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High-mobility group box 1 (HMGB1) is a multifunctional protein. Upon injury or infection, HMGB1 is passively released from necrotic and activated dendritic cells and macrophages, where it functions as a cytokine, acting as a ligand for RAGE, a major receptor of innate immunity stimulating inflammation responses including the pathogenesis of cerebral ischemia/reperfusion (I/R) injury. Blocking the HMGB1/RAGE axis offers a therapeutic approach to treating these inflammatory conditions. Here, we describe a synthetic antibody (SA), a copolymer nanoparticle (NP) that binds HMGB1. A lightly cross-linked N-isopropylacrylamide (NIPAm) hydrogel copolymer with nanomolar affinity for HMGB1 was selected from a small library containing trisulfated 3,4,6S-GlcNAc and hydrophobic N-tert-butylacrylamide (TBAm) monomers. Competition binding experiments with heparin established that the dominant interaction between SA and HMGB1 occurs at the heparin-binding domain. In vitro studies established that anti-HMGB1-SA inhibits HMGB1-dependent ICAM-1 expression and ERK phosphorylation of HUVECs, confirming that SA binding to HMGB1 inhibits the proteins' interaction with the RAGE receptor. Using temporary middle cerebral artery occlusion (t-MCAO) model rats, anti-HMGB1-SA was found to accumulate in the ischemic brain by crossing the blood-brain barrier. Significantly, administration of anti-HMGB1-SA to t-MCAO rats dramatically reduced brain damage caused by cerebral ischemia/reperfusion. These results establish that a statistical copolymer, selected from a small library of candidates synthesized using an "informed" selection of functional monomers, can yield a functional synthetic antibody. The knowledge gained from these experiments can facilitate the discovery, design, and development of a new category of drug.

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

The bilayer bending modulus (Kc) is one of the most important physical constants characterizing lipid membranes, but precisely measuring it is a challenge, both experimentally and computationally. Experimental measurements on chemically identical bilayers often differ depending upon the techniques employed, and robust simulation results have previously been limited to coarse-grained models (at varying levels of resolution). This Communication demonstrates the extraction of Kc from fully atomistic molecular dynamics simulations for three different single-component lipid bilayers (DPPC, DOPC, and DOPE). The results agree quantitatively with experiments that measure thermal shape fluctuations in giant unilamellar vesicles. Lipid tilt, twist, and compression moduli are also reported.

Human islet amyloid polypeptide (hIAPP or Amylin) is a 37 residue hormone that is cosecreted with insulin from the pancreatic islets. The aggregation of hIAPP plays a role in the progression of type 2 diabetes and contributes to the failure of islet cell grafts. Despite considerable effort, little is known about the mode of action of IAPP amyloid inhibitors, and this has limited rational drug design. Insulin is one of the most potent inhibitors of hIAPP fibril formation, but its inhibition mechanism is not understood. In this study, the aggregation of mixtures of hIAPP with insulin, as well as with the separate A and B chains of insulin, were characterized using ion mobility spectrometry-based mass spectrometry and atomic force microscopy. Insulin and the insulin B chain target the hIAPP monomer in its compact isoform and shift the equilibrium away from its extended isoform, an aggregation-prone conformation, and thus inhibit hIAPP from forming β-sheets and subsequently amyloid fibrils. All-atom molecular modeling supports these conclusions.

We describe a novel epitope discovery strategy for creating an affinity agent/peptide tag pair. A synthetic polymer nanoparticle (NP) was used as the "bait" to catch an affinity peptide tag. Biotinylated peptide tag candidates of varied sequence and length were attached to an avidin platform and screened for affinity against the polymer NP. NP affinity for the avidin/peptide tag complexes was used to provide insight into factors that contribute NP/tag binding. The identified epitope sequence with an optimized length (tMel-tag) was fused to two recombinant proteins. The tagged proteins exhibited higher NP affinity than proteins without tags. The results establish that a fusion peptide tag consisting of optimized 15 amino acid residues can provide strong affinity to an abiotic polymer NP. The affinity and selectivity of NP/tMel-tag interactions were exploited for protein purification in conjunction with immobilized metal ion/His6-tag interactions to prepare highly purified recombinant proteins. This strategy makes available inexpensive, abiotic synthetic polymers as affinity agents for peptide tags and provides alternatives for important applications where more costly affinity agents are used.

