Shaping the Place - A Digital Design Heuristics Tool to Support Creation of Urban Design Proposals by Non-professionals

This paper is exploring a solution to foster civic engagement in urban design projects by applying the concepts of creativity to ICT tools. We propose a framework to support interactions between non-professionals and professionals that will ease the understanding of urban design and creation of design proposals for non-trained people and, on the other hand, offer valuable propositions and inspiration to experts. This make tool should have the presented creativity affordances known as fluency, flexibility and originality during the divergent phase of the creation process. We propose to implement a 3D collage metaphor to facilitate creative expression with 3D models. An underlying technical challenge of our application is to provide an interactive 3D mesh cutting tool to help users to express their creative potential in urban design projects. We present a non-exhaustive survey of mesh segmentation and cutting methodologies and finally, first results of implementation of a cutting algorithm

Solid-state NMR investigations of the ATP binding cassette multidrug transporter LmrA

The development of resistance to multiple drugs is a major problem in treatment of number of infectious diseases and cancer. The phenomenon of multidrug resistance (MDR) is based on the synergetic interplay of a number of mechanisms such as target inactivation, target alteration, prevention of drug influx as well as active extrusion of drugs from the cell. The latter is mediated by over-expression of multidrug efflux pumps. The first discovered and the best characterized until now the human MDR transporter is P-glycoprotein. It is a member of the ATP binding cassette (ABC) superfamily and acts as an active transporter for a variety of anticancer agents using the energy released by ATP hydrolysis. The closest structure and functional homologue of P-glycoprotein found in bacteria is LmrA from Lactococcus lactis. The major goals of this work are to establish the selective isotope labelling of LmrA in Lactococcus lactis, to optimize LmrA sample preparation for solid-state NMR, and finally to perform first solidstate NMR investigations on LmrA shedding light on its catalytic cycle and substrate binding. For a long time the solid-state NMR applications to biological science has been limited to investigation of small molecules mostly. Recently, the solid-state NMR methods have shown potential for structuraland non-perturbing, site directed functional studies of large membrane proteins as well as ligands bound to them. However, to our knowledge neither selective isotope amino acid labelling of any ABC transporter, nor NMR investigations on full-length ABC transporter have been reported to date. Solidstate NMR experiments on a membrane protein require reconstitution of purified proteins into a membrane environment at a high density and either isotopic enrichment of the protein or bound drugs or inhibitors. Therefore, the large quantities of LmrA reconstituted at a high density in lipid membranes, sufficient for advanced NMR studies have been produced and its functional state in reconstituted form has been assessed. In the next step, a procedure for cost effective selective amino acids isotope labelling of LmrA in Lactococcus lactis has been established. Using this protocol deuterium alanine labelled LmrA reconstituted into E. coli liposomes has been prepared. Deuterium NMR has been used extensively to assess the proteins dynamics in past. However, it has never been applied to ABC transporter. Here, we report 2H NMR on selective alanine isotope labelled LmrA which has been used to shed light on the dynamics changes in the protein occurred under AMP-PNP, non-hydrolysable ATP analogue, binding and in ATP/ADP-Vanadate trapped state. It has been found that the major conformation changes affecting the protein motional characteristics occur in the ATP binding domains but not in the transmembrane domains. Additionally, the binding of several substrates to LmrA has been studied by fluorescence spectroscopy as well as by 19F and 31P solid-state NMR. The