A portable magneto-optical trap with prospects for atom interferometry in civil engineering.

The high precision and scalable technology offered by atom interferometry has the opportunity to profoundly affect gravity surveys, enabling the detection of features of either smaller size or greater depth. While such systems are already starting to enter into the commercial market, significant reductions are required in order to reach the size, weight and power of conventional devices. In this article, the potential for atom interferometry based gravimetry is assessed, suggesting that the key opportunity resides within the development of gravity gradiometry sensors to enable drastic improvements in measurement time. To push forward in realizing more compact systems, techniques have been pursued to realize a highly portable magneto-optical trap system, which represents the core package of an atom interferometry system. This can create clouds of 107 atoms within a system package of 20 l and 10 kg, consuming 80 W of power.This article is part of the themed issue 'Quantum technology for the 21st century'.

Quantifying the quantum gate fidelity of single-atom spin qubits in silicon by randomized benchmarking.

Building upon the demonstration of coherent control and single-shot readout of the electron and nuclear spins of individual (31)P atoms in silicon, we present here a systematic experimental estimate of quantum gate fidelities using randomized benchmarking of 1-qubit gates in the Clifford group. We apply this analysis to the electron and the ionized (31)P nucleus of a single P donor in isotopically purified (28)Si. We find average gate fidelities of 99.95% for the electron and 99.99% for the nuclear spin. These values are above certain error correction thresholds and demonstrate the potential of donor-based quantum computing in silicon. By studying the influence of the shape and power of the control pulses, we find evidence that the present limitation to the gate fidelity is mostly related to the external hardware and not the intrinsic behaviour of the qubit.

Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: structural analysis of the peptide complexes with SH2 domains.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide-SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide-SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide-protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide-SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region.

Deciphering common failures in molecular docking of ligand-protein complexes.

Common failures in predicting crystal structures of ligand-protein complexes are investigated for three ligand-protein systems by a combined thermodynamic and kinetic analysis of the binding energy landscapes. Misdocked predictions in ligand-protein docking are classified as 'soft' and 'hard' failures. While a soft failure arises when the search algorithm is unable to find the global energy minimum corresponding to the crystal structure, a hard failure results from a flaw of the energy function to qualify the crystal structure as the predicted lowest energy conformation in docking simulations. We find that neither the determination of a single structure with the lowest energy nor finding the most common binding mode is sufficient to predict crystal structures of the complexes, which belong to the category of hard failures. In a proposed hierarchical approach, structural similarity clustering of the conformations, generated from equilibrium simulations with the simplified energy function, is followed by energy refinement with the AMBER force field. This protocol, that involves a hierarchy of energy functions, resolves some common failures in ligand-protein docking and detects crystallographic binding modes that were not found during docking simulations.

Patient education and emergency room visits.

Patients visit emergency rooms for urgent and non-urgent care. Because emergency room visits are more costly than visits to primary care clinics and are less likely to involve preventive care, third party payers and institutions have always tried to shift patients away from the emergency room and towards primary care clinics where appropriate. Hypothesizes that an intervention based in an adult primary care clinic might enable this, especially if it involved patients who used both the clinic and the emergency room. Surveys patients to determine why they used the emergency room and to identify barriers to using the primary care clinic instead. Based on the survey results, an intervention was developed to facilitate use of the primary care clinic. Discusses the methodology used in the survey and analyses results. Concludes that it is difficult to change patient behaviour to fit the demands of the health care system. Possibly, it would be better to change the system to fit the behaviour patterns of the patients.

Use of an oral assessment tool to improve practice.

An oral assessment tool was developed alongside care plans in a neurosciences unit, including the rationale for interventions, to provide nurses with an evidence-based means of providing mouth care. Awareness and standards of mouth care improved as a result of introducing the tool and care plans.

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions.

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.

Glutathione peptidomimetic drug modulator of multidrug resistance-associated protein.

The peptidomimetic drug gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine diethyl ester (TER199) is an analog of glutathione designed to be an isozyme-specific inhibitor of GSTP1-1 protein1-1. This compound (and the de-esterified moiety) is shown to be an effective inhibitor of multidrug resistance-associated protein1 (MRP1)-mediated drug resistance. Kinetic analyses revealed that gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine reversibly inhibits the transport of 2,4-dinitrophenyl-S-glutathione with a K(i) of 752 microM. TER199 reversed the accumulation deficit of daunorubicin in MRP1-transfected NIH3T3 fibroblasts and maintained intracellular levels for >2 h after daunorubicin removal. Cytotoxicity assays revealed that TER199 significantly reversed the resistance of MRP1-transfected NIH3T3 cells for vincristine, doxorubicin, etoposide, and mitoxantrone. HL-60 cells made resistant to TER199 by chronic, long-term selection had increased mRNA and protein levels of multidrug resistance-associated protein, MRP1, and gamma-glutamyl cysteine synthetase heavy and light subunits (the rate-limiting enzyme in GSH synthesis). In spite of increased gamma-glutamyl cysteine synthetase, their glutathione content was reduced approximately 35% from that of parental HL-60 cells. These cells also exhibited a drug resistance profile commensurate with the previously described MRP1 overexpressing phenotype, with resistance to Vinca alkaloids, epipodophyllotoxins, and anthracyclines; additional cross-resistance to paclitaxel (Taxol), mitoxantrone, and 5-fluorouracil was observed.

