Modelling and optimisation of the one-pot, multi-enzymatic synthesis of chiral amino-alcohols based on microscale kinetic parameter determination

Advances in synthetic biology are facilitating the de novo design of complex, multi-step enzymatic conversions for industrial organic synthesis. This work describes the integration of multi-step enzymatic pathway construction with enzyme kinetics and bioreactor modelling, in order to optimise the synthesis of chiral amino-alcohols using engineered Escherichia coli transketolases (TK) and the Chromobacterium violaceum transaminase (TAm). The specific target products were (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT). Kinetic models and parameters for each of the enzymatic steps were first obtained using automated microwell experiments. These identified the TK-catalysed conversions as being up to 25 times faster than the subsequent TAm conversions and inhibition of TAm by the amino-donor used, (S)-(−)-α-methylbenzylamine (MBA), as limiting the overall conversion yields. In order to better ‘match’ the relative rates of the two enzymes an E. coli expression system, based on two compatible plasmids, was constructed to produce both enzymes in a single host. By control of induction time and temperature it was possible to produce six times more recombinant TAm than TK to help balance the reaction rates. To overcome MBA inhibition and an unfavourable reaction equilibrium, fed-batch addition of the amino-donor was introduced as well as the use of isopropylamine as an alternate amino-donor. Adopting these strategies, and using the kinetic models to optimise feeding strategies, the one pot syntheses of APD and ABT were successfully scaled-up to preparative scales. Excellent agreement was found between the kinetic profiles and yields predicted and those achieved experimentally at the larger scale. In this case the integration of these multi-disciplinary approaches enabled us to achieve up to a 6 fold greater yield using concentrations an order of magnitude higher than in previous preparative scale batch bioconversions carried out sequentially

Numerical approximations of second-order matrix differential equations using higher-degree splines

Many studies of mechanical systems in engineering are based on second-order matrix models. This work discusses the second-order generalization of previous research on matrix differential equations dealing with the construction of approximate solutions for non-stiff initial problems Y 00(x) = f(x, Y (x), Y 0 (x)) using higher-degree matrix splines without any dimensional increase. An estimation of the approximation error for some illustrative examples are presented by using Mathematica. Several MatLab functions have also been developed, comparing, under equal conditions, accuracy and execution times with built-in MatLab functions. Experimental results show the advantages of solving the above initial problem by using the implemented MatLab functions.The authors wish to thank for financial support by the Universidad Politecnica de Valencia [grant number PAID-06-11-2020].Defez Candel, E.; Tung ., MM.; Solis Lozano, FJ.; Ibáñez González, JJ. (2015). Numerical approximations of second-order matrix differential equations using higher-degree splines. Linear and Multilinear Algebra. 63(3):472-489. https://doi.org/10.1080/03081087.2013.873427S472489633Loscalzo, F. R., & Talbot, T. D. (1967). Spline Function Approximations for Solutions of Ordinary Differential Equations. SIAM Journal on Numerical Analysis, 4(3), 433-445. doi:10.1137/0704038Al-Said, E. A. (2001). The use of cubic splines in the numerical solution of a system of second-order boundary value problems. Computers & Mathematics with Applications, 42(6-7), 861-869. doi:10.1016/s0898-1221(01)00204-8Al-Said, E. A., & Noor, M. A. (2003). Cubic splines method for a system of third-order boundary value problems. Applied Mathematics and Computation, 142(2-3), 195-204. doi:10.1016/s0096-3003(02)00294-1Kadalbajoo, M. K., & Patidar, K. C. (2002). Numerical solution of singularly perturbed two-point boundary value problems by spline in tension. Applied Mathematics and Computation, 131(2-3), 299-320. doi:10.1016/s0096-3003(01)00146-1Micula, G., & Revnic, A. (2000). An implicit numerical spline method for systems for ODEs. Applied Mathematics and Computation, 111(1), 121-132. doi:10.1016/s0096-3003(98)10111-xDefez, E., Soler, L., Hervás, A., & Santamaría, C. (2005). Numerical solution ofmatrix differential models using cubic matrix splines. Computers & Mathematics with Applications, 50(5-6), 693-699. doi:10.1016/j.camwa.2005.04.012Defez, E., Hervás, A., Soler, L., & Tung, M. M. (2007). Numerical solutions of matrix differential models using cubic matrix splines II. Mathematical and Computer Modelling, 46(5-6), 657-669. doi:10.1016/j.mcm.2006.11.027Ascher, U., Pruess, S., & Russell, R. D. (1983). On Spline Basis Selection for Solving Differential Equations. SIAM Journal on Numerical Analysis, 20(1), 121-142. doi:10.1137/0720009Brunner, H. (2004). On the Divergence of Collocation Solutions in Smooth Piecewise Polynomial Spaces for Volterra Integral Equations. BIT Numerical Mathematics, 44(4), 631-650. doi:10.1007/s10543-004-3828-5Tung, M. M., Defez, E., & Sastre, J. (2008). Numerical solutions of second-order matrix models using cubic-matrix splines. Computers & Mathematics with Applications, 56(10), 2561-2571. doi:10.1016/j.camwa.2008.05.022Defez, E., Tung, M. M., Ibáñez, J. J., & Sastre, J. (2012). Approximating and computing nonlinear matrix differential models. Mathematical and Computer Modelling, 55(7-8), 2012-2022. doi:10.1016/j.mcm.2011.11.060Claeyssen, J. R., Canahualpa, G., & Jung, C. (1999). A direct approach to second-order matrix non-classical vibrating equations. Applied Numerical Mathematics, 30(1), 65-78. doi:10.1016/s0168-9274(98)00085-3Froese, C. (1963). NUMERICAL SOLUTION OF THE HARTREE–FOCK EQUATIONS. Canadian Journal of Physics, 41(11), 1895-1910. doi:10.1139/p63-189Marzulli, P. (1991). Global error estimates for the standard parallel shooting method. Journal of Computational and Applied Mathematics, 34(2), 233-241. doi:10.1016/0377-0427(91)90045-lShore, B. W. (1973). Comparison of matrix methods applied to the radial Schrödinger eigenvalue equation: The Morse potential. The Journal of Chemical Physics, 59(12), 6450-6463. doi:10.1063/1.1680025ZHANG, J. F. (2002). OPTIMAL CONTROL FOR MECHANICAL VIBRATION SYSTEMS BASED ON SECOND-ORDER MATRIX EQUATIONS. Mechanical Systems and Signal Processing, 16(1), 61-67. doi:10.1006/mssp.2001.1441Flett, T. M. (1980). Differential Analysis. doi:10.1017/cbo978051189719

