Collective cell migration is fundamental throughout development and in many diseases. Spatial confinement using micropatterns has been shown to promote collective cell migration in vitro, but its ...effect in vivo remains unclear. Combining computational and experimental approaches, we show that the in vivo collective migration of neural crest cells (NCCs) depends on such confinement. We demonstrate that confinement may be imposed by the spatiotemporal distribution of a nonpermissive substrate provided by versican, an extracellular matrix molecule previously proposed to have contrasting roles: barrier or promoter of NCC migration. We resolve the controversy by demonstrating that versican works as an inhibitor of NCC migration and also acts as a guiding cue by forming exclusionary boundaries. Our model predicts an optimal number of cells in a given confinement width to allow for directional migration. This optimum coincides with the width of neural crest migratory streams analyzed across different species, proposing an explanation for the highly conserved nature of NCC streams during development.
Collective cell migration is a fundamental process, occurring during embryogenesis and cancer metastasis. Neural crest cells exhibit such coordinated migration, where aberrant motion can lead to ...fatality or dysfunction of the embryo. Migration involves at least two complementary mechanisms: contact inhibition of locomotion (a repulsive interaction corresponding to a directional change of migration upon contact with a reciprocating cell), and co-attraction (a mutual chemoattraction mechanism). Here, we develop and employ a parameterized discrete element model of neural crest cells, to investigate how these mechanisms contribute to long-range directional migration during development. Motion is characterized using a coherence parameter and the time taken to reach, collectively, a target location. The simulated cell group is shown to switch from a diffusive to a persistent state as the response-rate to co-attraction is increased. Furthermore, the model predicts that when co-attraction is inhibited, neural crest cells can migrate into restrictive regions. Indeed, inhibition of co-attraction in vivo and in vitro leads to cell invasion into restrictive areas, confirming the prediction of the model. This suggests that the interplay between the complementary mechanisms may contribute to guidance of the neural crest. We conclude that directional migration is a system property and does not require action of external chemoattractants.
DNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of ...which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution. Here we present a new method to model the combined activation of non-homologous end joining, single strand annealing and alternative end joining, following exposure to ionising radiation. We use Bayesian statistics to integrate eight biological data sets of double-strand break repair curves under varying genetic knockouts and confirm that our model is predictive by re-simulating and comparing to additional data. Analysis of the model suggests that there are at least three disjoint modes of repair, which we assign as fast, slow and intermediate. Our results show that when multiple data sets are combined, the rate for intermediate repair is variable amongst genetic knockouts. Further analysis suggests that the ratio between slow and intermediate repair depends on the presence or absence of DNA-PKcs and Ku70, which implies that non-homologous end joining and alternative end joining are not independent. Finally, we consider the proportion of double-strand breaks within each mechanism as a time series and predict activity as a function of repair rate. We outline how our insights can be directly tested using imaging and sequencing techniques and conclude that there is evidence of variable dynamics in alternative repair pathways. Our approach is an important step towards providing a unifying theoretical framework for the dynamics of DNA repair processes.
The engineering of transcriptional networks presents many challenges due to the inherent uncertainty in the system structure, changing cellular context, and stochasticity in the governing dynamics. ...One approach to address these problems is to design and build systems that can function across a range of conditions; that is they are robust to uncertainty in their constituent components. Here we examine the parametric robustness landscape of transcriptional oscillators, which underlie many important processes such as circadian rhythms and the cell cycle, plus also serve as a model for the engineering of complex and emergent phenomena. The central questions that we address are: Can we build genetic oscillators that are more robust than those already constructed? Can we make genetic oscillators arbitrarily robust? These questions are technically challenging due to the large model and parameter spaces that must be efficiently explored. Here we use a measure of robustness that coincides with the Bayesian model evidence, combined with an efficient Monte Carlo method to traverse model space and concentrate on regions of high robustness, which enables the accurate evaluation of the relative robustness of gene network models governed by stochastic dynamics. We report the most robust two and three gene oscillator systems, plus examine how the number of interactions, the presence of autoregulation, and degradation of mRNA and protein affects the frequency, amplitude, and robustness of transcriptional oscillators. We also find that there is a limit to parametric robustness, beyond which there is nothing to be gained by adding additional feedback. Importantly, we provide predictions on new oscillator systems that can be constructed to verify the theory and advance design and modeling approaches to systems and synthetic biology.
