218 research outputs found
From natural to artificial metalloproteins – Challenges and opportunities
Organometallic catalysts are used in a wide range of industrially important reactions. By immobilizing such catalysts to a protein (Fig. 1), we can bring them into aqueous media, fine-tune activities and selectivities and overcome the challenges associated with trace metal removal in the product fraction [1, 2]. Trace metal removal is particularly important for the synthesis of pharmaceutical compounds. Typically, the transition metal content should be below 10 ppm. We have employed two types of metal catalysts, a Ru-based Grubbs-Hoveyda-type catalyst for olefin metathesis and a Rh-catalyst for phenylacetylene polymerization. These catalysts were covalently attached to either nitrobindin (NB) [3] or ferric hydroxamate uptake protein component: A (FhuA) [4] β-barrel proteins, yielding biohybrid catalysts (also denoted artificial metalloproteins) that can be immersed in aqueous reaction media either in their free form or immobilized to bacterial cells. Moreover, we could show that the metal catalysts can be immobilized on surfaces consisting of silica or polypropylene via peptide-based adhesion promoters, thereby enabling “green” surface immobilization strategies with the potential of catalyst recycling [5]. Either strategy yielded highly active catalysts that show great promise for single or sequential one-pot reactions. Separation of products and the catalysts was readily achieved by extraction. With the potential to tune reaction efficiencies and selectivities by modifying either the metal catalyst or the protein surrounding, biohybrid catalysts bear great potential to amend or even substitute the repertoire of reactions available by organic synthesis and, likewise, biocatalysis.
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Engineered living hydrogels for robust biocatalysis in pure organic solvents
Engineered living hydrogels that can protect cells from harsh environments have achieved preliminary successes in biomedicine and environmental remediation. However, their biocatalytic applications in pure organic solvents have not been explored. Here, living hydrogels were engineered by integrating genetically modified Escherichia coli cells into alginate hydrogels for robust biocatalysis in pure organic solvents. The biocompatible hydrogels could not only support cell growth and diminish cell escape but could also act as protective matrices to improve organic solvent tolerance, thereby prolonging catalytic activity of whole-cell biocatalysts. Moreover, the influence of hydrogel microenvironments on biocatalytic efficiency was thoroughly investigated. Importantly, the versatility of engineered living hydrogels paves the way to achieve robust biocatalytic efficiency in a variety of pure organic co-solvents. Overall, we are able to engineer living hydrogels for regio-selective synthesis in pure organic solvents, which may be particularly useful for the innovation of living hydrogels in biocatalysis
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A hydroquinone-specific screening system for directed P450 evolution
The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). © 2018, The Author(s)
Attracting states in frontal cortex networks associated with working memory and decision making
Unzipping Kinetics of Double-Stranded DNA in a Nanopore
We studied the unzipping kinetics of single molecules of double-stranded DNA
by pulling one of their two strands through a narrow protein pore. PCR analysis
yielded the first direct proof of DNA unzipping in such a system. The time to
unzip each molecule was inferred from the ionic current signature of DNA
traversal. The distribution of times to unzip under various experimental
conditions fit a simple kinetic model. Using this model, we estimated the
enthalpy barriers to unzipping and the effective charge of a nucleotide in the
pore, which was considerably smaller than previously assumed.Comment: 10 pages, 5 figures, Accepted: Physics Review Letter
Limitations of the MELD score in predicting mortality or need for removal from waiting list in patients awaiting liver transplantation
<p>Abstract</p> <p>Background</p> <p>Decompensated cirrhosis is associated with a poor prognosis and liver transplantation provides the only curative treatment option with excellent long-term results. The relative shortage of organ donors renders the allocation algorithms of organs essential. The optimal strategy based on scoring systems and/or waiting time is still under debate.</p> <p>Methods</p> <p>Data sets of 268 consecutive patients listed for single-organ liver transplantation for nonfulminant liver disease between 2003 and 2005 were included into the study. The Model for End-Stage Liver Disease (MELD) and Child-Turcotte-Pugh (CTP) scores of all patients at the time of listing were used for calculation. The predictive ability not only for mortality on the waiting list but also for the need for withdrawal from the waiting list was calculated for both scores. The Mann-Whitney-U Test was used for the univariate analysis and the AUC-Model for discrimination of the scores.</p> <p>Results</p> <p>In the univariate analysis comparing patients who are still on the waiting list and patients who died or were removed from the waiting list due to poor conditions, the serum albumin, bilirubin INR, and CTP and MELD scores as well as the presence of ascites and encephalopathy were significantly different between the groups (p < 0.05), whereas serum creatinine and urea showed no difference.</p> <p>Comparing the predictive abilities of CTP and MELD scores, the best discrimination between patients still alive on the waiting list and patients who died on or were removed from the waiting list was achieved at a CTP score of ≥9 and a MELD score of ≥14.4. The sensitivity and specificity to identify mortality or severe deterioration for CTP was 69.0% and 70.5%, respectively; for MELD, it was 62.1% and 72.7%, respectively. This result was supported by the AUC analysis showing a strong trend for superiority of CTP over MELD scores (AUROC 0.73 and 0.68, resp.; p = 0.091).</p> <p>Conclusion</p> <p>The long term prediction of mortality or removal from waiting list in patients awaiting liver transplantation might be better assessed by the CTP score than the MELD score. This might have implications for the development of new improved scoring systems.</p
Direct observation of Mg<sup>2+</sup> complexes in ionic liquid solutions by <sup>31</sup>Mg β-NMR spectroscopy
Prospective phase II study of preoperative short-course radiotherapy for rectal cancer with twice daily fractions of 2.9 Gy to a total dose of 29 Gy - Long-term results
Estimation and inference under economic restrictions
Estimation of economic relationships often requires imposition of constraints such as positivity or monotonicity on each observation. Methods to impose such constraints, however, vary depending upon the estimation technique employed. We describe a general methodology to impose (observation-specific) constraints for the class of linear regression estimators using a method known as constraint weighted bootstrapping. While this method has received attention in the nonparametric regression literature, we show how it can be applied for both parametric and nonparametric estimators. A benefit of this method is that imposing numerous constraints simultaneously can be performed seamlessly. We apply this method to Norwegian dairy farm data to estimate both unconstrained and constrained parametric and nonparametric models
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