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40. Here, we apply mass spectrometry and ion mobility to the study of Abeta42 and its Pro19 alloform. The Phe19 --> Pro19 substitution blocks fibril formation by [Pro19]Abeta42. Evidence indicates that solution-like structures of Abeta monomers are electrosprayed and characterized. Unfiltered solutions of Abeta42 produce only monomers and large oligomers, whereas [Pro19]Abeta42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers. When passed through a 10,000 amu filter and immediately sampled, Abeta42 solutions produce monomers, dimers, tetramers, hexamers, and an aggregate of two hexamers that may be the first step in protofibril formation. These results are consistent with recently published photochemical cross-linking data and lend support to recent aggregation mechanisms proposed by Bitan, Teplow, and co-workers [J. Biol. Chem. 2003, 278, 34882-34889].

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40. Here, we apply mass spectrometry and ion mobility to the study of Abeta42 and its Pro19 alloform. The Phe19 --> Pro19 substitution blocks fibril formation by [Pro19]Abeta42. Evidence indicates that solution-like structures of Abeta monomers are electrosprayed and characterized. Unfiltered solutions of Abeta42 produce only monomers and large oligomers, whereas [Pro19]Abeta42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers. When passed through a 10,000 amu filter and immediately sampled, Abeta42 solutions produce monomers, dimers, tetramers, hexamers, and an aggregate of two hexamers that may be the first step in protofibril formation. These results are consistent with recently published photochemical cross-linking data and lend support to recent aggregation mechanisms proposed by Bitan, Teplow, and co-workers [J. Biol. Chem. 2003, 278, 34882-34889].

Various NMR spectroscopic studies are conducted to demonstrate the role of C-boron enolates on the reaction of borane (B[H,sub.3]) and ethyl diazoacetate (EDA), which gives rise to several dimers, trimer and oligomers of EDA. The analysis reveals that the C-boron enolate species are much more stable than its isomeric O-enolates that normally leads to the formation of a doubly generated enolate as the side product of the reaction.

Research reveals a new, living polymerization of dimethylsulfoxonium methylide and describes the chemistry of the reaction. Results indicate that triorganoboranes mediate the polymerization reaction with one mole of dimethyl sulfoxide produced for each mole of ylide consumed. The product of the reaction is a 3-arm star polymethylene borane.

A range of imprinted polymers were synthesized using structurally related chiral benzodiazepines as templates.

a study was conducted on tetraanionic molecular squares, macrocycles that contain for pentacoordinate siliconate groups, to investigate their molecular structure and solution conformation. An ion exchange synthesis resulted in an ion pair consisting of tetraanionic siliconate macrocycle and cyclobis(paraquat-p-phenylene) tetracation. An analysis of the novel tetraanion-tetracation ion pair reveals that the cyclic bisparaquat selectively intercalates around the phenyl ring of the tetraanion's C2 face, which is then engulfed by the interior of the tetraanion.

Imprinted polymers formed at low temperatures produce optimal rebinding figures. Improved chromatographic retention is thought to be connected to a rise in high-fidelity binding sites at polymerization. The best rebinding figures were achieved when the 9-EA imprinted polymer was solvated in a mobile phase like the porogen of polymerization. Binding sites on the polymer change to accommodate the solvation of the porogen. The substrate also affects the binding affinity and specificity of the polymer.

An internal doping method facilitates the growth of nano-sized chromium clusters in a porous amorphous hybrid xerogel. Sol-gel processing of 1,4-bis(triethoxysilyl)benzene and chromium tricarbonyl(triethoxysilyl) benzene help prepare of xerogel, a polysilsesquioxane. Transmission electron microscopic images help determine the size of the metal particles, and energy-dispersive X-ray diffraction and electron diffraction experiments help estimate the elemental composition and examine the morphology of metal clusters. A combination of internal and external doping methodologies help prepare mixtures of nanophases with CdS and chromium metal.

Bridgehead olefin/bridgehead lactams were synthesized using the type 2 intramolecular imino Diels-Alder reaction. X-ray crystallographic studies were performed on the bridgehead olefin/bridgehead lactams to analyze their crystal structures. Results reveal the presence of torsional distortions in the carbon-carbon double bond to the quasi double bond of the structures' amide linkage.

Hydrolysis and condensation of a new series of molecular building blocks composed of bis(triethoxysilyl)aryl and -ethynyl monomers led to the production of aryl- and ethynyl-bridged polysilsesquioxanes adopting xerogel forms. Structural characterization of these hybrid organic and inorganic network materials proceeded with the application of gas sorption porosimetry, atomic force microscopy and nuclear magnetic resonance spectroscopy. Aryl bridges exhibited high thermal and structural stability while ethynyl bridges collapsed toward Si-C cleavage under sol-gel reactions.