Autonomous underwater vehicles (AUVs) are frequently used for deep-water ocean applications such as surveying and cable laying, where accurate control of vehicle depth and attitude is needed. The ...water level in the onboard ballast tanks is typically manually set for neutral buoyancy before each mission, while the vehicle is on the surface. The ballast tank contents are not normally adjusted to control vehicle depth and orientation while the AUV is in operation. As a result, vehicle trajectory and orientation is exclusively controlled using the vehicle's control surfaces during a mission. The challenges with controlling the depth and trim of an underwater vehicle include nonlinear hydrodynamic forces, as well as inherent time delays (latencies) associated with water tank level changes and valve adjustments. Furthermore, small changes in the location of the vehicle's center of gravity (i.e., due to the deployment of the AUV's payload equipment) can reduce the control authority of the AUV's control surfaces. To meet these challenges, this paper proposes two unique variable ballast system (VBS) control approaches. The first proposed VBS controller changes the weight of the AUV to help control vehicle depth and vertical (inertial) velocity. The second proposed VBS controller attempts to shift the center of gravity x G along the body-fixed x -(longitudinal)-axis to reduce depth and pitch angle error while restoring control authority to the bowplane and sternplane deflection fins. The ballasting system consists of two water tanks positioned aft and forward of amidships. The ballast tanks are then automatically filled or emptied of ocean water as desired. Numerical simulations have been carried out on a 2-D underwater vehicle simulator to test and compare the performance of the proposed ballast and fin deflection control systems. The simulation results show that, for the assumptions and conditions tested, the proposed controllers are capable of achieving a setpoint depth and pitch angle with minimal error by effectively utilizing the ballast tanks and deflection fins. As a result, the work presented in this paper helps increase the autonomy of large AUVs on long-duration missions.
Genetic switches exhibit multistability, form the basis of epigenetic memory, and are found in natural decision making systems, such as cell fate determination in developmental pathways. Synthetic ...genetic switches can be used for recording the presence of different environmental signals, for changing phenotype using synthetic inputs and as building blocks for higher-level sequential logic circuits. Understanding how multistable switches can be constructed and how they function within larger biological systems is therefore key to synthetic biology.
Here we present a new computational tool, called StabilityFinder, that takes advantage of sequential Monte Carlo methods to identify regions of parameter space capable of producing multistable behaviour, while handling uncertainty in biochemical rate constants and initial conditions. The algorithm works by clustering trajectories in phase space, and iteratively minimizing a distance metric. Here we examine a collection of models of genetic switches, ranging from the deterministic Gardner toggle switch to stochastic models containing different positive feedback connections. We uncover the design principles behind making bistable, tristable and quadristable switches, and find that rate of gene expression is a key parameter. We demonstrate the ability of the framework to examine more complex systems and examine the design principles of a three gene switch. Our framework allows us to relax the assumptions that are often used in genetic switch models and we show that more complex abstractions are still capable of multistable behaviour.
Our results suggest many ways in which genetic switches can be enhanced and offer designs for the construction of novel switches. Our analysis also highlights subtle changes in correlation of experimentally tunable parameters that can lead to bifurcations in deterministic and stochastic systems. Overall we demonstrate that StabilityFinder will be a valuable tool in the future design and construction of novel gene networks.
We show that a binary oncolytic/helper-dependent adenovirus (CAdVEC) that both lyses tumor cells and locally expresses the proinflammatory cytokine IL-12 and PD-L1 blocking antibody has potent ...antitumor activity in humanized mouse models. On the basis of these preclinical studies, we treated four patients with a single intratumoral injection of an ultralow dose of CAdVEC (NCT03740256), representing a dose of oncolytic adenovirus more than 100-fold lower than used in previous trials. While CAdVEC caused no significant toxicities, it repolarized the tumor microenvironment with increased infiltration of CD8 T cells. A single administration of CAdVEC was associated with both locoregional and abscopal effects on metastases and, in combination with systemic administration of immune checkpoint antibodies, induced sustained antitumor responses, including one complete and two partial responses. Hence, in both preclinical and clinical studies, CAdVEC is safe and even at extremely low doses is sufficiently potent to induce significant tumor control through oncolysis and immune repolarization.
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Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as ...the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.
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