. For this reason, we included the possibility to use CMA-ES, a state-of-the-art optimization algorithm that will automatically evolve parameters chosen by the user to try to match a specified behaviour. Finally, because all possible functionality cannot be captured by a single software, DACCAD includes the possibility to export a system in the synthetic biology markup language, a widely used language for describing biological reaction systems. DACCAD can be downloaded online at http://www.yannick-rondelez.com/downloads/.     

A constitutive model for muscle properties in a soft-bodied arthropod

In this paper, we examine the mechanical properties of muscles in a soft-bodied arthropod under both passive and stimulated conditions. In particular, we examine the ventral interior lateral muscle of the tobacco hornworm caterpillar, Manduca sexta, and show that its response is qualitatively similar to the behaviour of particle-reinforced rubber. Both materials are capable of large nonlinear elastic deformations, show a hysteretic behaviour and display stress softening during the first few cycles of repeated loading. The Manduca muscle can therefore be considered as different elastic materials during loading and unloading and is best described using the theory of pseudo-elasticity. We summarize the basic equations for transversely isotropic pseudo-elastic materials, first for general deformations and then for the appropriate uniaxial specialization. The constitutive relation proposed is in good agreement with the experimental data for both the passive and the stimulated conditions.     

A dynamical model for the Utricularia trap

We propose a model that captures the dynamics of a carnivorous plant, Utricularia inflata. This plant possesses tiny traps for capturing small aquatic animals. Glands pump water out of the trap, yielding a negative pressure difference between the plant and its surroundings. The trap door is set into a meta-stable state and opens quickly as an extra pressure is generated by the displacement of a potential prey. As the door opens, the pressure difference sucks the animal into the trap. We write an ODE model that captures all the physics at play. We show that the dynamics of the plant is quite similar to neuronal dynamics and we analyse the effect of a white noise on the dynamics of the trap.     

Multiphasic construct studied in an ectopic osteochondral defect model

In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.     

Maintenance of phenotypic diversity within a set of virulence encoding genes of the malaria parasite Plasmodium falciparum

Infection by the human malaria parasite Plasmodium falciparum results in a broad spectrum of clinical outcomes, ranging from severe and potentially life-threatening malaria to asymptomatic carriage. In a process of naturally acquired immunity, individuals living in malaria-endemic regions build up a level of clinical protection, which attenuates infection severity in an exposure-dependent manner. Underlying this shift in the immunoepidemiology as well as the observed range in malaria pathogenesis is the var multigene family and the phenotypic diversity embedded within. The var gene-encoded surface proteins Plasmodium falciparum erythrocyte membrane protein 1 mediate variant-specific binding of infected red blood cells to a diverse set of host receptors that has been linked to specific disease manifestations, including cerebral and pregnancy-associated malaria. Here, we show that cross-reactive immune responses, which minimize the within-host benefit of each additionally expressed gene during infection, can cause selection for maximum phenotypic diversity at the genome level. We further show that differential functional constraints on protein diversification stably maintain uneven ratios between phenotypic groups, in line with empirical observation. Our results thus suggest that the maintenance of phenotypic diversity within P. falciparum is driven by an evolutionary trade-off that optimizes between within-host parasite fitness and between-host selection pressure.     

Transmission dynamics of methicillin-resistant Staphylococcus aureus in a medical intensive care unit

Intensive care units (ICUs) play an important role in the epidemiology of methicillin-resistant Staphyloccocus aureus (MRSA). Although successful interventions are multi-modal, the relative efficacy of single measures remains unknown. We developed a discrete time, individual-based, stochastic mathematical model calibrated on cross-transmission observed through prospective surveillance to explore the transmission dynamics of MRSA in a medical ICU. Most input parameters were derived from locally acquired data. After fitting the model to the 46 observed cross-transmission events and performing sensitivity analysis, several screening and isolation policies were evaluated by simulating the number of cross-transmissions and isolation-days. The number of all cross-transmission events increased from 54 to 72 if only patients with a past history of MRSA colonization are screened and isolated at admission, to 75 if isolation is put in place only after the results of the admission screening become available, to 82 in the absence of admission screening and with a similar reactive isolation policy, and to 95 when no isolation policy is in place. The method used (culture or polymerase chain reaction) for admission screening had no impact on the number of cross-transmissions. Systematic regular screening during ICU stay provides no added-value, but aggressive admission screening and isolation effectively reduce the number of cross-transmissions. Critically, colonized healthcare workers may play an important role in MRSA transmission and their screening should be reinforced.     