Thermodynamics and kinetics of ligand-protein binding studied with the weighted histogram analysis method and simulated annealing.

The thermodynamics of ligand-protein molecular recognition is investigated by the energy landscape approach for two systems: methotrexate(MTX)--dihydrofolate reductase(DHFR) and biotin-streptavidin. The temperature-dependent binding free energy profile is determined using the weighted histogram analysis method. Two different force fields are employed in this study: a simplified model of ligand-protein interactions and the AMBER force field with a soft core smoothing component, used to soften the repulsive part of the potential. The results of multiple docking simulations are rationalized from the shape of the binding free energy profile that characterizes the thermodynamics of the binding process.

The timing of XIST replication: dominance of the domain.

Contiguous replicons are coordinately replicated and may be organized in temporal-spatial domains with early replication domains containing expressed genes and late ones carrying silent genes. XIST is silent on the active, early replicating X chromosome and expressed from the inactive, late replicating homolog. These circumstances potentially deviate from the aforementioned generalization and make studies of replication timing for XIST of special interest. Although earlier investigations of XIST replication in fibroblasts based on analysis of extracted DNA from cells at different stages of the cell cycle suggested that the silent gene does replicate before the expressed allele, studies using FISH technology produced the opposite results. Because the FISH replication studies could not directly distinguish between the active and inactive X chromosomes in the same cell, we undertook a re-investigation of this question utilizing FISH analysis under conditions that allowed us to make that distinction using cells sorted into different cell cycle stages by flow cytometry. The findings reported here indicate that the silent XIST gene on the active X chromosome does replicate before the expressed allele on the inactive X, supporting the view that the time of a gene's replication is determined by the large, multi-replicon domain in which it is located and not necessarily its expression state.

Expression and secretion of the Candida wickerhamii extracellular beta-glucosidase gene, bglB, in Saccharomyces cerevisiae.

The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.

Properties of an intracellular beta-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii.

An intracellular beta-glucosidase was isolated from the cellobiose-fermenting yeast, Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth, with the highest level of production in a medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94 KDa. It appeared to exist as a dimeric structure with a native molecular mass of about 180 KDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl beta-D-glucopyranoside (NpGlc) as a substrate. The optimal temperature for short-term (15-min) assays was 35 degrees C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28 degrees C and above. Using NpGlc as a substrate, the enzyme was estimated to have a Km of 0.28 mM and a Vmax of 525 mumol product min-1 mg protein-1. Similar to the extracellular beta-glucosidase produced by C. wickerhamii, this enzyme resisted end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. The activity of the enzyme was highest against aryl beta-1,4-glucosides. However, p-nitrophenyl xylopyranoside, lactose, cellobiose, and trehalose also served as substrates for the purified protein. Activity of the enzyme was stimulated by long-chain n-alkanols and inhibited by ethanol, 2-propanol, and 2-butanol. The amino acid sequence, obtained by Edman degradation analysis, suggests that this beta-glucosidase is related to the family-3 glycosyl hydrolases.

Exploring the energy landscapes of molecular recognition by a genetic algorithm: analysis of the requirements for robust docking of HIV-1 protease and FKBP-12 complexes.

Energy landscapes of molecular recognition are explored by performing "semi-rigid" docking of FK-506 and rapamycin with the Fukisawa binding protein (FKBP-12), and flexible docking simulations of the Ro-31-8959 and AG-1284 inhibitors with HIV-1 protease by a genetic algorithm. The requirements of a molecular recognition model to meet thermodynamic and kinetic criteria of ligand-protein docking simultaneously are investigated using a family of simple molecular recognition energy functions. The critical factor that determines the success rate in predicting the structure of ligand-protein complexes is found to be the roughness of the binding energy landscape, in accordance with a minimal frustration principle. The results suggest that further progress in structure prediction of ligand-protein complexes can be achieved by designing molecular recognition energy functions that generate binding landscapes with reduced frustration.

Production of beta-glucosidase and diauxic usage of sugar mixtures by Candida molischiana.

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).