A socio-environmental geodatabase for integrative research in the transboundary Rio Grande/Río Bravo basin

Integrative research on water resources requires a wide range of socio-environmental datasets to better understand human-water interactions and inform decision-making. However, in transboundary watersheds, integrating cross-disciplinary and multinational datasets is a daunting task due to the disparity of data sources and the inconsistencies in data format, content, resolution, and language. This paper introduces a socio-environmental geodatabase that transcends political and disciplinary boundaries in the Rio Grande/Río Bravo basin (RGB). The geodatabase aggregates 145 GIS data layers on five main themes: (i) Water & Land Governance, (ii) Hydrology, (iii) Water Use & Hydraulic Infrastructures, (iv) Socio-Economics, and (v) Biophysical Environment. Datasets were primarily collected from public open-access data sources, processed with ArcGIS, and documented through the FGCD metadata standard. By synthesizing a broad array of datasets and mapping public and private water governance, we expect to advance interdisciplinary research in the RGB, provide a replicable approach to dataset compilation for transboundary watersheds, and ultimately foster transboundary collaboration for sustainable resource management.The project was funded by Grant No. G15AP00132 from the United States Geological Survey. Open Access fees paid for in whole or in part by the University of Oklahoma Libraries.Ye

Effect of Cr(V) on reproductive organ morphology and sperm parameters: An experimental study in mice

BACKGROUND: Cr(V) species are formed during the intracellular reduction of Cr(VI), a ubiquitous environmental pollutant. In this study, the acute toxicity of a physiologically stable Cr(V) compound, [Cr(V)-BT](2- )(BT = bis(hydroxyethyl)aminotris(hydroxymethyl)methane) was investigated in the male reproductive system of sexually mature 60-day-old male ICR-CD1 mice. METHODS: Eight-week-old animals were subcutaneously injected daily with a dose of ca 8 μmol of Cr/mouse, during 5 days. The control group was injected with 0.5 mL of BT buffer. Testis and epididymis morphology was evaluated using light and transmission electron microscopy. Epididymal sperm counts, motility and acrosome integrity were also assayed using standard methods. RESULTS: Seminiferous epithelium abnormalities were detected in the Cr(V)-BT experimental group, including intraepithelial vacuolation, and remarkable degeneration of Sertoli cells, spermatocytes and spermatids. The premature release of germ cells into the tubular lumen was also evident. Histological evaluation of epididymal compartments revealed apparently normal features. However, the epididymal epithelium presented vacuolation. [Cr(V)-BT](2- )induced a reduction in sperm acrosome integrity. However, sperm motility and density were not significantly affected. CONCLUSION: This in vivo study using a Cr(V) compound, provides evidence for the potential reproductive hazards caused on male reproductive system by species containing chromium in intermediate oxidation states

Primary Language and Receipt of Recommended Health Care Among Hispanics in the United States

