Processes, Volume 5, Issue 2 (June 2017) – 19 articles

Metabolic acclimation to photosynthesis-associated stresses was examined in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 using integrated computational and photobioreactor analyses. A genome-enabled metabolic model, complete with measured biomass composition, was analyzed using ecological resource allocation theory to predict and interpret metabolic acclimation to irradiance, O₂, and nutrient stresses. Reduced growth efficiency, shifts in photosystem utilization, changes in photorespiration strategies, and differing byproduct secretion patterns were predicted to occur along culturing stress gradients. These predictions were compared with photobioreactor physiological data and previously published transcriptomic data and found to be highly consistent with observations, providing a systems-based rationale for the culture phenotypes. The analysis also indicated that cyanobacterial stress acclimation strategies created niches for heterotrophic organisms and that heterotrophic activity could enhance cyanobacterial stress tolerance by removing inhibitory metabolic byproducts. This study provides mechanistic insight into stress acclimation strategies in photoautotrophs and establishes a framework for predicting, designing, and engineering both axenic and photoautotrophic-heterotrophic systems as a function of controllable parameters. Full article

We have developed a closed-loop, high-throughput system that applies electrical stimulation and optical recording to facilitate the rapid characterization of extracellular, stimulus-evoked neuronal activity. In our system, a microelectrode array delivers current pulses to a dissociated neuronal culture treated with a calcium-sensitive fluorescent dye; automated real-time image processing of high-speed digital video identifies the neuronal response; and an optimized search routine alters the applied stimulus to achieve a targeted response. Action potentials are detected by measuring the post-stimulus, calcium-sensitive fluorescence at the neuronal somata. The system controller performs directed searches within the strength–duration (SD) stimulus-parameter space to build probabilistic neuronal activation curves. This closed-loop system reduces the number of stimuli needed to estimate the activation curves when compared to the more commonly used open-loop approach. This reduction allows the closed-loop system to probe the stimulus regions of interest in the multi-parameter waveform space with increased resolution. A sigmoid model was fit to the stimulus-evoked activation data in both current (strength) and pulse width (duration) parameter slices through the waveform space. The two-dimensional analysis results in a set of probability isoclines corresponding to each neuron–electrode pair. An SD threshold model was then fit to the isocline data. We demonstrate that a closed-loop methodology applied to our imaging and micro-stimulation system enables the study of neuronal excitation across a large parameter space. Full article

Abstract: Dynamic modelling is a powerful tool for studying biological networks. Reachability (controllability), observability, and structural identifiability are classical system-theoretic properties of dynamical models. A model is structurally identifiable if the values of its parameters can in principle be determined from observations of its outputs. If model parameters are considered as constant state variables, structural identifiability can be studied as a generalization of observability. Thus, it is possible to assess the identifiability of a nonlinear model by checking the rank of its augmented observability matrix. When such rank test is performed symbolically, the result is of general validity for almost all numerical values of the variables. However, for special cases, such as specific values of the initial conditions, the result of such test can be misleading—that is, a structurally unidentifiable model may be classified as identifiable. An augmented observability rank test that specializes the symbolic states to particular numerical values can give hints of the existence of this problem. Sometimes it is possible to find such problematic values analytically, or via optimization. This manuscript proposes procedures for performing these tasks and discusses the relation between loss of identifiability and loss of reachability, using several case studies of biochemical networks. Full article

In chemical industries, process operations are usually comprised of several discrete operating regions with distributions that drift over time. These complexities complicate outlier detection in the presence of intrinsic process dynamics. In this article, we consider the problem of detecting univariate outliers in dynamic systems with multiple operating points. A novel method combining the time series Kalman filter (TSKF) with the pruned exact linear time (PELT) approach to detect outliers is proposed. The proposed method outperformed benchmark methods in outlier removal performance using simulated data sets of dynamic systems with mean shifts. The method was also able to maintain the integrity of the original data set after performing outlier removal. In addition, the methodology was tested on industrial flaring data to pre-process the flare data for discriminant analysis. The industrial test case shows that performing outlier removal dramatically improves flare monitoring results through Partial Least Squares Discriminant Analysis (PLS-DA), which further confirms the importance of data cleaning in process data analytics. Full article

