Encoding combinatorial libraries: A novel application of fluorescent silica colloids

A major challenge associated with using large chemical libraries synthesized on microscopic solid support beads is the rapid discrimination of individual compounds in these libraries. This challenge can be overcome by encoding the beads with 1 mum silica colloidal particles (reporters) that contain specific and identifiable combinations of fluorescent byes. The colored bar code generated on support beads during combinatorial library synthesis can be easily, rapidly, and inexpensively decoded through the use of fluorescence microscopy. All reporters are precoated with polyelectrolytes [poly(acrylic acid), PAA, poly(sodium 4-styrenesulfonate PSSS, polyethylenimine, PEI, and/or poly(diallyldimethylammonium chloride), PDADMAC] with the aim of enhancing surface charge, promoting electrostatic attraction to the bead, and facilitating polymer bridging between the bead and reporter for permanent adhesion. As shown in this article, reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to quantity of attached reporters per bead (54 +/- 23 in 2500 mum(2) area for PEI/PAA coated and 11 +/- 6 for uncoated reporters) and minimization of cross-contamination (1 red reporter in 2500 mum(2) area of green-labeled bead for PEI/PAA coated and 26 +/- 15 red reporters on green-labeled beads for uncoated reporters after 10 days). Examination of various polyelectrolyte systems shows that the magnitude of the xi -potential of polyelectrolyte-coated reporters (-64 mV for PDADMAC/PSSS and -42 mV for PEI/PAA-coated reporters) has no correlation with the number of reporters that adhere to the solid support beads (21 +/- 16 in 2500 mum(2) area for PDADMAC/PSSS and 54 +/- 23 for PEI/PAA-coated reporters). The contribution of polymer bridging to the adhesion has a far greater influence than electrostatic attraction and is demonstrated by modification of the polyelectrolyte multilayers using gamma irradiation of precoated reporters either in aqueous solution or in polyelectrolyte solution.

Modelling cement grinding circuits

Modelling and simulation studies were carried out at 26 cement clinker grinding circuits including tube mills, air separators and high pressure grinding rolls in 8 plants. The results reported earlier have shown that tube mills can be modelled as several mills in series, and the internal partition in tube mills can be modelled as a screen which must retain coarse particles in the first compartment but not impede the flow of drying air. In this work the modelling has been extended to show that the Tromp curve which describes separator (classifier) performance can be modelled in terms of d(50)(corr), by-pass, the fish hook, and the sharpness of the curve. Also the high pressure grinding rolls model developed at the Julius Kruttschnitt Mineral Research Centre gives satisfactory predictions using a breakage function derived from impact and compressed bed tests. Simulation studies of a full plant incorporating a tube mill, HPGR and separators showed that the models could successfully predict the performance of the another mill working under different conditions. The simulation capability can therefore be used for process optimization and design. (C) 2001 Elsevier Science Ltd. All rights reserved.

Multi-fluorescent silica colloids for encoding large combinatorial libraries

Large chemical libraries can be synthesized on solid-support beads by the combinatorial split-and-mix method. A major challenge associated with this type of library synthesis is distinguishing between the beads and their attached compounds. A new method of encoding these solid-support beads, 'colloidal bar-coding', involves attaching fluorescent silica colloids ('reporters') to the beads as they pass through the compound synthesis, thereby creating a fluorescent bar code on each bead. In order to obtain sufficient reporter varieties to bar code extremely large libraries, many of the reporters must contain multiple fluorescent dyes. We describe here the synthesis and spectroscopic analysis of various mono- and multi-fluorescent silica particles for this purpose. It was found that by increasing the amount of a single dye introduced into the particle reaction mixture, mono- fluorescent silica particles of increasing intensities could be prepared. This increase was highly reproducible and was observed for six different fluorescent dyes. Multi-fluorescent silica particles containing up to six fluorescent dyes were also prepared. The resultant emission intensity of each dye in the multi-fluorescent particles was found to be dependent upon a number of factors; the hydrolysis rate of each silane-dye conjugate, the magnitude of the inherent emission intensity of each dye within the silica matrix, and energy transfer effects between dyes. We show that by varying the relative concentration of each silane-dye conjugate in the synthesis of multi-fluorescent particles, it is possible to change and optimize the resultant emission intensity of each dye to enable viewing in a fluorescence detection instrument.

Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca2+-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (∼1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability. © 2002 Elsevier Science B.V. All rights reserved.

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.

Countering cooperative effects in protease inhibitors using constrained b-strand-mimicking templates in focused combinatorial libraries

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

A probabilistic switch model of information flow in social networks: Application for virtual circuits to organizational design

Network building and exchange of information by people within networks is crucial to the innovation process. Contrary to older models, in social networks the flow of information is noncontinuous and nonlinear. There are critical barriers to information flow that operate in a problematic manner. New models and new analytic tools are needed for these systems. This paper introduces the concept of virtual circuits and draws on recent concepts of network modelling and design to introduce a probabilistic switch theory that can be described using matrices. It can be used to model multistep information flow between people within organisational networks, to provide formal definitions of efficient and balanced networks and to describe distortion of information as it passes along human communication channels. The concept of multi-dimensional information space arises naturally from the use of matrices. The theory and the use of serial diagonal matrices have applications to organisational design and to the modelling of other systems. It is hypothesised that opinion leaders or creative individuals are more likely to emerge at information-rich nodes in networks. A mathematical definition of such nodes is developed and it does not invariably correspond with centrality as defined by early work on networks.

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The struclural features that contribute to their remarkable stability are described ill this review. (c) 2006 Wiley Periodicals, Inc.

Double-heater-wire circuits and heat-and-moisture exchangers and the risk of ventilator-associated pneumonia

Objective: To compare the incidence of ventilator-associated pneumonia (VAP) in patients ventilated in intensive care by means of circuits humidified with a hygroscopic heat-and-moisture exchanger with a bacterial viral filter (HME) or hot-water humidification with a heater wire in both inspiratory and expiratory circuit limbs (DHW) or the inspiratory limb only (SHW). Design: A prospective, randomized trial. Setting: A metropolitan teaching hospital's general intensive care unit. Patients: Three hundred eighty-one patients requiring a minimum period of mechanical ventilation of 48 hrs. Interventions: Patients were randomized to humidification with use of an HME (n = 190), SHW (n = 94), or DHW (n = 97). Measurements and Main Results. Study end points were VAP diagnosed on the basis of Clinical Pulmonary Infection Score (CPIS) (1), HME resistance after 24 hrs of use, endotracheal tube resistance, and HME use per patient. VAP occurred with similar frequency in all groups (13%, HME; 14%, DHW; 10%, SHW; p = 0.61) and was predicted only by current smoking (adjusted odds ratio [AOR], 2.1; 95% confidence interval [CI], 1.1-3.9; p =.03) and ventilation days (AOR, 1.05; 95% Cl, 1.0-1.2; p =.001); VAP was less likely for patients with an admission diagnosis of pneumonia (AOR, 0.40; 95% Cl, 0.4-0.2; p =.04). HME resistance after 24 hrs of use measured at a gas flow of 50 L/min was 0.9 cm H2O (0.4-2.9). Endotracheal tube resistance was similar for all three groups (16-19 cm H2O min/L; p =.2), as were suction frequency, secretion thickness, and blood on suctioning (p =.32, p =.06, and p =.34, respectively). The HME use per patient per day was 1.13. Conclusions: Humidification technique does not influence either VAP incidence or secretion characteristics, but HMEs may have air-flow resistance higher than manufacturer specifications after 24 hrs of use.