BackgroundDisparities in health care services between Hispanics and whites in the United States are well documented.ObjectiveThe objective of the study was to determine whether language spoken at home identifies Hispanics at risk for not receiving recommended health care services.DesignThe design of the study was cross-sectional, nationally representative survey of households.PatientsThe patients were non-Hispanic white and Hispanic adults participating in the 2003 Medical Expenditure Panel Survey.MeasurementsWe compared receipt of ten recommended health care services by ethnicity and primary language adjusting for demographic and socioeconomic characteristics, health status, and access to care.ResultsThe sample included 12,706 whites and 5,500 Hispanics. In bivariate comparisons, 57.0% of whites received all eligible health care services compared to 53.6% for Hispanics who spoke English at home, 44.9% for Hispanics who did not speak English at home but who were comfortable speaking English, and 35.0% for Hispanics who did not speak English at home and were uncomfortable speaking English (p < .001). In multivariate logistic models, compared to non-Hispanic whites, Hispanics who did not speak English at home were less likely to receive all eligible health care services, whether they were comfortable speaking English (risk ratio [RR] 0.88, 95% confidence interval [CI] 0.74-0.97) or not (RR 0.84, 95% CI 0.68-0.95).ConclusionsSpeaking a language other than English at home identified Hispanics at risk for not receiving recommended health care services, whether they were comfortable in speaking English or not. Identifying the mechanism for disparities by language usage may lead to interventions to reduce ethnic disparities

Characterization of Synaptically Connected Nuclei in a Potential Sensorimotor Feedback Pathway in the Zebra Finch Song System

Birdsong is a learned behavior that is controlled by a group of identified nuclei, known collectively as the song system. The cortical nucleus HVC (used as a proper name) is a focal point of many investigations as it is necessary for song production, song learning, and receives selective auditory information. HVC receives input from several sources including the cortical area MMAN (medial magnocellular nucleus of the nidopallium). The MMAN to HVC connection is particularly interesting as it provides potential sensorimotor feedback to HVC. To begin to understand the role of this connection, we investigated the physiological relation between MMAN and HVC activity with simultaneous multiunit extracellular recordings from these two nuclei in urethane anesthetized zebra finches. As previously reported, we found similar timing in spontaneous bursts of activity in MMAN and HVC. Like HVC, MMAN responds to auditory playback of the bird's own song (BOS), but had little response to reversed BOS or conspecific song. Stimulation of MMAN resulted in evoked activity in HVC, indicating functional excitation from MMAN to HVC. However, inactivation of MMAN resulted in no consistent change in auditory responses in HVC. Taken together, these results indicate that MMAN provides functional excitatory input to HVC but does not provide significant auditory input to HVC in anesthetized animals. We hypothesize that MMAN may play a role in motor reinforcement or coordination, or may provide modulatory input to the song system about the internal state of the animal as it receives input from the hypothalamus

Limits on WWZ and WW\gamma couplings from p\bar{p}\to e\nu jj X events at \sqrt{s} = 1.8 TeV

We present limits on anomalous WWZ and WW-gamma couplings from a search for WW and WZ production in p-bar p collisions at sqrt(s)=1.8 TeV. We use p-bar p -> e-nu jjX events recorded with the D0 detector at the Fermilab Tevatron Collider during the 1992-1995 run. The data sample corresponds to an integrated luminosity of 96.0+-5.1 pb^(-1). Assuming identical WWZ and WW-gamma coupling parameters, the 95% CL limits on the CP-conserving couplings are -0.33<lambda<0.36 (Delta-kappa=0) and -0.43<Delta-kappa<0.59 (lambda=0), for a form factor scale Lambda = 2.0 TeV. Limits based on other assumptions are also presented.Comment: 11 pages, 2 figures, 2 table

The Dijet Mass Spectrum and a Search for Quark Compositeness in bar{p}p Collisions at sqrt{s} = 1.8 TeV

Using the DZero detector at the 1.8 TeV pbarp Fermilab Tevatron collider, we have measured the inclusive dijet mass spectrum in the central pseudorapidity region |eta_jet| < 1.0 for dijet masses greater than 200 Gev/c^2. We have also measured the ratio of spectra sigma(|eta_jet| < 0.5)/sigma(0.5 < |eta_jet| < 1.0). The order alpha_s^3 QCD predictions are in good agreement with the data and we rule out models of quark compositeness with a contact interaction scale < 2.4 TeV at the 95% confidence level.Comment: 11 pages, 4 figures, 2 tables, submitted to Phys. Rev. Let

Search For Heavy Pointlike Dirac Monopoles

We have searched for central production of a pair of photons with high transverse energies in $p\bar p$ collisions at $\sqrt{s} = 1.8$ TeV using $70 pb^{-1}$ of data collected with the D\O detector at the Fermilab Tevatron in 1994--1996. If they exist, virtual heavy pointlike Dirac monopoles could rescatter pairs of nearly real photons into this final state via a box diagram. We observe no excess of events above background, and set lower 95% C.L. limits of $610, 870, or 1580 GeV/c^2$ on the mass of a spin 0, 1/2, or 1 Dirac monopole.Comment: 12 pages, 4 figure