binding constants for several LmrA substrates have been obtained by fitting the concentration dependant tryptophan intrinsic fluorescence quenching curves. Based on the fluorescence studies and solid-state NMR data, the conformation changes in LmrA under substrate binding have been discussed. In addition, the preferable location of nine LmrA and P-glycoprotein substrates within the model membrane has been studied via 1H-MAS-NOESY-NMR. The results have been interpreted with respect to LmrA and P-glycoprotein binding site accessibility from the membrane interface region.Die Entstehung von Resistenzen gegenüber mehrerer Wirkstoffe ist ein großes Problem bei der Behandlung einiger ansteckender Krankheiten und Krebs. Multiwirkstoff Resistenzen (MDR) basieren auf einem synergetischen Zusammenspiel mehrerer Mechanismen wie der Inaktivierung oder Veränderung des Angriffziels, Verhinderung des Wirkstoffeinstroms ebenso wie der aktive Transport von Wirkstoffen aus der Zelle heraus. Der letzere wird durch die Überexpression von Multidrug Effluxpumpe vermittelt. Der erst entdeckte und am besten charakterisierte ist der menschliche MDR Transporter P-Glycoprotein. Er gehört zur Superfamilie der ATP Bindekassetten (ABC) und wirkt als aktiver Transporter für mehrere Anti-Krebs Stoffe. Die Energie wird durch ATP Hydrolyse gewonnen. Die ähnlichste Struktur und funktionelles Homolog von P-Glykoprotein, das in Bakterien gefunden wurde, ist LmrA von Lactococcus lactis. Das Hauptziel dieser Arbeit ist es, LmrA in Lactococcus lactis aminosäureselektiv mit Isotopen zu markieren, die Probenpräperation für Festkörper NMR zu optimieren und letztendlich erste Festkörper NMR Untersuchungen an LmrA durchzuführen, um den katalytischen Zyklus und die Substrat-Bindung aufzuklären. Seit längerer Zeit waren die Anwendungen der Festkörper NMR im Bereich der Biologie meist auf die Untersuchung kleiner Moleküle beschränkt. Kürzlich haben die Methoden der Festkörper NMR Potential für strukturelle und nicht-störende ortsspezifische funktionale Studien großer Membranproteine sowie ihrer Liganden gezeigt. Es gibt bis jetzt jedoch nach unserem Wissen weder amonosäureselektive Isotopen-Markierung von ABC Transportern, noch NMR Untersuchungen an einem gesamten ABC Transporter. Festkörper NMR Experimente an einem Membranprotein erfordern die Rekonstitution mit einer hohen Konzentration und entweder Anreicherung von Isotopen des Proteins oder gebundener Wirkstoffe oder Inhibitoren. Deshalb wurden große Mengen von LmrA mit einer hohen Konzentration in Lipidmembranen für Festkörper-NMR rekonstituiert. Im nächsten Schritt wurde ein Verfahren für effiziente selektive Markierung von Aminosäuren von LmrA in Lactococcus lactis etabliert. Unter Verwendung dieses Protokolles wurde LmrA, das mit deuteriertem Alanin markiert war und in E. coli Liposomen rekonstituiert war, hergestellt. Deuterium NMR wurde in der Vergangenheit umfangreich genutzt um die Dynamik der Proteine zu untersuchen. Sie wurde jedoch niemals auf ABC Transporter angewendet. Hier werden 2H NMR experimente an Alanin-Isotopen markiertem LmrA vorgestellt, um die Veränderungen der Dynamik in dem Protein bei Bindung von AMP-PNP, einem nicht hydrolysierbares ATP Analogon, und in einem im ATP/ ADP-Vandat eingefangenen Zustand zu zeigen. Es stellte sich heraus, dass die wichtigsten Konformationsänderungen, die die Bewegung des Proteins beeinflussen, in der ATP-Binde-Domäne auftreten, nicht aber in den Transmembran-Domänen. Zusätzlich wurde die Bindung mehrerer Substrate an LmrA mit Fluoreszenz Spektroskopie untersucht als auch mit 19F und 31P Festkörper NMR. Die Bindungs-Konstanten für mehrere LmrA Substrate wurden mittels Tryptophan Fluoreszenz–Quenching erhalten. Bezogen auf die Fluoreszenz-Untersuchungen und Festkörper NMR Daten wurden die Konformations-Änderungen von LmrA bei der Substrat Bindung diskutiert. Zusatzlich wurde die bevorzugte Lokalisierung von neun Substraten von LmrA und P-Glykoprotein innerhalb der Modell-Membran durch 1H-MAS-NOESY-NMR untersucht. Die Ergebnisse wurden in Bezug auf die Zugänglichkeit der Bindungs-Stelle in LmrA und P-Glykoprotein von der Membranzwischenschicht interpretiert