There is a debate in the literature on whether or not polymer networks synthesized by reversible deactivation radical polymerization (RDRP) processes, such as reversible addition-fragmentation radical transfer (RAFT) copolymerization of vinyl/divinyl monomers, are less heterogeneous than those synthesized by conventional free radical copolymerization (FRP). In this contribution, the syntheses by FRP and RAFT of hydrogels based on 2-hydroxyethylene methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) in supercritical carbon dioxide (scCO₂), using Krytox 157 FSL as the dispersing agent, and the properties of the materials produced, are compared. The materials were characterized by differential scanning calorimetry (DSC), swelling index (SI), infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Studies on ciprofloxacin loading and release rate from hydrogels were also carried out. The combined results show that the hydrogels synthesized by FRP and RAFT are significantly different, with apparently less heterogeneity present in the materials synthesized by RAFT copolymerization. A ratio of experimental (Mc_exp) to theoretical (Mc_theo) molecular weight between crosslinks was established as a quantitative tool to assess the degree of heterogeneity of a polymer network. Full article

Cross-linked starch nanocapsules (NCs) were synthesized by interfacial polymerization carried out using the inverse mini-emulsion technique. 2,4-toluene diisocyanate (TDI) was used as the cross-linker. The influence of TDI concentrations on the polymeric shell, particle size, and encapsulation efficiency of a hydrophilic dye, sulforhodamine 101 (SR 101), was investigated by Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS), and fluorescence measurements, respectively. The final NC morphology was confirmed by scanning electron microscopy. The leakage of SR 101 through the shell of NCs was monitored at 37 °C for seven days, and afterwards the NCs were redispersed in water. Depending on cross-linker content, permeable and impermeable NCs shell could be designed. Enzyme-triggered release of SR 101 through impermeable NC shells was investigated using UV spectroscopy with different α-amylase concentrations. Impermeable NCs shell were able to release their cargo upon addition of amylase, being suitable for a drug delivery system of hydrophilic compounds. Full article

Water-soluble polymers with high molecular weights are known to decrease the frictional drag in turbulent flow very effectively at concentrations of tens or hundreds of ppm. This drag reduction efficiency of water-soluble polymers is well known to be closely associated with the flow conditions and rheological, physical, and/or chemical characteristics of the polymers added. Among the many promising polymers introduced in the past several decades, this review focuses on recent progress in the drag reduction capability of various water-soluble macromolecules in turbulent flow including both synthetic and natural polymers such as poly(ethylene oxide), poly(acrylic acid), polyacrylamide, poly(N-vinyl formamide), gums, and DNA. The polymeric species, experimental parameters, and numerical analysis of these water-soluble polymers in turbulent drag reduction are highlighted, along with several existing and potential applications. The proposed drag reduction mechanisms are also discussed based on recent experimental and numerical researches. This article will be helpful to the readers to understand better the complex behaviors of a turbulent flow with various water-soluble polymeric additives regarding experimental conditions, drag reduction mechanisms, and related applications. Full article

Water-soluble, carboxylic acid monomers are known to exhibit peculiar kinetics when polymerized in aqueous solution. Namely, their free-radical polymerization rate is affected by several parameters such as monomer concentration, ionic strength, and pH. Focusing on methacrylic acid (MAA), even though this monomer has been largely addressed, a systematic investigation of the effects of the above-mentioned parameters on its polymerization rate is missing, in particular in the fully ionized case. In this work, the kinetics of non-ionized and fully ionized MAA are characterized by in-situ nuclear magnetic resonance (NMR). Such accurate monitoring of the reaction rate enables the identification of relevant but substantially different effects of the monomer and electrolyte concentration on polymerization rate in the two ionization cases. For non-ionized MAA, the development of a kinetic model based on literature rate coefficients allows us to nicely simulate the experimental data of conversion versus time at a high monomer concentration. For fully ionized MAA, a novel propagation rate law accounting for the electrostatic interactions is proposed: the corresponding model is capable of predicting reasonably well the electrolyte concentration effect on polymerization rate. Nevertheless, further kinetic information in a wider range of monomer concentrations would be welcome to increase the reliability of the model predictions. Full article