Disparity and convergence in bipedal archosaur locomotion

This study aims to investigate functional disparity in the locomotor apparatus of bipedal archosaurs. We use reconstructions of hindlimb myology of extant and extinct archosaurs to generate musculoskeletal biomechanical models to test hypothesized convergence between bipedal crocodile-line archosaurs and dinosaurs. Quantitative comparison of muscle leverage supports the inference that bipedal crocodile-line archosaurs and non-avian theropods had highly convergent hindlimb myology, suggesting similar muscular mechanics and neuromuscular control of locomotion. While these groups independently evolved similar musculoskeletal solutions to the challenges of parasagittally erect bipedalism, differences also clearly exist, particularly the distinct hip and crurotarsal ankle morphology characteristic of many pseudosuchian archosaurs. Furthermore, comparative analyses of muscle design in extant archosaurs reveal that muscular parameters such as size and architecture are more highly adapted or optimized for habitual locomotion than moment arms. The importance of these aspects of muscle design, which are not directly retrievable from fossils, warns against over-extrapolating the functional significance of anatomical convergences. Nevertheless, links identified between posture, muscle moments and neural control in archosaur locomotion suggest that functional interpretations of osteological changes in limb anatomy traditionally linked to postural evolution in Late Triassic archosaurs could be constrained through musculoskeletal modelling.     

Bio-electrospraying and aerodynamically assisted bio-jetting the model eukaryotic Dictyostelium discoideum: assessing stress and developmental competency post treatment

Bio-electrospraying (BES) and aerodynamically assisted bio-jetting (AABJ) have recently been established as important novel biospray technologies for directly manipulating living cells. To elucidate their potential in medical and clinical sciences, these bio-aerosol techniques have been subjected to increasingly rigorous investigations. In parallel to these studies, we wish to introduce these unique biotechnologies for use in the basic biological sciences, for handling a wide range of cell types and systems, thus increasing the range and the scope of these techniques for modern research. Here, the authors present the analysis of the new use of these biospray techniques for the direct handling of the simple eukaryotic biomedical model organism Dictyostelium discoideum. These cells are widely used as a model for immune cell chemotaxis and as a simple model for development. We demonstrate that AABJ of these cells did not cause cell stress, as defined by the stress-gene induction, nor affect cell development. Furthermore, although BES induced the increased expression of one stress-related gene (gapA), this was not a generalized stress response nor did it affect cell development. These data suggest that these biospray techniques can be used to directly manipulate single cells of this biomedical model without inducing a generalized stress response or perturbing later development.     

Directional scattering from the glossy flower of Ranunculus: how the buttercup lights up your chin

The bright and glossy appearance of the flowers of Ranunculus repens was investigated spectroscopically and the optical results were correlated with the layered anatomy of the petal. The highly directional reflected light arises from the partially transparent, pigment-bearing epidermal layer, while a more diffused yellow colour is the result of scattering from the lower starch layer. This directionality of the light reflections causes the unusually intense gloss of the buttercup flower and the strong yellow reflection evident when holding the flower under the chin.     

Suitability of European climate for the Asian tiger mosquito Aedes albopictus: recent trends and future scenarios

The Asian tiger mosquito (Aedes albopictus) is an invasive species that has the potential to transmit infectious diseases such as dengue and chikungunya fever. Using high-resolution observations and regional climate model scenarios for the future, we investigated the suitability of Europe for A. albopictus using both recent climate and future climate conditions. The results show that southern France, northern Italy, the northern coast of Spain, the eastern coast of the Adriatic Sea and western Turkey were climatically suitable areas for the establishment of the mosquito during the 1960–1980s. Over the last two decades, climate conditions have become more suitable for the mosquito over central northwestern Europe (Benelux, western Germany) and the Balkans, while they have become less suitable over southern Spain. Similar trends are likely in the future, with an increased risk simulated over northern Europe and slightly decreased risk over southern Europe. These distribution shifts are related to wetter and warmer conditions favouring the overwintering of A. albopictus in the north, and drier and warmer summers that might limit its southward expansion.     

Metapopulation dynamics of Escherichia coli O157 in cattle: an exploratory model.

Livestock movement is thought to be a risk factor for the transmission of infectious diseases of farm animals. Simple mathematical models were constructed for the transmission of Escherichia coli serogroup O157 between Scottish cattle farms, and the models were used in a preliminary exploration of factors contributing to the levels of infection reported in the field. The results suggest that cattle movement can make a significant contribution to the observed prevalence of E. coli O157-positive farms, but is not by itself sufficient for the persistence of E. coli O157. The results also suggest that cattle movements involving infected farms with cattle shedding an exceptional amount of E. coli O157, 'super-shedders', also make a substantial contribution to the prevalence of infected farms. Simulations indicate that E. coli O157 could have reached the currently observed prevalence levels in less than a decade. Implications and findings from our models are discussed in relation to possible control of E. coli O157 in Scottish cattle.     

Insights into mucosal innate responses to Escherichia coli O157 : H7 colonization of cattle by mathematical modelling of excretion dynamics

Mathematical model-based statistical inference applied to within-host dynamics of infectious diseases can help dissect complex interactions between hosts and microbes. This work has applied advances in model-based inference to understand colonization of cattle by enterohaemorrhagic Escherichia coli O157 : H7 at the terminal rectum. A mathematical model was developed based on niche replication and transition rates at this site. A nested-model comparison, applied to excretion curves from 25 calves, was used to reduce complexity while maintaining integrity. We conclude that, 5–9 days post inoculation, the innate immune response negates bacterial replication on the epithelium and either reduces attachment to or increases detachment from the epithelium of the terminal rectum. Thus, we provide a broadly applicable model that gives novel insights into bacterial replication rates in vivo and the timing and impact of host responses.     

Understanding macroalgal dispersal in a complex hydrodynamic environment: a combined population genetic and physical modelling approach

Gene flow in macroalgal populations can be strongly influenced by spore or gamete dispersal. This, in turn, is influenced by a convolution of the effects of current flow and specific plant reproductive strategies. Although several studies have demonstrated genetic variability in macroalgal populations over a wide range of spatial scales, the associated current data have generally been poorly resolved spatially and temporally. In this study, we used a combination of population genetic analyses and high-resolution hydrodynamic modelling to investigate potential connectivity between populations of the kelp Laminaria digitata in the Strangford Narrows, a narrow channel characterized by strong currents linking the large semi-enclosed sea lough, Strangford Lough, to the Irish Sea. Levels of genetic structuring based on six microsatellite markers were very low, indicating high levels of gene flow and a pattern of isolation-by-distance, where populations are more likely to exchange migrants with geographically proximal populations, but with occasional long-distance dispersal. This was confirmed by the particle tracking model, which showed that, while the majority of spores settle near the release site, there is potential for dispersal over several kilometres. This combined population genetic and modelling approach suggests that the complex hydrodynamic environment at the entrance to Strangford Lough can facilitate dispersal on a scale exceeding that proposed for L. digitata in particular, and the majority of macroalgae in general. The study demonstrates the potential of integrated physical–biological approaches for the prediction of ecological changes resulting from factors such as anthropogenically induced coastal zone changes.     