Empirical free energy calculations of ligand-protein crystallographic complexes. I. Knowledge-based ligand-protein interaction potentials applied to the prediction of human immunodeficiency virus 1 protease binding affinity.

The steadily increasing number of high-resolution human immunodeficiency virus (HIV) 1 protease complexes has been the impetus for the elaboration of knowledge-based mean field ligand-protein interaction potentials. These potentials have been linked with the hydrophobicity and conformational entropy scales developed originally to explain protein folding and stability. Empirical free energy calculations of a diverse set of HIV-1 protease crystallographic complexes have enabled a detailed analysis of binding thermodynamics. The thermodynamic consequences of conformational changes that HIV-1 protease undergoes upon binding to all inhibitors, and a substantial concomitant loss of conformational entropy by the part of HIV-1 protease that forms the ligand-protein interface, have been examined. The quantitative breakdown of the entropy-driven changes occurring during ligand-protein association, such as the hydrophobic contribution, the conformational entropy term and the entropy loss due to a reduction of rotational and translational degrees of freedom, of a system composed to ligand, protein and crystallographic water molecules at the ligand-protein interface has been carried out. The proposed approach provides reasonable estimates of distinctions in binding affinity and gives an insight into the nature of enthalpyentropy compensation factors detected in the binding process.

Molecular recognition of the inhibitor AG-1343 by HIV-1 protease: conformationally flexible docking by evolutionary programming.

BACKGROUND: An important prerequisite for computational structure-based drug design is prediction of the structures of ligand-protein complexes that have not yet been experimentally determined by X-ray crystallography or NMR. For this task, docking of rigid ligands is inadequate because it assumes knowledge of the conformation of the bound ligand. Docking of flexible ligands would be desirable, but requires one to search an enormous conformational space. We set out to develop a strategy for flexible docking by combining a simple model of ligand-protein interactions for molecular recognition with an evolutionary programming search technique. RESULTS: We have developed an intermolecular energy function that incorporates steric and hydrogen-bonding terms. The parameters in this function were obtained by docking in three different protein systems. The effectiveness of this method was demonstrated by conformationally flexible docking of the inhibitor AG-1343, a potential new drug against AIDS, into HIV-1 protease. For this molecule, which has nine rotatable bonds, the crystal structure was reproduced within 1.5 A root-mean-square deviation 34 times in 100 simulations, each requiring eight minutes on a Silicon Graphics R4400 workstation. The energy function correctly evaluates the crystal structure as the global energy minimum. CONCLUSIONS: We believe that a solution of the docking problem may be achieved by matching a simple model of molecular recognition with an efficient search procedure. The necessary ingredients of a molecular recognition model include only steric and hydrogen-bond interaction terms. Although these terms are not necessarily sufficient to predict binding affinity, they describe ligand-protein interactions faithfully enough to enable a docking program to predict the structure of the bound ligand. This docking strategy thus provides an important tool for the interdisciplinary field of rational drug design.

De novo design of enzyme inhibitors by Monte Carlo ligand generation.

A new computational method for the in situ generation of small molecules within the binding site of a protein is described. The method has been evaluated using two well-studied systems, dihydrofolate reductase and thymidylate synthase. The method has also been used to guide improvements to inhibitors of HIV-1 protease. One such improvement resulted in a compound selected for preclinical studies as an antiviral agent against AIDS.

Cloning and characterization of a gene encoding a cell-bound, extracellular beta-glucosidase in the yeast Candida wickerhamii.

The ability of yeasts to ferment cellodextrins is rare. Candida wickerhamii is able to use these sugars for alcohol production because of a cell-bound, extracellular, beta-glucosidase that is unusual by not being inhibited by glucose. A cDNA expression library in lambda phage was prepared with mRNA isolated from cellobiose-grown C. wickerhamii. Immunological screening of the library with polyclonal antibodies against purified C. wickerhamii cell-bound, extracellular beta-glucosidase yielded 12 positive clones. Restriction endonuclease analysis and sequence data revealed that the clones could be divided into two groups, bglA and bglB, which were shown to be genetically distinct by Southern hybridization analyses. Efforts were directed at the study of bglB since it appeared to code for the cell-bound beta-glucosidase. Sequence data from both cDNA and genomic clones showed the absence of introns in bglB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of cell lysates from Escherichia coli bglB clones confirmed the presence of an expressed protein with an apparent molecular mass of 72 kDa, which is consistent with that expected for an unglycosylated form of the enzyme. Amino acid comparisons of BglB with other beta-glucosidase sequences suggest that it is a member of family 1 glycosyl hydrolases but is unusual in that it contains an additional 100 to 130 amino acids at the N terminus. This sequence did not have homologies to other known protein sequences and may impart unique properties to this beta-glucosidase.