Influence of the Accepted Concrete Deformation Diagrams on the Results of Calculating Bending Reinforced Concrete Elements

The article describes the influence of various types of concrete deformation diagrams on the accuracy of the deformational analysis of a bending concrete element. By analyzing different forms of diagrams, we get results with the help of the software "Beta 4.2 (5.0)". After getting the results, conclusions are drawn, according to particular qualities concerning the investigated element

Concerning the Question of Calculating a Beam with a Hinge-Rod Chain

We consider the calculation of a statically indeterminate combined system consisting of a beam and a hinge-rod chain of a general form for the action of an arbitrary vertical load. The qualitative regularities of the internal forces in the rods of the hinge-rod chain are established. Depending on the scheme of interaction of the chain with the beam, the calculation of combined systems with and without a horizontal reaction support is considered. Finite formulas are obtained for determining the internal forces in such systems

An Allosteric Inhibitor of Protein Arginine Methyltransferase 3

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets

The reconstituted Escherichia coli MsbA protein displays lipid flippase activity

The MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria. This 65 kDa membrane protein is thought to function as a homodimeric ATP-dependent lipid translocase or flippase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. We have previously shown that purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Bacterial membrane vesicles isolated from E. coli overexpressing His6-tagged MsbA displayed ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Purified MsbA was reconstituted into proteoliposomes of E. coli lipid and its ability to translocate NBD-labelled lipid derivatives was characterized. In this system, the protein displayed maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for an acyl chain-labelled PE (phosphatidylethanolamine) derivative. The protein showed the highest rates of flippase activity when reconstituted into an E. coli lipid mixture. Substantial flippase activity was also observed for a variety of other NBD-labelled phospholipids and glycolipids, including molecules labelled on either the headgroup or the acyl chain. Lipid flippase activity required ATP hydrolysis, and was dependent on the concentration of ATP and NBD–lipid. Translocation of NBD–PE was inhibited by the presence of the putative physiological substrate lipid A. The present paper represents the first report of a direct measurement of the lipid flippase activity of purified MsbA in a reconstituted system

Characterization of the Interactions between Fluoroquinolone Antibiotics and Lipids: a Multitechnique Approach

Probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research and membrane biophysics. Previous studies showed differences in accumulation and intracellular activity between two fluoroquinolones, ciprofloxacin and moxifloxacin, that may actually result from their differential susceptibility to efflux by the ciprofloxacin transporter. In view of the critical role of lipids for the drug cellular uptake and differences observed for the two closely related fluoroquinolones, we investigated the interactions of these two antibiotics with lipids, using an array of complementary techniques. Moxifloxacin induced, to a greater extent than ciprofloxacin, an erosion of the DPPC domains in the DOPC fluid phase (atomic force microscopy) and a shift of the surface pressure-area isotherms of DOPC/DPPC/fluoroquinolone monolayer toward lower area per molecule (Langmuir studies). These effects are related to a lower propensity of moxifloxacin to be released from lipid to aqueous phase (determined by phase transfer studies and conformational analysis) and a marked decrease of all-trans conformation of acyl-lipid chains of DPPC (determined by ATR-FTIR) without increase of lipid disorder and change in the tilt between the normal and the germanium surface (also determined by ATR-FTIR). All together, differences of ciprofloxacin as compared to moxifloxacin in their interactions with lipids could explain differences in their cellular accumulation and susceptibility to efflux transporters

The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites

MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA–MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA–MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35–10 μM. Lipid A and daunorubicin were able to bind to MsbA–MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter

Discovery of a 2,4-Diamino-7-aminoalkoxyquinazoline as a Potent and Selective Inhibitor of Histone Lysine Methyltransferase G9a

SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the G9a-8 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. The co-crystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling