Reply to the letter to the editor: Could the use of bone morphogenetic proteins in fracture healing do more harm than good to our patients?

Surger

Icemaker^(TM): an excel-based environment for collaborative design

The creative process of team design can be rapid and powerful when focused, yet complex designs, such as spacecrafit, can slow and quench the essential elements of this process. Concurrent Engineering techniques partially address this problem, but a fuller realization of their benefits require an approach centering on the human aspects of teamwork. ICEMaker^(TM) is a Microsoft Excel® based software tool that facilitates closer-to-ideal collaboration within teams employing the new Integrated Concurrent Engineering (ICE) methodology. ICE is a generic approach that emphasizes focused collaborative design in a single-room context, and is now employed at several aerospace organizations to increase the productivity of design teams defining complex early development-phase products. By way of introduction, this paper describes the basic elements of ICE needed to understand ICEMaker and its application. We present the design approach, philosophy, and client-server architecture of the ICEMaker system, as well as a simplified user scenario. NASA's Jet Propulsion Laboratory (JPL) has recently adopted ICEMaker for its primary early-phase space mission and system advanced project design team, Team-X. We describe Team-X's experience with ICEMaker and report on the lessons learned, and qualitative product improvements, resulting from JPL's implementation of ICEMaker

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C‐C Bond‐Forming Enzyme

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins

Using Mutability Landscapes To Guide Enzyme Thermostabilization

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature Tm (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in Tm value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes

Guidance for laboratories performing molecular pathology for cancer patients

Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here

Structural basis for the catalytic mechanism of ethylenediamine-N,N′-disuccinic acid lyase, a carbon-nitrogen bond-forming enzyme with broad substrate scope

The natural aminocarboxylic acid product ethylenediamine-N,N′-disuccinic acid [(S,S)-EDDS] is able to form a stable complex with metal ions, making it an attractive biodegradable alternative for the synthetic metal chelator ethylenediamine tetraacetic acid (EDTA), which is currently used at large scale in numerous applications. Previous studies have demonstrated that biodegradation of (S,S)-EDDS may be initiated by an EDDS lyase, converting (S,S)-EDDS via the intermediate N-(2-aminoethyl)aspartic acid (AEAA) into ethylenediamine and two molecules of fumarate. However, current knowledge of this enzyme is limited due to the absence of structural data. Here, we describe the identification and characterization of an EDDS lyase from Chelativorans sp. BNC1, which has a broad substrate scope, accepting various mono- and diamines for addition to fumarate. We report crystal structures of the enzyme in an unliganded state and in complex with formate, succinate, fumarate, AEAA and (S,S)-EDDS. The structures reveal a tertiary and quaternary fold that is characteristic of the aspartase/fumarase superfamily and support a mechanism that involves general base-catalyzed, sequential two-step deamination of (S,S)-EDDS. This work broadens our understanding of mechanistic diversity within the aspartase/fumarase superfamily and will aid in the optimization of EDDS lyase for asymmetric synthesis of valuable (metal-chelating) aminocarboxylic acids

Monitoren van kleine landschapselementen met IKONOS satellietbeelden

In deze studie is nagegaan in hoeverre zeer hoge-resolutie satellietbeelden (ZHRS), lees IKONOS, een toegevoegde waarde kunnen hebben ten opzichte van luchtfoto¿s en de Top10-vector in het Meetnet Landschap. Alle aandacht binnen deze studie was gericht op kleine landschapselementen. Voor twee studiegebieden zijn IKONOS satellietbeelden vergeleken met kleurenluchtfoto¿s en de informatie in de Top10-vector. Daarnaast is er gekeken naar de temporele dynamiek van kleine landschapselementen en naar de actualiteit en nauwkeurigheid van de Top10-vector voor deze landschapselementen. Uit deze studie is gebleken dat IKONOS satellietbeelden en luchtfoto¿s, naast de Top10-vector, belangrijke informatie kunnen verschaffen voor monitoring van kleine landschapselementen. Het bleek zelfs dat panchromatische IKONOS satellietbeelden zich beter lenen voor het detecteren van opgaande beplantingen dan de gebruikte (true color) kleurenluchtfoto¿s met een 1m resolutie. Dit komt door het feit dat de panchromatische IKONOS beelden ook in het nabij-infrarood meten. Ten tweede kunnen structuurparameters (bv. breedte, continuïteit, homogeniteit en exacte begrenzingen) uit de ZHRS en /of luchtfoto¿s worden gehaald die niet in de Top10-vector aanwezig zijn. Ten derde blijkt dat de Top10-vector vaak te onbetrouwbaar was wat betreft kleine landschappelijke elementen. Er moet dus voor veldwerk en/of regionale studies ook gebruik worden gemaakt van ZHRS en/of luchtfoto¿s. Als actualiteit een belangrijke voorwaarde is dan is dit een extra argument om gebruik te maken van recente IKONOS satellietbeelden en/of luchtfoto¿s. Dit ook omdat uit deze en andere studies is gebleken dat de dynamiek (lees achteruitgang) van vele kleine landschapselementen zeer hoog is. Uit deze studie is verder gebleken data visuele interpretatie nu gedeeltelijk kan worden vervangen door semi-automatische classificatie m.b.v. het softwarepakket eCognition. Objectgerichte classificatie m.b.v. eCognition en IKONOS satellietbeelden gaf in deze studie goede resultaten voor het vastleggen van kleine landschapselementen (hier in het geval van bomen). Echter voor verdere inbedding is een goed satellietbeeldenarchief nodig waaruit gebruikers snel data kunnen downloaden. Bovendien is een gunstiger prijsbeleid een essentiële voorwaarde voor het succes van IKONOS satellietbeelden

Computational Redesign of an ω-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines

[Image: see text] ω-Transaminases (ω-TA) are attractive biocatalysts for the production of chiral amines from prochiral ketones via asymmetric synthesis. However, the substrate scope of ω-TAs is usually limited due to steric hindrance at the active site pockets. We explored a protein engineering strategy using computational design to expand the substrate scope of an (S)-selective ω-TA from Pseudomonas jessenii (PjTA-R6) toward the production of bulky amines. PjTA-R6 is attractive for use in applied biocatalysis due to its thermostability, tolerance to organic solvents, and acceptance of high concentrations of isopropylamine as amino donor. PjTA-R6 showed no detectable activity for the synthesis of six bicyclic or bulky amines targeted in this study. Six small libraries composed of 7–18 variants each were separately designed via computational methods and tested in the laboratory for ketone to amine conversion. In each library, the vast majority of the variants displayed the desired activity, and of the 40 different designs, 38 produced the target amine in good yield with >99% enantiomeric excess. This shows that the substrate scope and enantioselectivity of PjTA mutants could be predicted in silico with high accuracy. The single mutant W58G showed the best performance in the synthesis of five structurally similar bulky amines containing the indan and tetralin moieties. The best variant for the other bulky amine, 1-phenylbutylamine, was the triple mutant W58M + F86L + R417L, indicating that Trp58 is a key residue in the large binding pocket for PjTA-R6 redesign. Crystal structures of the two best variants confirmed the computationally predicted structures. The results show that computational design can be an efficient approach to rapidly expand the substrate scope of ω-TAs to produce enantiopure bulky amines