Tuberculosis reinfection rate as a proportion of total infection rate correlates with the logarithm of the incidence rate: a mathematical model

In a significant number of instances, an episode of tuberculosis can be attributed to a reinfection event. Because reinfection is more likely in high incidence regions than in regions of low incidence, more tuberculosis (TB) cases due to reinfection could be expected in high-incidence regions than in low-incidence regions. Empirical data from regions with various incidence rates appear to confirm the conjecture that, in fact, the incidence rate due to reinfection only, as a proportion of all cases, correlates with the logarithm of the incidence rate, rather than with the incidence rate itself. A theoretical model that supports this conjecture is presented. A Markov model was used to obtain a relationship between incidence and reinfection rates. It was assumed in this model that the rate of reinfection is a multiple, ρ (the reinfection factor), of the rate of first-time infection, λ. The results obtained show a relationship between the proportion of cases due to reinfection and the rate of incidence that is approximately logarithmic for a range of values of the incidence rate typical of those observed in communities across the globe. A value of ρ is determined such that the relationship between the proportion of cases due to reinfection and the logarithm of the incidence rate closely correlates with empirical data. From a purely theoretical investigation, it is shown that a simple relationship can be expected between the logarithm of the incidence rates and the proportions of cases due to reinfection after a prior episode of TB. This relationship is sustained by a rate of reinfection that is higher than the rate of first-time infection and this latter consideration underscores the great importance of monitoring recovered TB cases for repeat disease episodes, especially in regions where TB incidence is high. Awareness of this may assist in attempts to control the epidemic.     

Cerebrospinal fluid dynamics in the human cranial subarachnoid space: an overlooked mediator of cerebral disease. II. In vitro arachnoid outflow model

The arachnoid membrane (AM) and granulations (AGs) are important in cerebrospinal fluid (CSF) homeostasis, regulating intracranial pressure in health and disease. We offer a functional perspective of the human AM''s transport mechanism to clarify the role of AM in the movement of CSF and metabolites. Using cultures of human AG cells and a specialized perfusion system, we have shown that this in vitro model mimics the in vivo characteristics of unidirectional fluid transport and we present the first report of serum-free permeability values (92.5 µl min⁻¹ mm Hg⁻¹ cm⁻²), which in turn are in agreement with the CSF outflow rates derived from a dynamic, in vivo magnetic resonance imaging-based computational model of the subarachnoid cranial space (130.9 µl min⁻¹ mm Hg⁻¹ cm⁻²). Lucifer yellow permeability experiments have verified the maintenance of tight junctions by the arachnoidal cells with a peak occurring around 21 days post-seeding, which is when all perfusion experiments were conducted. Addition of ruthenium red to the perfusate, and subsequent analysis of its distribution post-perfusion, has verified the passage of perfusate via both paracellular and transcellular mechanisms with intracellular vacuoles of approximately 1 µm in diameter being the predominant transport mechanism. The comparison of the computational and in vitro models is the first report to measure human CSF dynamics functionally and structurally, enabling the development of innovative approaches to modify CSF outflow and will change concepts and management of neurodegenerative diseases resulting from CSF stagnation.     

Transient dynamic phenotypes as criteria for model discrimination: fold-change detection in Rhodobacter sphaeroides chemotaxis

The chemotaxis pathway of the bacterium Rhodobacter sphaeroides shares many similarities with that of Escherichia coli. It exhibits robust adaptation and has several homologues of the latter''s chemotaxis proteins. Recent theoretical results have correctly predicted that the E. coli output behaviour is unchanged under scaling of its ligand input signal; this property is known as fold-change detection (FCD). In the light of recent experimental results suggesting that R. sphaeroides may also show FCD, we present theoretical assumptions on the R. sphaeroides chemosensory dynamics that can be shown to yield FCD behaviour. Furthermore, it is shown that these assumptions make FCD a property of this system that is robust to structural and parametric variations in the chemotaxis pathway, in agreement with experimental results. We construct and examine models of the full chemotaxis pathway that satisfy these assumptions and reproduce experimental time-series data from earlier studies. We then propose experiments in which models satisfying our theoretical assumptions predict robust FCD behaviour where earlier models do not. In this way, we illustrate how transient dynamic phenotypes such as FCD can be used for the purposes of discriminating between models that reproduce the same experimental time-series data.     

