Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

Kinetic characterization for pretreatment of timber varieties and switchgrass using diluted acid hydrolysis

In recent years, growing attention has been devoted to the use of lignocellulosic biomass as a feedstock to produce renewable carbohydrates as a source of energy products, including liquid alternatives to fossil fuels. The benefits of developing woody biomass to ethanol technology are to increase the long-term national energy security, reduce fossil energy consumption, lower greenhouse gas emissions, use renewable rather than depletable resources, and create local jobs. Currently, research is driven by the need to reduce the cost of biomass-ethanol production. One of the preferred methods is to thermochemically pretreat the biomass material and subsequently, enzymatically hydrolyze the pretreated material to fermentable sugars that can then be converted to ethanol using specialized microorganisms. The goals of pretreatment are to remove the hemicellulose fraction from other biomass components, reduce bioconversion time, enhance enzymatic conversion of the cellulose fraction, and, hopefully, obtain a higher ethanol yield. The primary goal of this research is to obtain kinetic detailed data for dilute acid hydrolysis for several timber species from the Upper Peninsula of Michigan and switchgrass. These results will be used to identify optimum reaction conditions to maximize production of fermentable sugars and minimize production of non-fermentable byproducts. The structural carbohydrate analysis of the biomass species used in this project was performed using the procedure proposed by National Renewable Energy Laboratory (NREL). Subsequently, dilute acid-catalyzed hydrolysis of biomass, including aspen, basswood, balsam, red maple, and switchgrass, was studied at various temperatures, acid concentrations, and particle sizes in a 1-L well-mixed batch reactor (Parr Instruments, ii Model 4571). 25 g of biomass and 500 mL of diluted acid solution were added into a 1-L glass liner, and then put into the reactor. During the experiment, 5 mL samples were taken starting at 100°C at 3 min intervals until reaching the targeted temperature (160, 175, or 190°C), followed by 4 samples after achieving the desired temperature. The collected samples were then cooled in an ice bath immediately to stop the reaction. The cooled samples were filtered using 0.2 μm MILLIPORE membrane filter to remove suspended solids. The filtered samples were then analyzed using High Performance Liquid Chromatography (HPLC) with a Bio-Rad Aminex HPX-87P column, and refractive index detection to measure monomeric and polymeric sugars plus degradation byproducts. A first order reaction model was assumed and the kinetic parameters such as activation energy and pre-exponential factor from Arrhenius equation were obtained from a match between the model and experimental data. The reaction temperature increases linearly after 40 minutes during experiments. Xylose and other sugars were formed from hemicellulose hydrolysis over this heat up period until a maximum concentration was reached at the time near when the targeted temperature was reached. However, negligible amount of xylose byproducts and small concentrations of other soluble sugars, such as mannose, arabinose, and galactose were detected during this initial heat up period. Very little cellulose hydrolysis yielding glucose was observed during the initial heat up period. On the other hand, later in the reaction during the constant temperature period xylose was degraded to furfural. Glucose production from cellulose was increased during this constant temperature period at later time points in the reaction. The kinetic coefficient governing the generation of xylose from hemicellulose and the generation of furfural from xylose presented a coherent dependence on both temperature and acid concentration. However, no effect was observed in the particle size. There were three types of biomass used in this project; hardwood (aspen, basswood, and red maple), softwood (balsam), and a herbaceous crop (switchgrass). The activation energies and the pre-exponential factors of the timber species and switchgrass were in a range of 49 - 180 kJ/mol and from 7.5x104 - 2.6x1020 min-1, respectively, for the xylose formation model. In addition, for xylose degradation, the activation energies and the preexponential factors ranged from 130 - 170 kJ/mol and from 6.8x1013 - 3.7x1017 min-1, respectively. The results compare favorably with the literature values given by Ranganathan et al, 1985. Overall, up to 92 % of the xylose was able to generate from the dilute acid hydrolysis in this project.

