A versatile pathway for the direct assembly of organo-functional mesostructures from sodium silicate

Silica mesostructures with well-expressed hexagonal and wormhole framework structures and up to 50% organo-functionalization of the framework silicon sites have been directly assembled from sodium silicate.     

A pairwise chemical genetic screen identifies new inhibitors of glucose transport

Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.     

Facile synthesis, assembly, and immobilization of ordered arrays of monodisperse magnetic nanoparticles on silicon substrates

This paper outlines the preparation of monodisperse MnFe(2)O(4) nanoparticles modified with omega-alkenyl moieties in a one-pot reaction, requiring no ligand-exchange step, followed by deposition of the resulting surfactant-coated nanoparticles onto a hydrogen-terminated silicon (111) wafer and covalent anchoring to the surface via UV-initiated bonding, creating a stable two-dimensional array of monodisperse magnetic nanoparticles.     

A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function

Riboswitches are RNA-based genetic control elements that regulate gene expression in a ligand-dependent fashion without the need for proteins. The ability to create synthetic riboswitches that control gene expression in response to any desired small-molecule ligand will enable the development of sensitive genetic screens that can detect the presence of small molecules, as well as designer genetic control elements to conditionally modulate cellular behavior. Herein, we present an automated high-throughput screening method that identifies synthetic riboswitches that display extremely low background levels of gene expression in the absence of the desired ligand and robust increases in expression in its presence. Mechanistic studies reveal how these riboswitches function and suggest design principles for creating new synthetic riboswitches. We anticipate that the screening method and design principles will be generally useful for creating functional synthetic riboswitches.     

A comparison of a direct search method and a genetic algorithm for conformational searching

We present results from the application of two conformational searching methods: genetic algorithms (GA) and direct search methods for finding low energy conformations of organic molecules. GAs are in a class of biologically motivated optimization methods that evolve a population of individuals in which individuals who are more “fit” have a higher probability of surviving into subsequent generations. The parallel direct search method (PDS) is a type of pattern search method that uses an adaptive grid to search for minima. Both methods found energies equal to or lower than the energy of the relaxed crystal structure in all cases, at a relatively small cost in CPU time. We suggest that either method would be a good candidate to find 3-D conformations in a large scale screening application. © 1996 by John Wiley & Sons, Inc.     

A solid phase linker strategy for the direct synthesis of EDANS-labelled peptide substrates

A novel linker strategy for the efficient synthesis of peptides C-terminally labelled with the EDANS fluorophore is described. Using this support, FRET peptide substrates bearing EDANS/Dabcyl fluorescent donor/acceptor groups can be readily prepared using standard Fmoc solid phase methods.     

A flexible data analysis tool for chemical genetic screens

High-throughput assays generate immense quantities of data that require sophisticated data analysis tools. We have created a freely available software tool, SLIMS (Small Laboratory Information Management System), for chemical genetics which facilitates the collection and analysis of large-scale chemical screening data. Compound structures, physical locations, and raw data can be loaded into SLIMS. Raw data from high-throughput assays are normalized using flexible analysis protocols, and systematic spatial errors are automatically identified and corrected. Various computational analyses are performed on tested compounds, and dilution-series data are processed using standard or user-defined algorithms. Finally, published literature associated with active compounds is automatically retrieved from Medline and processed to yield potential mechanisms of actions. SLIMS provides a framework for analyzing high-throughput assay data both as a laboratory information management system and as a platform for experimental analysis.     

A direct route toward assembly of nanoparticle-carbon nanotube composite materials

The preparation of nanocomposite materials from carbon nanotubes (CNTs) and metal or metal oxide nanoparticles has important implications to the development of advanced catalytic and sensory materials. This paper reports findings of an investigation of the preparation of nanoparticle-coated carbon nanotube composite materials. Our approach involves molecularly mediated assembly of monolayer-capped nanoparticles on multiwalled CNTs via a combination of hydrophobic and hydrogen-bonding interactions between the capping/mediating shell and the CNT surface. The advantage of this route is that it does not require tedious surface modification of CNTs. We have demonstrated its simplicity and effectiveness for assembling alkanethiolate-capped gold nanoparticles of 2-5 nm core sizes onto CNTs with controllable coverage and spatially isolated character. The loading and distribution of the nanoparticles on CNTs depend on the relative concentrations of gold nanoparticles, CNTs, and mediating or linking agents. The composite nanomaterials can be dispersed in organic solvent, and the capping/linking shells can be removed by thermal treatment to produce controllable nanocrystals on the CNT surfaces. The nanocomposite materials are characterized using transmission electron microscopy and Fourier transform infrared spectroscopy techniques. The results will be discussed in terms of developing advanced catalytic and sensory nanomaterials.     

A substituent effects study reveals the kinetic pathway for an interfacial reaction

This paper describes the use of a substituent effects study to understand the mechanistic basis for an interfacial Diels-Alder reaction that does not proceed with standard second-order kinetics. Cyclopentadiene (Cp) undergoes a Diels-Alder reaction with a chemisorbed mercaptobenzoquinone to yield an immobilized Diels-Alder adduct. The pseudo-first-order rate constants are not linearly related to the concentration of diene, but they reach a limiting value with increasing concentrations of diene. The results of a substituent effects study support a mechanism wherein the electrochemical oxidation of hydroquinone produces two states of quinone. The first form, Q*, either reacts with Cp or isomerizes to Q, a form that is significantly less reactive with the diene. The interfacial reaction reaches a maximum rate when the concentration of diene is sufficiently high so that Q* undergoes complete Diels-Alder reaction and does not isomerize to Q. This work provides an example of the use of physical organic chemistry to understand an interfacial reaction.     

Nanoparticle-driven assembly of block copolymers: a simple route to ordered hybrid materials

The addition of nanoparticles that selectively hydrogen bond with one of the segments of a block copolymer is shown to induce order in otherwise disordered systems. This enables the fabrication of well-ordered hybrid materials with spherical, cylindrical, or lamellar domains at particle loadings of more than 40%, as evidenced by TEM and SAXS. The approach described is simple and applicable to a wide range of nanoparticles and block copolymers, and it lays the groundwork for the design of cooperatively assembled functional devices.     

Determination of tannic acid by direct chemiluminescence in a FIA assembly

The determination of tannic acid is performed in a FIA assembly on the basis of the analytical output obtained by oxidation of the acid. The analyte solution was daily prepared in a mixture of quinine as sensitiser and perchloric acid and it was injected into a pure water stream acting as a carrier. This solution merges with the mixture potassium permanganate in perchloric medium and the resulting chemiluminescence is monitored. The method was applied over the range 0.5–20 mg l⁻¹ of tannic acid with a LOD 100 μg l⁻¹. The reproducibility was 2.1% and the sample throughput 54 h⁻¹. The influence of foreign substances was studied and the new method is applied to the determination of tannic acid in pharmaceutical and galenic formulations in human urine and surface waters.     

First direct assembly of molecular helical complexes into a coordination polymer

Luminescent triple-stranded helicates, formed between Tb(iii) ions and bis-acylpyrazolones, were directly assembled into a 1-D polymeric system.     

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.     

Designed fabrication of ordered porous au/ag nanostructured films for surface-enhanced Raman scattering substrates

This article reports the designed preparation of two different kinds of novel porous metal nanostructured films, namely, an ordered macroporous Au/Ag nanostructured film and an ordered hollow Au/Ag nanostructured film. Different from previous reports, the presently proposed method can be conveniently used to control film structures by simply varying the experimental conditions. The morphology of these films has been characterized by scanning electron microscopy (SEM), and their performance as surface-enhanced Raman scattering (SERS) substrates has been evaluated by using rhodamine 6G (R6G) as a probe molecule. We show that such porous nanostructured films consisting of larger interconnected aggregates are highly desirable as SERS substrates in terms of high Raman intensity enhancement, excellent stability, and reproducibility. The interconnected nanostructured aggregate, long-range ordering porosity, and nanoscale roughness are important factors responsible for this large SERS enhancement ability.     

A membrane electrode assembly with high fuel coulombic efficiency for passive direct borohydride fuel cells

Here, we report the development of a new membrane electrode assembly (MEA) structure for passive direct borohydride fuel cells (DBFCs). The anode of this type of MEA includes upper and lower parts for the electro-oxidation of borohydride and hydrogen, respectively. In comparison to conventional MEAs, the maximum power of this MEA is increased by 28.1%, and the anode polarization is decreased due to the current contribution of hydrogen electro-oxidation. The hydrogen generated from borohydride hydrolysis can be oxidized inside the cell, and the fuel coulombic efficiency reaches 100%. Therefore, high fuel utilization and cell safety can be obtained by employing this novel MEA in DBFCs.     

Single particle tracking reveals corralling of a transmembrane protein in a double-cushioned lipid bilayer assembly

A predominate question associated with supported bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT(3A), has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT(3A) receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bilayer assemblies include a simple POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported planar bilayer, a “single-cushion” POPC bilayer with a PEG (poly(ethylene glycol)) layer between membrane and support, and a “double-cushion” POPC bilayer with both a PEG layer and a layer of BSA (bovine serum albumin). Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble fluorescence recovery after photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT(3A) molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains ( 450 nm). Comparisons with the simple POPC membrane and the single-cushion POPC?PEG membrane reveal that BSA both serves to minimize interactions with the solid support and creates the corrals that reduce the long-range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion assemblies proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.     

Programmable assembly of a metabolic pathway enzyme in a pre-packaged reusable bioMEMS device

We report a biofunctionalization strategy for the assembly of catalytically active enzymes within a completely packaged bioMEMS device, through the programmed generation of electrical signals at spatially and temporally defined sites. The enzyme of a bacterial metabolic pathway, S-adenosylhomocysteine nucleosidase (Pfs), is genetically fused with a pentatyrosine "pro-tag" at its C-terminus. Signal responsive assembly is based on covalent conjugation of Pfs to the aminopolysaccharide, chitosan, upon biochemical activation of the pro-tag, followed by electrodeposition of the enzyme-chitosan conjugate onto readily addressable sites in microfluidic channels. Compared to traditional physical entrapment and surface immobilization approaches in microfluidic environments, our signal-guided electrochemical assembly is unique in that the enzymes are assembled under mild aqueous conditions with spatial and temporal programmability and orientational control. Significantly, the chitosan-mediated enzyme assembly can be reversed, making the bioMEMS reusable for repeated assembly and catalytic activity. Additionally, the assembled enzymes retain catalytic activity over multiple days, demonstrating enhanced enzyme stability. We envision that this assembly strategy can be applied to rebuild metabolic pathways in microfluidic environments for antimicrobial drug discovery.