Design of oscillatory dynamics in numerical simulations of compartment-based enzyme systems.

Enzymatic reactions that yield non-neutral products are known to involve feedback due to the bell-shaped pH-rate curve of the enzyme. Compartmentalizing the reaction has been shown to lead to transport-driven oscillations in theory; however, there have been few reproducible experimental examples. Our objective was to determine how the conditions could be optimized to achieve pH oscillations. We employed numerical simulations to investigate the hydrolysis of ethyl acetate in a confined esterase enzyme system, examining the influence of key factors on its behavior. Specific parameter ranges that lead to bistability and self-sustained pH oscillations and the importance of fast base transport for oscillations in this acid-producing system are highlighted. Suggestions are made to expand the parameter space for the occurrence of oscillations, including modifying the maximum of the enzyme pH-rate curve and increasing the negative feedback rate. This research not only sheds light on the programmable nature of enzyme-driven pH regulation but also furthers knowledge on the optimal design of such feedback systems for experimentalists.

A microfluidic double emulsion platform for spatiotemporal control of pH and particle synthesis.

The temporal control of pH in microreactors such as emulsion droplets plays a vital role in applications including biomineralisation and microparticle synthesis. Typically, pH changes are achieved either by passive diffusion of species into a droplet or by acid/base producing reactions. Here, we exploit an enzyme reaction combined with the properties of a water-oil-water (W/O/W) double emulsion to control the pH-time profile in the droplets. A microfluidic platform was used for production of ∼100-200 µm urease-encapsulated double emulsions with a tuneable mineral oil shell thickness of 10-40 µm. The reaction was initiated on-demand by addition of urea and a pulse in base (ammonia) up to pH 8 was observed in the droplets after a time lag of the order of minutes. The pH-time profile can be manipulated by the diffusion timescale of urea and ammonia through the oil layer, resulting in a steady state pH not observed in bulk reactive solutions. This approach may be used to regulate the formation of pH sensitive materials under mild conditions and, as a proof of concept, the reaction was coupled to calcium phosphate precipitation in the droplets. The oil shell thickness was varied to select for either brushite microplatelets or hydroxyapatite particles, compared to the mixture of different precipitates obtained in bulk.

Changing the paradigm in modelling the Bray-Liebhafsky oscillatory chemical reaction.

The Bray-Liebhafsky (BL) reaction is one of the simplest chemical oscillators consisting initially of only three components. Despite this, its mechanism is unknown for more than 100 years due to the absence of selective, sensitive, and fast experimental techniques for following all of the involved intermediates. The modeling of the BL mechanism assumes presumably mass action kinetics "adjustable" to oscillatory solutions by the application of mathematical stability analysis and treating the system as homogeneous. Such a basically mathematical approach need not suggest physically realistic kinetic parameters and is not unique since a number of models can be proposed. Based on recent experimental and computational results, a new model of the BL oscillatory reaction mechanism is constructed by including heterogeneous processes occurring in the system. The same set of equations is able to demonstrate not only the oscillatory evolution but also mixing effects on the oscillatory dynamics, and non-oscillatory stepwise-iodine oxidation and can rationalize other effects described in literature. Thus, the paradigm of treating the BL oscillatory system as a homogeneous one, described by formal kinetics only, is extended for a better understanding of the chemistry of this apparently simple system. The introduced ideas of energy redistribution may contribute to establishing an improved conceptual base for considering other complex oscillators in various fields of science.

Influence of Oxygen on Chemoconvective Patterns in the Iodine Clock Reaction.

There is increasing interest in using chemical clock reactions to drive material formation; however, these reactions are often subject to chemoconvective effects, and control of such systems remains challenging. Here, we show how the transfer of oxygen at the air-water interface plays a crucial role in the spatiotemporal behavior of the iodine clock reaction with sulfite. A kinetic model was developed to demonstrate how the reaction of oxygen with sulfite can control a switch from a low-iodine to high-iodine state under well-stirred conditions and drive the formation of transient iodine gradients in unstirred solutions. In experiments in thin layers with optimal depths, the reaction couples with convective instability at the air-water interface forming an extended network-like structure of iodine at the surface that develops into a spotted pattern at the base of the layer. Thus, oxygen drives the spatial separation of iodine states essential for patterns in this system and may influence pattern selection in other clock reaction systems with sulfite.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen's ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

Coupling between nucleation and chemical reactions and its importance in understanding the oxidation of iodine by hydrogen peroxide.