Comparison of 3D kinetic and hydrodynamic models to ROSINA-COPS measurements of the neutral coma of 67P/Churyumov-Gerasimenko

67P/Churyumov-Gerasimenko (67P) is a Jupiter-family comet and the object of investigation of the European Space Agency mission Rosetta. This report presents the first full 3D simulation results of 67P’s neutral gas coma. In this study we include results from a direct simulation Monte Carlo method, a hydrodynamic code, and a purely geometric calculation which computes the total illuminated surface area on the nucleus. All models include the triangulated 3D shape model of 67P as well as realistic illumination and shadowing conditions. The basic concept is the assumption that these illumination conditions on the nucleus are the main driver for the gas activity of the comet. As a consequence, the total production rate of 67P varies as a function of solar insolation. The best agreement between the model and the data is achieved when gas fluxes on the night side are in the range of 7% to 10% of the maximum flux, accounting for contributions from the most volatile components. To validate the output of our numerical simulations we compare the results of all three models to in situ gas number density measurements from the ROSINA COPS instrument. We are able to reproduce the overall features of these local neutral number density measurements of ROSINA COPS for the time period between early August 2014 and January 1 2015 with all three models. Some details in the measurements are not reproduced and warrant further investigation and refinement of the models. However, the overall assumption that illumination conditions on the nucleus are at least an important driver of the gas activity is validated by the models. According to our simulation results we find the total production rate of 67P to be constant between August and November 2014 with a value of about 1 × 10²⁶ molecules s⁻¹.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

Upper-ocean microstructure data from the tropical Atlantic

SST variability within the Atlantic cold tongue (ACT) region is of climatic relevance for the surrounding continents. A multi cruise data set of microstructure observations is used to infer regional as well as seasonal variability of upper ocean mixing and diapycnal heat flux within the ACT region. The variability in mixing intensity is related to the variability in large scale background conditions, which were additionally observed during the cruises. The observations indicate fundamental differences in background conditions in terms of shear and stratification below the mixed layer (ML) for the western and eastern equatorial ACT region causing critical Froude numbers (Fr) to be more frequently observed in the western equatorial ACT. The distribution of critical Fr occurrence below the ML reflects the regional and seasonal variability of mixing intensity. Turbulent dissipation rates (?) at the equator (2°N-2°S) are strongly increased in the upper thermocline compared to off-equatorial locations. In addition, ? is elevated in the western equatorial ACT compared to the east from May to November, whereas boreal summer appears as the season of highest mixing intensities throughout the equatorial ACT region, coinciding with ACT development. Diapycnal heat fluxes at the base of the ML in the western equatorial ACT region inferred from ? and stratification range from a maximum of 90 Wm-2 in boreal summer to 55 Wm-2 in September and 40 Wm-2 in November. In the eastern equatorial ACT region maximum values of about 25 Wm-2 were estimated during boreal summer reducing to about 5 Wm-2 towards the end of the year. Outside the equatorial region, inferred diapycnal heat fluxes are comparably low rarely exceeding 10 Wm-2. Integrating the obtained heat flux estimates in the ML heat budget at 10°W on the equator accentuates the diapycnal heat flux as the largest ML cooling term during boreal summer and early autumn. In the western equatorial ACT elevated meridional velocity shear in the upper thermocline contributes to the enhanced diapycnal heat flux within this region during boreal summer and autumn. The elevated meridional velocity shear appears to be associated with intra-seasonal wave activity.

Physical oceanography, current meter data, and microstructure measurements 180 km downstream of Denmark Strait sill during Maria S. Merian cruise MSM21/1b

The main objectives of the cruise MSM21/1b were to characterize the spatio-temporal variability of the Denmark Strait overflow and to identify processes responsible for the exchange of the overflow plume with ambient water downstream of Denmark Strait. A multi-platform approach was taken to achieve the goals, based on moorings, an autonomous underwater vehicle (AUV), as well as lowered and vessel-mounted observations. From these platforms, measurements of temperature, salinity, dissolved oxygen, current velocity, dissipation of turbulent kinetic energy, and bottom pressure were obtained.