A discrete element model (DEM) has been developed for an industrial batch bin blender in which three different types of materials are mixed. The mixing dynamics have been evaluated from a model-based study with respect to the blend critical quality attributes (CQAs) which are relative standard deviation (RSD) and segregation intensity. In the actual industrial setup, a sensor mounted on the blender lid is used to determine the blend composition in this region. A model-based analysis has been used to understand the mixing efficiency in the other zones inside the blender and to determine if the data obtained near the blender-lid region are able to provide a good representation of the overall blend quality. Sub-optimal mixing zones have been identified and other potential sampling locations have been investigated in order to obtain a good approximation of the blend variability. The model has been used to study how the mixing efficiency can be improved by varying the key processing parameters, i.e., blender RPM/speed, fill level/volume and loading order. Both segregation intensity and RSD reduce at a lower fill level and higher blender RPM and are a function of the mixing time. This work demonstrates the use of a model-based approach to improve process knowledge regarding a pharmaceutical mixing process. The model can be used to acquire qualitative information about the influence of different critical process parameters and equipment geometry on the mixing dynamics. Full article

Hydrogels based on poly(2-hydroxyethyl methacrylate) (PHEMA) are a very important class of biomaterials with several applications mainly in tissue engineering and contacts lenses. Although the polymerization kinetics of HEMA have been investigated in the literature, the development of a model, accounting for both the chemical reaction mechanism and diffusion-controlled phenomena and valid over the whole conversion range, has not appeared so far. Moreover, research on the synthesis of nanocomposite materials based on a polymer matrix has grown rapidly recently because of the improved mechanical, thermal and physical properties provided by the polymer. In this framework, the objective of this research is two-fold: to provide a kinetic model for the polymerization of HEMA with accurate estimations of the kinetic and diffusional parameters employed and to investigate the effect of adding various types and amounts of nano-additives to the polymerization rate. In the first part, experimental data are provided from Differential Scanning Calorimetry (DSC) measurements on the variation of the reaction rate with time at several polymerization temperatures. These data are used to accurately evaluate the kinetic rate constants and diffusion-controlled parameters. In the second part, nanocomposites of PHEMA are formed, and the in situ bulk radical polymerization kinetics is investigated with DSC. It was found that the inclusion of nano-montmorillonite results in a slight enhancement of the polymerization rate, while the inverse holds when adding nano-silica. These results are interpreted in terms of noncovalent interactions, such as hydrogen bonding between the monomer and polymer or the nano-additive. X-Ray Diffraction (XRD) and Fourier Transform Infra-Red (FTIR) measurements were carried out to verify the results. Full article

While many perceive mammalian cell culture-based manufacturing for biopharmaceuticals an established technology, numerous open questions remain to be solved. Genetic diversity and mutation rates in CHO cells have been underestimated since progeny of a clonal CHO cell become genetically diverse with each cell division. This is an important issue since products are made in bioreactors containing up to 10¹⁴ cells that have divided for weeks. Regulatory interest in “Proof of Clonality” is a misguided and misunderstood concern in this context. We revisit decades of research on scope and rate of genetic changes in CHO cells and suggest approaches to minimize trends for genomic instability when establishing reliable manufacturing processes. A concept is proposed for transfection-derived cell populations containing relatively stable (The term “stable” will always be used in a context of relative stability—considering time frames of weeks to months at best.) “CHO master sequence” genomes (containing the desired DNAs of interest). Stable cell populations are to be selected for and maintained for the various phases of manufacturing under specific culture conditions reducing trends for the selection of diverse subpopulations. Such conditions are based on insights gained from population genetics, evolutionary landscape fitness principles, and a 40-year old model for evolution of error prone replicating systems—the Quasi-Species concept. Full article