Regulatory principles and experimental approaches to the circadian control of starch turnover

In many plants, starch is synthesized during the day and degraded during the night to avoid carbohydrate starvation in darkness. The circadian clock participates in a dynamic adjustment of starch turnover to changing environmental condition through unknown mechanisms. We used mathematical modelling to explore the possible scenarios for the control of starch turnover by the molecular components of the plant circadian clock. Several classes of plausible models were capable of describing the starch dynamics observed in a range of clock mutant plants and light conditions, including discriminating circadian protocols. Three example models of these classes are studied in detail, differing in several important ways. First, the clock components directly responsible for regulating starch degradation are different in each model. Second, the intermediate species in the pathway may play either an activating or inhibiting role on starch degradation. Third, the system may include a light-dependent interaction between the clock and downstream processes. Finally, the clock may be involved in the regulation of starch synthesis. We discuss the differences among the models’ predictions for diel starch profiles and the properties of the circadian regulators. These suggest additional experiments to elucidate the pathway structure, avoid confounding results and identify the molecular components involved.     

The control of sea lice in Atlantic salmon by selective breeding

Sea lice threaten the welfare of farmed Atlantic salmon and the sustainability of fish farming across the world. Chemical treatments are the major method of control but drug resistance means that alternatives are urgently needed. Selective breeding can be a cheap and effective alternative. Here, we combine experimental trials and diagnostics to provide a practical protocol for quantifying resistance to sea lice. We then combined quantitative genetics with epidemiological modelling to make the first prediction of the response to selection, quantified in terms of reduced need for chemical treatments. We infected over 1400 young fish with Lepeophtheirus salmonis, the most important species in the Northern Hemisphere. Mechanisms of resistance were expressed early in infection. Consequently, the number of lice per fish and the ranking of families were very similar at 7 and 17 days post infection, providing a stable window for assessing susceptibility to infection. The heritability of lice numbers within this time window was moderately high at 0.3, confirming that selective breeding is viable. We combined an epidemiological model of sea lice infection and control on a salmon farm with genetic variation in susceptibility among individuals. We simulated 10 generations of selective breeding and examined the frequency of treatments needed to control infection. Our model predicted that substantially fewer chemical treatments are needed to control lice outbreaks in selected populations and chemical treatment could be unnecessary after 10 generations of selection. Selective breeding for sea lice resistance should reduce the impact of sea lice on fish health and thus substantially improve the sustainability of Atlantic salmon production.     

Measurements of the wall shear stress distribution in the outflow tract of an embryonic chicken heart

In order to study the role of blood–tissue interaction in the developing chicken embryo heart, detailed information about the haemodynamic forces is needed. In this study, we present the first in vivo measurements of the three-dimensional distribution of wall shear stress (WSS) in the outflow tract (OFT) of an embryonic chicken heart. The data are obtained in a two-step process: first, the three-dimensional flow fields are measured during the cardiac cycle using scanning microscopic particle image velocimetry; second, the location of the wall and the WSS are determined by post-processing flow velocity data (finding velocity gradients at locations where the flow approaches zero). The results are a three-dimensional reconstruction of the geometry, with a spatial resolution of 15–20 µm, and provides detailed information about the WSS in the OFT. The most significant error is the location of the wall, which results in an estimate of the uncertainty in the WSS values of 20 per cent.     

Investigating the rheological properties of native plant latex

Plant latex, the source of natural rubber, has been of interest to mankind for millennia, with much of the research on its rheological (flow) properties focused towards industrial application. However, little is known regarding the rheology of the native material as produced by the plant, a key factor in determining latex''s biological functions. In this study, we outline a method for rheological comparison between native latices that requires a minimum of preparatory steps. Our approach provides quantitative insights into the coagulation mechanisms of Euphorbia and Ficus latex allowing interpretation within a comparative evolutionary framework. Our findings reveal that in laboratory conditions both latices behave like non-Newtonian materials with the coagulation of Euphorbia latex being mediated by a slow evaporative process (more than 60 min), whereas Ficus appears to use additional biochemical components to increase the rate of coagulation (more than 30 min). Based on these results, we propose two different primary defensive roles for latex in these plants: the delivery of anti-herbivory compounds (Euphorbia) and rapid wound healing (Ficus).