We have investigated the reaction of iodine with hydrogen peroxide coupled to gas nucleation. A step-like increase in the nucleation rate with increasing amounts of dissolved oxygen can act as a trigger for the formation of highly reactive components and complete oxidation of iodine to iodate despite the large thermodynamic barrier for the whole process. Energetic coupling of nucleation with chemical reactions is based on local redistribution of energy by collapsing unstable nuclei. The developed model correctly describes the evolution of the measured reaction parameters. It offers a conceptual improvement of formal-kinetic models by drawing attention to important contributions of physical effects to the reaction mechanism.

Collective Behavior of Urease pH Clocks in Nano- and Microvesicles Controlled by Fast Ammonia Transport.

The transmission of chemical signals via an extracellular solution plays a vital role in collective behavior in cellular biological systems and may be exploited in applications of lipid vesicles such as drug delivery. Here, we investigated chemical communication in synthetic micro- and nanovesicles containing urease in a solution of urea and acid. We combined experiments with simulations to demonstrate that the fast transport of ammonia to the external solution governs the pH-time profile and synchronizes the timing of the pH clock reaction in a heterogeneous population of vesicles. This study shows how the rate of production and emission of a small basic product controls pH changes in active vesicles with a distribution of sizes and enzyme amounts, which may be useful in bioreactor or healthcare applications.

Moving Pharmacogenetics Into Practice: It's All About the Evidence!

The evidence for pharmacogenetics has grown rapidly in recent decades. However, the strength of evidence required for the clinical implementation of pharmacogenetics is highly debated. Therefore, the purpose of this review is to summarize different perspectives on the evidence required for the clinical implementation of pharmacogenetics. First, we present two patient cases that demonstrate how knowledge of pharmacogenetic evidence affected their care. Then we summarize resources that curate pharmacogenetic evidence, types of evidence (with an emphasis on randomized controlled trials [RCT]) and their limitations, and different perspectives from implementers, clinicians, and patients. We compare pharmacogenetics to a historical example (i.e., the evidence required for the clinical implementation of pharmacokinetics/therapeutic drug monitoring), and we provide future perspectives on the evidence for pharmacogenetic panels and the need for more education in addition to evidence. Although there are differences in the interpretation of pharmacogenetic evidence across resources, efforts for standardization are underway. Survey data illustrate the value of pharmacogenetic testing from the patient perspective, with their providers seen as key to ensuring maximum benefit from test results. However, clinicians and practice guidelines from medical societies often rely on RCT data to guide treatment decisions, which are not always feasible or ethical in pharmacogenetics. Thus, recognition of other types of evidence to support pharmacogenetic implementation is needed. Among pharmacogenetic implementers, consistent evidence of pharmacogenetic associations is deemed most critical. Ultimately, moving pharmacogenetics into practice will require consideration of multiple stakeholder perspectives, keeping particularly attuned to the voice of the ultimate stakeholder-the patient.

Reaction-diffusion hydrogels from urease enzyme particles for patterned coatings.

The reaction and diffusion of small molecules is used to initiate the formation of protective polymeric layers, or biofilms, that attach cells to surfaces. Here, inspired by biofilm formation, we present a general method for the growth of hydrogels from urease enzyme-particles by combining production of ammonia with a pH-regulated polymerization reaction in solution. We show through experiments and simulations how the propagating basic front and thiol-acrylate polymerization were continuously maintained by the localized urease reaction in the presence of urea, resulting in hydrogel layers around the enzyme particles at surfaces, interfaces or in motion. The hydrogels adhere the enzyme-particles to surfaces and have a tunable growth rate of the order of 10 µm min-1 that depends on the size and spatial distribution of particles. This approach can be exploited to create enzyme-hydrogels or chemically patterned coatings for applications in biocatalytic flow reactors.

Stop flailing: The impact of bicortically displaced rib fractures on pulmonary outcomes in patients with chest trauma - an American Association for the Surgery of Trauma multi-institutional study.