An in situ nuclear magnetic resonance spectroscopy (NMR) technique is used to monitor the aqueous-phase copolymerization kinetics of methacrylic acid (MAA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) macromonomers. In particular, the study analyses the effect of the number of ethylene glycol (EG) groups along the lateral chains of PEGMA and is carried out under fully ionized conditions of MAA at different initial monomer ratios and initial overall monomer concentrations (5–20 wt % in aqueous solution). The composition drift with conversion indicates that PEGMA macromonomer is more reactive than MAA. Individual monomer consumption rates show that the rates of consumption of both monomers are not first order with respect to overall concentration of the monomer. The reactivity ratios estimated from the copolymerization kinetics reveal, that for the short PEGMA, the reactivity ratios r_MAA and r_PEGMA increase with the solids content (SC). A totally different trend is obtained for the longer PEGMA, whose reactivity ratio (r_PEGMA23) decreases with solids content, whereas the reactivity ratio of MAA remains roughly constant. Full article

A series of P_DLLA-PEG_1k-P_DLLA tri-block co-polymers with various compositions, i.e., containing 2–10 lactoyl units, were prepared via ring opening polymerisation of d,l-lactide in the presence of poly (ethylene glycol) (PEG) (M_n = 1000 g·mol⁻¹) as the initiator and stannous 2-ethylhexanoate as the catalyst at different feed ratios. P_DLLA-PEG_1k-P_DLLA co-polymers were then functionalised with acrylate groups using acryloyl chloride under various reaction conditions. The diacrylated P_DLLA-PEG_1k-P_DLLA (diacryl-P_DLLA-PEG_1k-P_DLLA) were further polymerised to synthesize soluble hyperbranched polymers by either homo-polymerisation or co-polymerisation with poly(ethylene glycol) methyl ether methylacrylate (PEGMEMA) via free radical polymerisation. The polymer samples obtained were characterised by ¹H NMR (proton Nuclear Magnetic Resonance), FTIR (Fourier Transform Infra-red spectroscopy), and GPC (Gel Permeation Chromatography). Moreover, the diacryl-P_DLLA-PEG_1k-P_DLLA macromers were used for the preparation of biodegradable crosslinked hydrogels through the Michael addition reaction and radical photo-polymerisation with or without poly(ethylene glycol) methyl ether methylacrylate (PEGMEMA, M_n = 475 g·mol⁻¹) as the co-monomer. It was found that fine tuning of the diacryl-P_DLLA-PEG_1k-P_DLLA constituents and its combination with co-monomers resulted in hydrogels with tailored swelling properties. It is envisioned that soluble hyperbranched polymers and crosslinked hydrogels prepared from diacryl-P_DLLA-PEG_1k-P_DLLA macromers can have promising applications in the fields of nano-medicines and regenerative medicines. Full article

A new Automatic Continuous Online Monitoring of Polymerization reactions (ACOMP) system has been developed with multiple light scattering and viscosity detection stages in serial flow, where solution conditions are different at each stage. Solution conditions can include ionic strength (IS), pH, surfactants, concentration, and other factors. This allows behavior of a polymer under simultaneous, varying solution conditions to be monitored at each instant of its synthesis. The system can potentially be used for realtime formulation, where a solution formulation is built up additively in successive stages. It can also monitor the effect of solution conditions on stimuli responsive polymers, as their responsiveness changes during synthesis. In this first work, the new ACOMP system monitored light scattering and reduced viscosity properties of copolymeric polyelectrolytes under various IS during synthesis. Aqueous copolymerization of acrylamide (Am) and styrene sulfonate (SS) was used. Polyelectrolytes in solution expand as IS decreases, leading to increased intrinsic viscosity (η) and suppression of light scattering intensity due to electrostatically enhanced second and third virial coefficients, A₂ and A₃. At a fixed IS, the same effects occur if polyelectrolyte linear charge density (ξ) increases. This work presents polyelectrolyte response to a series of IS and changing ξ during chemical synthesis. Full article

