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.     

Towards electrically conductive, self-healing materials.

A novel class of organometallic polymers comprising N-heterocyclic carbenes and transition metals was shown to have potential as an electrically conductive, self-healing material. These polymers were found to exhibit conductivities of the order of 10(-3) S cm-1 and showed structurally dynamic characteristics in the solid-state. Thin films of these materials were cast onto silicon wafers, then scored and imaged using a scanning electron microscopy (SEM). The scored films were subsequently healed via thermal treatment, which enabled the material to flow via a unique depolymerization process, as determined by SEM and surface profilometry. A method for incorporating these features into a device that exhibits electrically driven, self-healing functions is proposed.     

Co-culture systems and technologies: taking synthetic biology to the next level

Co-culture techniques find myriad applications in biology for studying natural or synthetic interactions between cell populations. Such techniques are of great importance in synthetic biology, as multi-species cell consortia and other natural or synthetic ecology systems are widely seen to hold enormous potential for foundational research as well as novel industrial, medical and environmental applications with many proof-of-principle studies in recent years. What is needed for co-cultures to fulfil their potential? Cell–cell interactions in co-cultures are strongly influenced by the extracellular environment, which is determined by the experimental set-up, which therefore needs to be given careful consideration. An overview of existing experimental and theoretical co-culture set-ups in synthetic biology and adjacent fields is given here, and challenges and opportunities involved in such experiments are discussed. Greater focus on foundational technology developments for co-cultures is needed for many synthetic biology systems to realize their potential in both applications and answering biological questions.     

Population structure in the Neisseria, and the biological significance of fuzzy species

Phenotypic and genetic variation in bacteria can take bewilderingly complex forms even within a single genus. One of the most intriguing examples of this is the genus Neisseria, which comprises both pathogens and commensals colonizing a variety of body sites and host species, and causing a range of disease. Complex relatedness among both named species and previously identified lineages of Neisseria makes it challenging to study their evolution. Using the largest publicly available collection of bacterial sequence data in combination with a population genetic analysis and experiment, we probe the contribution of inter-species recombination to neisserial population structure, and specifically whether it is more common in some strains than others. We identify hybrid groups of strains containing sequences typical of more than one species. These groups of strains, typical of a fuzzy species, appear to have experienced elevated rates of inter-species recombination estimated by population genetic analysis and further supported by transformation experiments. In particular, strains of the pathogen Neisseria meningitidis in the fuzzy species boundary appear to follow a different lifestyle, which may have considerable biological implications concerning distribution of novel resistance elements and meningococcal vaccine development. Despite the strong evidence for negligible geographical barriers to gene flow within the population, exchange of genetic material still shows directionality among named species in a non-uniform manner.     

The influence of surface lubricity on the adhesion of Navicula perminuta and Ulva linza to alkanethiol self-assembled monolayers

The settlement and adhesion of Navicula perminuta and Ulva linza to methyl-terminated alkanethiol self-assembled monolayers (SAMs) of increasing chain length has been investigated. Organisms were allowed to settle onto the monolayers and were subsequently exposed to hydrodynamic shear stress in order to determine their adhesion strength. Results show that as the SAM structure changes from amorphous to crystalline (C14), there is a marked change in the adhesion of N. perminuta and U. linza. Given that the SAMs in the series all exhibit similar contact angle behaviour and surface energy, it is hypothesized that the lubricity of the surface plays a role in determining the surface adhesion.     

The dynamic influence of genetic variation on the susceptibility of sheep to gastrointestinal nematode infection.

The interaction between sheep and the nematode Teladorsagia circumcincta is one of the best understood of all host-parasite interactions. Following infection, there is considerable variation among lambs in the number of nematode eggs produced, the number of early fourth-stage larvae and the number of adult worms in the mucosa. These traits have a high variance to mean ratio (i.e. they are overdispersed or aggregated among hosts), they are skewed and approximately negative binomially distributed. The sources of overdispersion are differences among lambs in the ingestion of infective larvae and the immune response. Both forces can produce aggregation but their relative importance is unknown. The key components of variation can be identified by variance analysis. The sum of the average effects of polymorphic genes is known as additive genetic variation and this increases essentially from zero at one month of age to quite high values at six months of age. The major mechanism underlying genetic variation appears to be the differences among individuals in immune responses. Two of the major sources of variation in immune responses are differences in antigen recognition and differences in the type of cytokines produced. Genes that influence both these sources of variation are associated with differences in resistance to nematode infection. Therefore, much of the heterogeneity among animals in parasite transmission appears to be due to genetic variation in immune responsiveness.     