The absolute reaction rates of picryl azide addition in CHCl3 with cyclic olefins are measured via molecular mechanics calculations and their products identified to determine the relationship between chemical reactivity and strain. The conformational analysis of their zwitterionic intermediates allows for the establishment of the mechanism of formation. Results indicate that the strain involved in an olefin alters both reactivity and stereochemistry.

The kinetic consequences of torsional distortions in carbon-carbon double bonds are evaluated. The rates of epoxidation of various isosteric series of double bonds are measured. Results reveal that strained bridgehead alkenes and trans-cycloalkenes has a faster reaction rates than nonstrained cis-cycloalkenes. Analysis of kinetic data showed that the steric energy reflects strain energy relief in the transition state.

The chair and boat transition states of the Cope rearrangement are investigated by the introduction of substituents at positions 2 and 5. Both d,l- and meso-2,2'-bis(1-methylene-1,2,3,4-tetrahydronaphthalenes) undergo a (3.3) sigmatropic rearrangement, with the former constrained to the chair conformation and the latter to the boat. A quantification of the effect of the 2,5-diphenyl substituents on the energy differential between the boat and chair conformations is reported. Moreover, results show that the enthalpy of activation of both reactions is decreased by similar amounts.

Multifunctional polymer nanoparticles approximately the size of a large protein is purified by using an affinity chromatography strategy. The purified particles have shown much stronger affinity and a narrower affinity distribution than the average of particles before purification at room temperature but can release the peptide by changing the temperature.

A functional monomer optimization strategy was combined with molecular-imprinting nanoparticle synthesis to prepare protein-size organic polymer nanoparticles (NPs) with a binding affinity and selectivity comparable to those of natural antibodies. The plastic antibodies with biocompatibility and nontoxic characteristics offer a potential use for neutralizing a wide range of biomacromolecules in vivo.

Combination of ion mobility spectrometry-mass spectrometry (IMS-MS) and replica exchange molecular dynamics (REMD) are used to examine the monomeric and oligomeric structures of normal prion protein PrP106-126 and two nonaggregating forms of the peptide. The data obtained suggested that the oligomerization observed only for the normal peptide and not for the oxidized peptide resulted from the association of ordered [beta]-hairpin monomers rather than disordered monomers.

The structural features of the human islet amyloid polypeptide (IAPP) monomer are probed by using a combination of ion mobility mass spectrometry (IMS-MS) and all-atom replica exchange molecular dynamics (REMD) simulations. The studies have provided evidence of a significant population of [beta]-hairpin conformers for the IAPP peptide.

The synthesis, properties and the applications of the highly versatile rare earth tantalate pyrochlore nanophosphors are discussed. These materials are shown to be widely used in numerous energy and environment related applications, including the batteries and the fuel cells.

High affinity and selective polymer nanoparticles (NPs) are prepared for the biotoxin melittin (Mel) by an imprinting precipitation polymerization. The optimum NP formulation is selected by quartz crystal microbalance (QCM) evaluation of a small combinatorial library of functional monomers and the bonding affinity and size of the Mel imprinted polymer (MIP) NPs are similar to those of natural antibodies.

A synthetic molecular imprinted polymer (MIP) receptor for the recognition of WAY-100635, an antagonist of the serotonin (5-HT) receptor subtype 5-[HT.sub.1A] and its analogues is developed. A relationship is observed to exist between the 5-[HT.sub.1A] receptor and MIP affinities with both showing limited tolerance for variations at the amino pyridine position in a series of benzamido analogues of WAY-100635.

The first synthesis of bridged polysilsesquioxane (BP) spherical nanoparticles is described. The photocleavage of these bridged dimers have resulted in softening and deformation of nanoparticles by direct irradiation with an ultraviolet (UV) light.

A dual function catalyst that serves to generate the reactive Diels-Alder precursor as well as to induce asymmetry in the subsequent cycloaddition step is reported. It is concluded that low enantioselectivity for the intermolecular reaction is consistent with the proposal since the Diels-Alder step involves a bimolecular cycloaddition that would be competitive with the bimolecular reoxidation of Ru(II).

Mass spectrometry and ion mobility is applied to the study of A(beta)42 and its Pro(super 19) alloform. Unfiltered solutions of A(beta)42 produce only monomers and large oligomers, whereas [Pro(super 19)]A(beta)42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers.