Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4‐Oxalocrotonate Tautomerase More Effectively than Distant Mutations

The enzyme 4‐oxalocrotonate tautomerase (4‐OT), which catalyzes enol–keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon–carbon bond‐forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4‐OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000‐fold improvement in catalytic efficiency (k_cat/K_m) and a >10⁷‐fold change in reaction specificity, by exploring small libraries in which only “hotspots” are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4‐OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.     

pOVEX vector: prokaryotic expression and purification of onchocerciasis vaccine candidate antigens as fusion proteins with the 24 kD Onchocerca volvulus glutathione S-transferase

We describe a 42-year-old male patient with a primitive peripheral neuroectodermal tumor (PPNET) arising from the kidney. The patient was treated sequentially with surgery, radiotherapy, experimental cytokine treatment (rhIL-6), combination chemotherapy and consolidated in complete remission with high-dose chemotherapy, supported by autologous stem cells (AST) and hematopoietic growth factors. The patient remains in complete remission at 34+ months after AST and is surviving disease-free for five years after initial presentation. The case presented here is unusual because of the renal origin and the long remission duration and survival when compared with the usual outcome of adult metastatic PPNET. High-dose chemotherapy may have favorably influenced the outcome in this patient. The case is discussed in the context of the general treatment of metastatic PPNET.     

Qualit?t in der Analytik

Zusammenfassung Analysenergebnissen ist deren Qualität nicht anzusehen. Hierfür sind vielmehr das Zustandekommen der Daten und der Versuchsplan maßgebend, der an der Fragestellung und am Vorwissen auszurichten ist. Wenn der Analytiker keinen Einfluß auf den Versuchsplan hatte und nur Proben vorgelegt bekommt, kann die chemische Analytik unter Umständen in Schwierigkeiten geraten. Eine unvollständige Problemanalyse oder ein ungeeigneter Versuchsplan kann dazu führen, daß bei aller Sorgfalt der Bestimmung sowie hervorragender Richtigkeit und Präzision des analytischen Verfahrens unbrauchbare Daten erhalten werden. Die rezeptartige Anwendung statistischer Auswertungsmethoden liefert in solchen Fällen allenfalls Verzierungen.

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Quality in analytical chemistry

Summary Quality of analytical results cannot be recognized directly. For this purpose the way of obtaining the data and the experimental design is the crucial point. The design should be a consequence of the kind of question to be answered and of prior knowledge. Analytical chemistry can lead to great difficulties, if the analytical chemist had no influence on the design and only received the samples taken. Despite of all his endeavors and of an excellent accuracy and precision of the analytical method, uselessdata might be obtained as a consequence of incomplete planning and unsuited experimental design. Under such circumstances the formal application of statistical methods would provide decoration only.

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In Anlehnung an einen Plenarvortrag bei der ANALYTICA 84 in München     

Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self‐Condensations of Linear Aliphatic Aldehydes

The enzyme 4‐oxalocrotonate tautomerase (4‐OT) from Pseudomonas putida mt‐2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa‐2,4‐dienedioate into 2‐oxohexa‐3‐enedioate. This tautomerase can also promiscuously catalyze carbon–carbon bond‐forming reactions, including various types of aldol reactions, by using its amino‐terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4‐OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self‐condensation of propanal. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4‐OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self‐condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β‐unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self‐condensation of propanal rather than the cross‐condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self‐coupling of aliphatic aldehydes.     

Vertex angle and crossing angle resolution of leveled tree drawings

We study the problem of computing leveled tree drawings, i.e., straight-line drawings of trees where the vertices have distinct preassigned y-coordinates. Our optimization goal is to maximize the crossing angle resolution (i.e., the minimum angle formed by any two crossing edges) and/or the vertex angle resolution (i.e., the minimum angle formed by two edges incident to the same vertex) of the drawing. We provide tight and almost tight worst case bounds for the crossing angle resolution and for the total angle resolution (i.e., the minimum of crossing and vertex angle resolution), respectively.     

Mindfulness-based cognitive therapy: Evaluating current evidence and informing future research.

Mindfulness-based cognitive therapy (MBCT) is a recently developed class-based program designed to prevent relapse or recurrence of major depression (Z. V. Segal, J. M. G. Williams, & J. Teasdale, 2002). Although research in this area is in its infancy, MBCT is generally discussed as a promising therapy in terms of clinical effectiveness. The aim of this review was to outline the evidence that contributes to this current viewpoint and to evaluate the strengths and weaknesses of this evidence to inform future research. By systematically searching 6 electronic databases and the reference lists of retrieved articles, the authors identified 4 relevant studies: 2 randomized clinical trials, 1 study based on a subset of 1 of these trials, and 1 nonrandomized trial. The authors evaluated these trials and discussed methodological issues in the context of future research. The current evidence from the randomized trials suggests that, for patients with 3 or more previous depressive episodes, MBCT has an additive benefit to usual care. However, because of the nature of the control groups, these findings cannot be attributed to MBCT-specific effects. Further research is necessary to clarify whether MBCT does have any specific effects. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Pyruvate Supplementation for Weight Loss: A Systematic Review and Meta-Analysis of Randomized Clinical Trials

Background : Several slimming aids being sold as food supplements are widely available. One of them is pyruvate. Its efficacy in causing weight reduction in humans has not been fully established. The objective of this systematic review was to examine the efficacy of pyruvate in reducing body weight.

Methods: Electronic and nonelectronic searches were conducted to identify all relevant human randomized clinical trials. The bibliographies of all located articles were also searched. No restrictions in language or time were applied. Two independent reviewers extracted the data according to predefined criteria. A fixed-effect model was used to calculate mean differences (MD) and 95% confidence interval (CI).

Results: Nine trials were identified and 6 were included. All had methodological weaknesses. The meta-analysis revealed a statistically significant difference in body weight with pyruvate compared to placebo (MD: ?0.72 kg; 95% CI: ?1.24 to ?0.20). The magnitude of the effect is small, and its clinical relevance is uncertain. Adverse events included gas, bloating, diarrhea, and increase in low-density lipoprotein (LDL) cholesterol.

Conclusion: The evidence from randomized clinical trials does not convincingly show that pyruvate is efficacious in reducing body weight. Limited evidence exists about the safety of pyruvate. Future trials involving the use of this supplement should be more rigorous and better reported.     

Test criteria and coverage measures for software integration testing

The aim of this paper is to introduce a systematic approach to integration testing of software systems. Various test data selection criteria for integration testing are presented, coverage measures are introduced, and interconnection between them are discussed. The main principle is to transfer and adapt test criteria and coverage measures which are useful for unit testing to the level of integration testing. Test criteria help the tester to organise the test process. They should be chosen in accordance with the available test effort. Test coverage measures are defined as a ratio between the test cases required for satisfying the criteria and those of these which have been executed. The measures are used to obtain information about the completeness of integration tests. The approach is described for data flow and control flow oriented criteria and measures. The intention is to enable the tester to specify integration tests in advance in terms of effort, and to evaluate the results in terms of test completeness.