In situ X-ray scattering evaluation of heat-induced ultrastructural changes in dental tissues and synthetic hydroxyapatite

Human dental tissues consist of inorganic constituents (mainly crystallites of hydroxyapatite, HAp) and organic matrix. In addition, synthetic HAp powders are frequently used in medical and chemical applications. Insights into the ultrastructural alterations of skeletal hard tissues exposed to thermal treatment are crucial for the estimation of temperature of exposure in forensic and archaeological studies. However, at present, only limited data exist on the heat-induced structural alterations of human dental tissues. In this paper, advanced non-destructive small- and wide angle X-ray scattering (SAXS/WAXS) synchrotron techniques were used to investigate the in situ ultrastructural alterations in thermally treated human dental tissues and synthetic HAp powders. The crystallographic properties were probed by WAXS, whereas HAp grain size distribution changes were evaluated by SAXS. The results demonstrate the important role of the organic matrix that binds together the HAp crystallites in responding to heat exposure. This is highlighted by the difference in the thermal behaviour between human dental tissues and synthetic HAp powders. The X-ray analysis results are supported by thermogravimetric analysis. The results concerning the HAp crystalline architecture in natural and synthetic HAp powders provide a reliable basis for deducing the heating history for dental tissues in the forensic and archaeological context, and the foundation for further development and optimization of biomimetic material design.     

Digital clocks: simple Boolean models can quantitatively describe circadian systems

The gene networks that comprise the circadian clock modulate biological function across a range of scales, from gene expression to performance and adaptive behaviour. The clock functions by generating endogenous rhythms that can be entrained to the external 24-h day–night cycle, enabling organisms to optimally time biochemical processes relative to dawn and dusk. In recent years, computational models based on differential equations have become useful tools for dissecting and quantifying the complex regulatory relationships underlying the clock''s oscillatory dynamics. However, optimizing the large parameter sets characteristic of these models places intense demands on both computational and experimental resources, limiting the scope of in silico studies. Here, we develop an approach based on Boolean logic that dramatically reduces the parametrization, making the state and parameter spaces finite and tractable. We introduce efficient methods for fitting Boolean models to molecular data, successfully demonstrating their application to synthetic time courses generated by a number of established clock models, as well as experimental expression levels measured using luciferase imaging. Our results indicate that despite their relative simplicity, logic models can (i) simulate circadian oscillations with the correct, experimentally observed phase relationships among genes and (ii) flexibly entrain to light stimuli, reproducing the complex responses to variations in daylength generated by more detailed differential equation formulations. Our work also demonstrates that logic models have sufficient predictive power to identify optimal regulatory structures from experimental data. By presenting the first Boolean models of circadian circuits together with general techniques for their optimization, we hope to establish a new framework for the systematic modelling of more complex clocks, as well as other circuits with different qualitative dynamics. In particular, we anticipate that the ability of logic models to provide a computationally efficient representation of system behaviour could greatly facilitate the reverse-engineering of large-scale biochemical networks.     

Fish responses to flow velocity and turbulence in relation to size,sex and parasite load

Riverine fish are subjected to heterogeneous flow velocities and turbulence and may use this to their advantage by selecting regions that balance energy expenditure for station holding while maximizing energy gain through feeding opportunities. This study investigated microhabitat selection by guppies Poecilia reticulata in terms of flow characteristics generated by hemisphere boulders in an open channel flume. Velocity and turbulence influenced the variation in swimming behaviour with respect to size, sex and parasite intensity. With increasing body length, fish swam further and more frequently between boulder regions. Larger guppies spent more time in the areas of high-velocity and low-turbulence regions beside the boulders, whereas smaller guppies frequented the low-velocity and high-turbulence regions directly behind the boulders. Male guppies selected the regions of low velocity, indicating possible reduced swimming ability owing to hydrodynamic drag imposed by their fins. With increasing Gyrodactylus turnbulli burden, fish spent more time in regions with moderate velocity and lowest turbulent kinetic energy which were the most spatially and temporally homogeneous in terms of velocity and turbulence. These findings highlight the importance of heterogeneous flow conditions in river channel design owing to the behavioural variability within a species in response to velocity and turbulence.     

Bone tissue reactions to biomimetic ion-substituted apatite surfaces on titanium implants

The aim of this study was to evaluate the bone tissue response to strontium- and silicon-substituted apatite (Sr-HA and Si-HA) modified titanium (Ti) implants. Sr-HA, Si-HA and HA were grown on thermally oxidized Ti implants by a biomimetic process. Oxidized implants were used as controls. Surface properties, i.e. chemical composition, surface thickness, morphology/pore characteristics, crystal structure and roughness, were characterized with various analytical techniques. The implants were inserted in rat tibiae and block biopsies were prepared for histology, histomorphometry and scanning electron microscopy analysis. Histologically, new bone formed on all implant surfaces. The bone was deposited directly onto the Sr-HA and Si-HA implants without any intervening soft tissue. The statistical analysis showed significant higher amount of bone–implant contact (BIC) for the Si-doped HA modification (P = 0.030), whereas significant higher bone area (BA) for the Sr-doped HA modification (P = 0.034), when compared with the non-doped HA modification. The differences were most pronounced at the early time point. The healing time had a significant impact for both BA and BIC (P < 0.001). The present results show that biomimetically prepared Si-HA and Sr-HA on Ti implants provided bioactivity and promoted early bone formation.     