BACKGROUND: Current evaluation of rib fractures focuses almost exclusively on flail chest with little attention on bicortically displaced fractures. Chest trauma that is severe enough to cause fractures leads to worse outcomes. An association between bicortically displaced rib fractures and pulmonary outcomes would potentially change patient care in the setting of trauma. We tested the hypothesis that bicortically displaced fractures were an important clinical marker for pulmonary outcomes in patients with nonflail rib fractures. METHODS: This nine-center American Association for the Surgery of Trauma multi-institutional study analyzed adults with two or more rib fractures. Admission computerized tomography scans were independently reviewed. The location, degree of rib fractures, and pulmonary contusions were categorized. Univariate and multivariate logistic regression analyses were performed to identify independent predictors of pneumonia, acute respiratory distress syndrome (ARDS), and tracheostomy. Analyses were performed in nonflail patients and also while controlling for flail chest to determine if bicortically displaced fractures were independently associated with outcomes. RESULTS: Of the 1,110 patients, 103 (9.3%) developed pneumonia, 78 (7.0%) required tracheostomy, and 30 (2.7%) developed ARDS. Bicortically displaced fractures were present in 277 (25%) of patients and in 206 (20.3%) of patients without flail chest. After adjusting for patient demographics, injury, and admission physiology, negative pulmonary outcomes occurred over twice as frequently in those with bicortically displaced fractures without flail chest (n = 206) when compared with those without bicortically displaced fractures-pneumonia (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.1-3.6), ARDS (OR, 2.6; 95% CI, 1.0-6.8), and tracheostomy (OR, 2.7; 95% CI, 1.4-5.2). When adjusting for the presence of flail chest, bicortically displaced fractures remained an independent predictor of pneumonia, tracheostomy, and ARDS. CONCLUSION: Patients with bicortically displaced rib fractures are more likely to develop pneumonia, ARDS, and need for tracheostomy even when controlling for flail chest. Future studies should investigate the utility of flail chest management algorithms in patients with bicortically displaced fractures. LEVEL OF EVIDENCE: Prognostic and epidemiological study, level III.

Correction: Common-variant associations with fragile X syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Periodic Nucleation of Calcium Phosphate in a Stirred Biocatalytic Reaction.

Highly ordered superstructures composed of inorganic nanoparticles appear in natural and synthetic systems, however the mechanisms of non-equilibrium self-organization that may be involved are still poorly understood. Herein, we performed a kinetic investigation of the precipitation of calcium phosphate using a process widely found in microorganisms: the hydrolysis of urea by enzyme urease. With high initial ratio of calcium ion to phosphate, periodic precipitation was obtained accompanied by pH oscillations in a well-stirred, closed reactor. We propose that an internal pH-regulated change in the concentration of phosphate ion is the driving force for periodicity. A simple model involving the biocatalytic reaction network coupled with burst nucleation of nanoparticles above a critical supersaturation reproduced key features of the experiments. These findings may provide insight to the self-organization of nanoparticles in biomineralization and improve design strategies of biomaterials for medical applications.

Modelling Bacteria-Inspired Dynamics with Networks of Interacting Chemicals.

One approach to understanding how life-like properties emerge involves building synthetic cellular systems that mimic certain dynamical features of living cells such as bacteria. Here, we developed a model of a reaction network in a cellular system inspired by the ability of bacteria to form a biofilm in response to increasing cell density. Our aim was to determine the role of chemical feedback in the dynamics. The feedback was applied through the enzymatic rate dependence on pH, as pH is an important parameter that controls the rates of processes in cells. We found that a switch in pH can be used to drive base-catalyzed gelation or precipitation of a substance in the external solution. A critical density of cells was required for gelation that was essentially independent of the pH-driven feedback. However, the cell pH reached a higher maximum as a result of the appearance of pH oscillations with feedback. Thus, we conclude that while feedback may not play a vital role in some density-dependent behavior in cellular systems, it nevertheless can be exploited to activate internally regulated cell processes at low cell densities.

Influence of reaction-induced convection on quorum sensing in enzyme-loaded agarose beads.

In theory, groups of enzyme-loaded particles producing an acid or base may show complex behavior including dynamical quorum sensing, the appearance of synchronized oscillations above a critical number or density of particles. Here, experiments were performed with the enzyme urease loaded into mm-sized agarose beads and placed in a solution of urea, resulting in an increase in pH. This behavior was found to be dependent upon the number of beads present in the array; however, reaction-induced convection occurred and plumes of high pH developed that extended to the walls of the reactor. The convection resulted in the motion of the mm-sized particles and conversion of the solution to high pH. Simulations in a simple model of the beads demonstrated the suppression of dynamical quorum sensing in the presence of flow.

Towards feedback-controlled nanomedicines for smart, adaptive delivery.

IMPACT STATEMENT: The timing and rate of release of pharmaceuticals from advanced drug delivery systems is an important property that has received considerable attention in the scientific literature. Broadly, these mostly fall into two classes: controlled release with a prolonged release rate or triggered release where the drug is rapidly released in response to an environmental stimulus. This review aims to highlight the potential for developing adaptive release systems that more subtlety modulate the drug release profile through continuous communication with its environment facilitated through feedback control. By reviewing the key elements of this approach in one place (fundamental principles of nanomedicine, enzymatic nanoreactors for medical therapies and feedback-controlled chemical systems) and providing additional motivating case studies in the context of chronobiology, we hope to inspire innovative development of novel "chrononanomedicines."