Iterative approach to computational enzyme design

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes

Chromatographic Properties of Different Methyl—Phenyl (1:1) Substituted Silicone Stationary Phases for Open-Tubular Gas Chromatography

The influence of different configurations of silicones having 50% methyl and 50% phenyl substitution on chromatographic properties, such as polarity and thermal stability, has been systematically investigated. Polysiloxanes composed of dimethyl and diphenyl units show very low levels of column bleed at temperatures up to 370°C, while polymers having methyl—phenyl substitution show severe bleeding at this temperature. The polarity of the latter polymers, as reflected by Kováts indices, is higher than for the polymers composed by dimethyl—diphenyl unit

Poloidal ULF oscillations in the dayside magnetosphere: a Cluster study

Three ULF wave events, all occurring in the dayside magnetopshere during magnetically quiet times, are studied using the Cluster satellites. The multi-point measurements obtained from Cluster are used to determine the azimuthal wave number for the events by means of the phase shift and the azimuthal separation between the satellites. Also, the polarisation of the electric and magnetic fields is examined in a field-aligned coordinate system, which, in turn, gives the mode of the oscillations. The large-inclination orbits of Cluster allow us to examine the phase relationship between the electric and magnetic fields along the field lines. The events studied have large azimuthal wave numbers (<i>m</i>~100), two of them have eastward propagation and all are in the poloidal mode, consistent with the large wave numbers. We also use particle data from geosynchronous satellites to look for signatures of proton injections, but none of the events show any sign of enhanced proton flux. Thus, the drift-bounce resonance instability seems unlikely to have played any part in the excitation of these pulsations. As for the drift-mirror instability we conclude that it would require an unreasonably high plasma pressure for the instability criterion to be satisfied.<br><br><b>Keywords.</b> Ionosphere (Wave propagation) – Magnetospheric physics (Plasma waves and instabilities; Instruments and techniques

Lending Cycles

We investigate the lending behavior of banks by exploiting a rich panel dataset on the contract terms of approximately two million commercial and industrial loans granted by 580 banks between 1977-1993. Using a Markov switching panel model we demonstrate that banks change their lending standards from tightness to laxity systematically over the cycle. We then use an efficient minimum chi-square estimator to examine the relationship between the cyclical component of aggregate unemployment and bank lending standards when both variables are jointly endogenously determined in a system of simultaneous equations with mixed, continuous/discrete dependent variables. The patterns we uncover suggest that lax lending standards that tend to occur during expansions exert considerable influence on the dynamics of aggregate fluctuations.

Surveillance of antimicrobial resistance at a tertiary hospital in Tanzania

BACKGROUND: Antimicrobial resistance is particularly harmful to infectious disease management in low-income countries since expensive second-line drugs are not readily available. The objective of this study was to implement and evaluate a computerized system for surveillance of antimicrobial resistance at a tertiary hospital in Tanzania. METHODS: A computerized surveillance system for antimicrobial susceptibility (WHONET) was implemented at the national referral hospital in Tanzania in 1998. The antimicrobial susceptibilities of all clinical bacterial isolates received during an 18 months' period were recorded and analyzed. RESULTS: The surveillance system was successfully implemented at the hospital. This activity increased the focus on antimicrobial resistance issues and on laboratory quality assurance issues. The study identified specific nosocomial problems in the hospital and led to the initiation of other prospective studies on prevalence and antimicrobial susceptibility of bacterial infections. Furthermore, the study provided useful data on antimicrobial patterns in bacterial isolates from the hospital. Gram-negative bacteria displayed high rates of resistance to common inexpensive antibiotics such as ampicillin, tetracycline and trimethoprim-sulfamethoxazole, leaving fluoroquinolones as the only reliable oral drugs against common Gram-negative bacilli. Gentamicin and third generation cephalosporins remain useful for parenteral therapy. CONCLUSION: The surveillance system is a low-cost tool to generate valuable information on antimicrobial resistance, which can be used to prepare locally applicable recommendations on antimicrobial use. The system pinpoints relevant nosocomial problems and can be used to efficiently plan further research. The surveillance system also functions as a quality assurance tool, bringing attention to methodological issues in identification and susceptibility testing

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells

<p>Abstract</p> <p>Background</p> <p>The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3⁺T-cells, including CD4⁺and CD8⁺subsets, using Affymetrix microarrays.</p> <p>Results</p> <p>The largest difference between Itk^-/-and Wt CD3⁺T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4⁺and CD8⁺T-cell subsets identified a greater differential expression than in total CD3⁺cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the <it>Bub1</it>, <it>IL7R, Ctla2a</it>, <it>Ctla2b</it>, and <it>Schlafen1 </it>genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found.</p> <p>Conclusion</p> <p>Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.</p

Electroexcitation of the Δ+ (1232) at Low Momentum Transfer

We report on new p(e, e\u27 p)π°. measurements at the Δ+(1232) resonance at the low momentum transfer region, where the mesonic cloud dynamics is predicted to be dominant and rapidly changing, offering a test bed for chiral effective field theory calculations. The new data explore the Q2 dependence of the resonant quadrupole amplitudes and for the first time indicate that the Electric and the Coulomb quadrupole amplitudes converge as Q2 -\u3e 0. The measurements of the Coulomb quadrupole amplitude have been extended to the lowest momentum transfer ever reached, and suggest that more than half of its magnitude is attributed to the mesonic cloud in this region. The new data disagree with predictions of constituent quark models and are in reasonable agreement with dynamical calculations that include pion cloud effects, chiral effective field theory and lattice calculations. The measurements indicate that improvement is required to the theoretical calculations and provide valuable input that will allow their refinements