Assessing Differences Between Results Determined According to the Guide to the Expression of Uncertainty in Measurement

In some metrology applications multiple results of measurement for a common measurand are obtained and it is necessary to determine whether the results agree with each other. A result of measurement based on the Guide to the Expression of Uncertainty in Measurement (GUM) consists of a measured value together with its associated standard uncertainty. In the GUM, the measured value is regarded as the expected value and the standard uncertainty is regarded as the standard deviation, both known values, of a state-of-knowledge probability distribution. A state-of-knowledge distribution represented by a result need not be completely known. Then how can one assess the differences between the results based on the GUM? Metrologists have for many years used the Birge chisquare test as ‘a rule of thumb’ to assess the differences between two or more measured values for the same measurand by pretending that the standard uncertainties were the standard deviations of the presumed sampling probability distributions from random variation of the measured values. We point out that this is misuse of the standard uncertainties; the Birge test and the concept of statistical consistency motivated by it do not apply to the results of measurement based on the GUM. In 2008, the International Vocabulary of Metrology, third edition (VIM3) introduced the concept of metrological compatibility. We propose that the concept of metrological compatibility be used to assess the differences between results based on the GUM for the same measurand. A test of the metrological compatibility of two results of measurement does not conflict with a pairwise Birge test of the statistical consistency of the corresponding measured values.     

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 dose and time response Markov model for the in-host dynamics of infection with intracellular bacteria following inhalation: with application to Francisella tularensis

In a novel approach, the standard birth–death process is extended to incorporate a fundamental mechanism undergone by intracellular bacteria, phagocytosis. The model accounts for stochastic interaction between bacteria and cells of the immune system and heterogeneity in susceptibility to infection of individual hosts within a population. Model output is the dose–response relation and the dose-dependent distribution of time until response, where response is the onset of symptoms. The model is thereafter parametrized with respect to the highly virulent Schu S4 strain of Francisella tularensis, in the first such study to consider a biologically plausible mathematical model for early human infection with this bacterium. Results indicate a median infectious dose of about 23 organisms, which is higher than previously thought, and an average incubation period of between 3 and 7 days depending on dose. The distribution of incubation periods is right-skewed up to about 100 organisms and symmetric for larger doses. Moreover, there are some interesting parallels to the hypotheses of some of the classical dose–response models, such as independent action (single-hit model) and individual effective dose (probit model). The findings of this study support experimental evidence and postulations from other investigations that response is, in fact, influenced by both in-host and between-host variability.     

Label-free magnetic resonance imaging to locate live cells in three-dimensional porous scaffolds

Porous scaffolds are widely tested materials used for various purposes in tissue engineering. A critical feature of a porous scaffold is its ability to allow cell migration and growth on its inner surface. Up to now, there has not been a method to locate live cells deep inside a material, or in an entire structure, using real-time imaging and a non-destructive technique. Herein, we seek to demonstrate the feasibility of the magnetic resonance imaging (MRI) technique as a method to detect and locate in vitro non-labelled live cells in an entire porous material. Our results show that the use of optimized MRI parameters (4.7 T; repetition time = 3000 ms; echo time = 20 ms; resolution 39 × 39 µm) makes it possible to obtain images of the scaffold structure and to locate live non-labelled cells in the entire material, with a signal intensity higher than that obtained in the culture medium. In the current study, cells are visualized and located in different kinds of porous scaffolds. Moreover, further development of this MRI method might be useful in several three-dimensional biomaterial tests such as cell distribution studies, routine qualitative testing methods and in situ monitoring of cells inside scaffolds.     

Cranial sutures work collectively to distribute strain throughout the reptile skull

The skull is composed of many bones that come together at sutures. These sutures are important sites of growth, and as growth ceases some become fused while others remain patent. Their mechanical behaviour and how they interact with changing form and loadings to ensure balanced craniofacial development is still poorly understood. Early suture fusion often leads to disfiguring syndromes, thus is it imperative that we understand the function of sutures more clearly. By applying advanced engineering modelling techniques, we reveal for the first time that patent sutures generate a more widely distributed, high level of strain throughout the reptile skull. Without patent sutures, large regions of the skull are only subjected to infrequent low-level strains that could weaken the bone and result in abnormal development. Sutures are therefore not only sites of bone growth, but could also be essential for the modulation of strains necessary for normal growth and development in reptiles.     