The realization of physiologically-relevant advanced in vitro models is not just related to the reproduction of a three-dimensional multicellular architecture, but also to the maintenance of a cell culture environment in which parameters, such as temperature, pH, and hydrostatic pressure are finely controlled. Tunable and reproducible culture conditions are crucial for the study of environment-sensitive cells, and can also be used for mimicking pathophysiological conditions related with alterations of temperature, pressure and pH. Here, we present the SUITE (Supervising Unit for In Vitro Testing) system, a platform able to monitor and adjust local environmental variables in dynamic cell culture experiments. The physical core of the control system is a mixing chamber, which can be connected to different bioreactors and acts as a media reservoir equipped with a pH meter and pressure sensors. The chamber is heated by external resistive elements and the temperature is controlled using a thermistor. A purpose-built electronic control unit gathers all data from the sensors and controls the pH and hydrostatic pressure by regulating air and CO₂ overpressure and flux. The system’s modularity and the possibility of imposing different pressure conditions were used to implement a model of portal hypertension with both endothelial and hepatic cells. The results show that the SUITE platform is able to control and maintain cell culture parameters at fixed values that represent either physiological or pathological conditions. Thus, it represents a fundamental tool for the design of biomimetic in vitro models, with applications in disease modelling or toxicity testing. Full article

Background: Magnetic fields can interact with liquid matter in a homogeneous and instantaneous way, without physical contact, independently of its temperature, pressure, and agitation degree, and without modifying recipes nor heat and mass transfer conditions. In addition, magnetic fields may affect the mechanisms of generation and termination of free radicals. This paper is devoted to the elucidation of the appropriate conditions needed to develop magnetic field effects for controlling the kinetics of polymerization of water soluble monomers. Methods: Thermal- and photochemically-initiated polymerizations were investigated at different initiator and monomer concentrations, temperatures, viscosities, and magnetic field intensities. Results: Significant magnetic field impact on the polymerization kinetics was only observed in photochemically-initiated polymerizations carried out in viscous media and performed at relatively low magnetic field intensity. Magnetic field effects were absent in polymerizations in low viscosity media and thermally-initiated polymerizations performed at low and high magnetic field intensities. The effects were explained in terms of the radical pair mechanism for intersystem crossing of spin states. Conclusion: Polymerization kinetics of water soluble monomers can be potentially controlled using magnetic fields only under very specific reaction conditions. Full article

Electrical stimulation is ubiquitous as a method for activating neuronal tissue, but there is still significant room for advancement in the ability of these electrical devices to implement smart stimulus waveform design to more selectively target populations of neurons. The capability of a device to encode more complicated and precise messages to a neuronal network greatly increases if the stimulus input space is broadened to include variable shaped waveforms and multiple stimulating electrodes. The relationship between a stimulating electrode and the activated population is unknown; a priori. For that reason, the population of excitable neurons must be characterized in real-time and for every combination of stimulating electrodes and neuronal populations. Our automated experimental system allows investigation into the stimulus-evoked neuronal response to a current pulse using dissociated neuronal cultures grown atop microelectrode arrays (MEAs). The studies presented here demonstrate that differential activation is achievable between two neurons using either multiple stimulating electrodes or variable waveform shapes. By changing the aspect ratio of a rectangular current pulse; the stimulus activated neurons in the strength–duration (SD) waveform space with differing probabilities. Additionally, in the case when two neuronal activation curves intersect each other in the SD space; one neuron can be selectively activated with short-pulse-width; high-current stimuli while the other can be selectively activated with long-pulse-width; low-current stimuli. Exploring the capabilities and limitations of electrical stimulation allows for improvements to the delivery of stimulus pulses to activate neuronal populations. Many state-of-the-art research and clinical stimulation solutions, including those using a single microelectrode, can benefit from waveform design methods to improve stimulus efficacy. These findings have even greater import into multi-electrode systems because spatially distributed electrodes further enhance accessibility to differential neuronal activation. Full article