Development of sputtered nanoscale titanium oxide coating on osseointegrated implant devices and their biological evaluation

Nanoscale titanium oxide (TiO₂) coating was deposited on titanium (Ti) disks and Ti dental implants using r.f. magnetron sputtering technique. The coating was characterized using grazing angle X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle measurement, profilometry and nano-scratch test. The coating also was evaluated with in vitro cell culture and in vivo dog femur model. Three groups of samples were prepared, including as-sputtered Ti (AS-Ti), sputtered Ti with a post-deposition heat treatment at 600 °C (SH-Ti) and machined Ti (MA-Ti) as controls. The AS-Ti and SH-Ti were dense surfaces consisting of nanoscale grains of 40 nm and 80 nm, respectively. Post-deposition heat treatment increased the coating adhesion. The SH-Ti and AS-Ti significantly decreased the water contact angles compared to the MA-Ti. The nanoscale AS-Ti and SH-Ti significantly improved cell adhesion within the first hour of incubation compared with the MA-Ti. No significant differences were observed in osteoblast proliferation and differentiation in vitro as well as reverse torque and histology in vivo among the three groups. In the present study, it was not observed that the nanoscale dense TiO₂ coating improved the osseointegration compared to the microscale dense Ti.     

Logic programming to predict cell fate patterns and retrodict genotypes in organogenesis

Caenorhabditis elegans vulval development is a paradigm system for understanding cell differentiation in the process of organogenesis. Through temporal and spatial controls, the fate pattern of six cells is determined by the competition of the LET-23 and the Notch signalling pathways. Modelling cell fate determination in vulval development using state-based models, coupled with formal analysis techniques, has been established as a powerful approach in predicting the outcome of combinations of mutations. However, computing the outcomes of complex and highly concurrent models can become prohibitive. Here, we show how logic programs derived from state machines describing the differentiation of C. elegans vulval precursor cells can increase the speed of prediction by four orders of magnitude relative to previous approaches. Moreover, this increase in speed allows us to infer, or ‘retrodict’, compatible genomes from cell fate patterns. We exploit this technique to predict highly variable cell fate patterns resulting from dig-1 reduced-function mutations and let-23 mosaics. In addition to the new insights offered, we propose our technique as a platform for aiding the design and analysis of experimental data.     

In-roads to the spread of antibiotic resistance: regional patterns of microbial transmission in northern coastal Ecuador

The evolution of antibiotic resistance (AR) increases treatment cost and probability of failure, threatening human health worldwide. The relative importance of individual antibiotic use, environmental transmission and rates of introduction of resistant bacteria in explaining community AR patterns is poorly understood. Evaluating their relative importance requires studying a region where they vary. The construction of a new road in a previously roadless area of northern coastal Ecuador provides a valuable natural experiment to study how changes in the social and natural environment affect the epidemiology of resistant Escherichia coli. We conducted seven bi-annual 15 day surveys of AR between 2003 and 2008 in 21 villages. Resistance to both ampicillin and sulphamethoxazole was the most frequently observed profile, based on antibiogram tests of seven antibiotics from 2210 samples. The prevalence of enteric bacteria with this resistance pair in the less remote communities was 80 per cent higher than in more remote communities (OR = 1.8 [1.3, 2.3]). This pattern could not be explained with data on individual antibiotic use. We used a transmission model to help explain this observed discrepancy. The model analysis suggests that both transmission and the rate of introduction of resistant bacteria into communities may contribute to the observed regional scale AR patterns, and that village-level antibiotic use rate determines which of these two factors predominate. While usually conceived as a main effect on individual risk, antibiotic use rate is revealed in this analysis as an effect modifier with regard to community-level risk of resistance.     

Correlation of the dynamics of native human acetylcholinesterase and its inhibited huperzine A counterpart from sub-picoseconds to nanoseconds

It is a long debated question whether catalytic activities of enzymes, which lie on the millisecond timescale, are possibly already reflected in variations in atomic thermal fluctuations on the pico- to nanosecond timescale. To shed light on this puzzle, the enzyme human acetylcholinesterase in its wild-type form and complexed with the inhibitor huperzine A were investigated by various neutron scattering techniques and molecular dynamics simulations. Previous results on elastic neutron scattering at various timescales and simulations suggest that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. In the work presented here, the focus was laid on quasi-elastic (QENS) and inelastic neutron scattering (INS). These techniques give access to different kinds of individual diffusive motions and to the density of states of collective motions at the sub-picoseconds timescale. Hence, they permit going beyond the first approach of looking at mean square displacements. For both samples, the autocorrelation function was well described by a stretched-exponential function indicating a linkage between the timescales of fast and slow functional relaxation dynamics. The findings of the QENS and INS investigation are discussed in relation to the results of our earlier elastic incoherent neutron scattering and molecular dynamics simulations.     

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.