Immunogenicity, Efficacy, and Safety of a Novel Synthetic Microparticle Pre-Erythrocytic Malaria Vaccine in Multiple Host Species.

We previously reported a protective antibody response in mice immunized with synthetic microparticle vaccines made using layer-by-layer fabrication (LbL-MP) and containing the conserved T1BT* epitopes from the circumsporozoite protein. To further optimize the vaccine candidate, a benchtop tangential flow filtration method (LbL-by-TFF) was developed and utilized to produce vaccine candidates that differed in the status of base layer crosslinking, inclusion of a TLR2 ligand in the antigenic peptide, and substitution of serine or alanine for an unpaired cysteine residue in the T* epitope. Studies in mice revealed consistent superiority of the Pam3Cys-modified candidates and a modest benefit of base layer crosslinking, as evidenced by higher and more persistent antibody titers (up to 18 months post-immunization), a qualitative improvement of T-cell responses toward a Th1 phenotype, and greater protection from live parasite challenges compared to the unmodified prototype candidate.

Mechanics of Emulsion Electrospun Porous Carbon Fibers as Building Blocks of Multifunctional Materials.

Many multifunctional composite structures incorporate porosity at various length scales to increase the available surface area of a functional component. One material system of particular interest is activated or porous carbon fibers and nanofibers that can serve as structural reinforcement as well as providing active surface for added functionality. A key question in the design and manufacture of these fibers is to what degree the induced pore affects the mechanical properties by inducing discontinuities in the material.

Performance Comparison of Three Chemical Vapor Deposited Aminosilanes in Peptide Synthesis: Effects of Silane on Peptide Stability and Purity.

Silicon oxide substrates underwent gas-phase functionalization with various aminosilanes, and the resulting surfaces were evaluated for their suitability as a solid support for solid phase peptide synthesis (SPPS). APTES (3-aminopropyltriethoxysilane), APDEMS (3-aminopropyldiethoxymethylsilane), and APDIPES (3-aminopropyldiisopropylethoxysilane) were individually applied to thermal oxide-terminated silicon substrates via gas-phase deposition. Coated surfaces were characterized by spectroscopic ellipsometry (SE), contact angle goniometry, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and spectrophotometry.

Mechanically Strong Graphene/Aramid Nanofiber Composite Electrodes for Structural Energy and Power.

Structural energy and power systems offer both mechanical and electrochemical performance in a single multifunctional platform. These are of growing interest because they potentially offer reduction in mass and/or volume for aircraft, satellites, and ground transportation. To this end, flexible graphene-based supercapacitors have attracted much attention due to their extraordinary mechanical and electrical properties, yet they suffer from poor strength.

Porous fibres with encapsulated functional materials and tunable release.

A novel sequential processing approach to fabricate versatile fibres containing encapsulated materials is presented. It is based on developing highly porous fibres, to be filled with functional materials and coated with protective layers. Applicability of the approach to develop porous poly(methyl methacrylate) (PMMA) fibres with encapsulated antibacterial salts within a coating is demonstrated.

Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children; however no effective treatment or vaccine is currently available. Previous studies have shown that therapeutic treatment with a monoclonal antibody (clone 131-2G) specific to the RSV G glycoprotein CX3C motif, mediates virus clearance and decreases leukocyte trafficking to the lungs of RSV-infected mice. In this study, we show that vaccination with layer-by-layer nanoparticles (LbL-NP) carrying the G protein CX3C motif induces blocking antibodies that prevent the interaction of the RSV G protein with the fractalkine receptor (CX3CR1) and protect mice against RSV replication and disease pathogenesis.

Nanoparticle vaccines encompassing the respiratory syncytial virus (RSV) G protein CX3C chemokine motif induce robust immunity protecting from challenge and disease.

Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge.

Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses.

Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease.

Deflection and pull-in instability of nanoscale beams in liquid electrolytes.

An elastic beam suspended horizontally over a substrate in liquid electrolyte was subjected to electric, osmotic, and van der Waals forces. The problem, which is governed by four non-dimensional parameters, was solved using the finite element method. The sum of the electric and osmotic forces, the electrochemical force, is usually attractive.

Synthetic nanoparticle vaccines produced by layer-by-layer assembly of artificial biofilms induce potent protective T-cell and antibody responses in vivo.

Nanoparticle vaccines induce potent immune responses in the absence of conventional adjuvant due to the recognition by immune cells of the particle structures, which mimic natural pathogens such as viruses and bacteria. Nanoparticle vaccines were fabricated by constructing artificial biofilms using layer-by-layer (LbL) deposition of oppositely charged polypeptides and target designed peptides on CaCO(3) cores. LbL nanoparticles were efficiently internalized by dendritic cells in vitro by a mechanism that was at least partially phagocytic, and induced DC maturation without triggering secretion of inflammatory cytokines.

Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection.

The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected.

Design, synthesis, and pharmacology of fluorescently labeled analogs of serotonin: application to screening of the 5-HT2C receptor.

Novel fluorescent derivatives of serotonin have been synthesized and used as tracers for the development of a 5-HT2C fluorescence polarization assay. Serotonin analogs that feature a fluorescent probe attached through an ether linkage at the tryptamine 5-position have high affinity for the 5-HT2C receptor, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 5a, which has 10-fold higher 5-HT2C affinity relative to serotonin (Kd=0.

Binding to the low-density lipoprotein receptor accelerates futile catalytic cycling in PCSK9 and raises the equilibrium level of intramolecular acylenzyme.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) on target cells and lowers the level of receptor by impeding its recycling. PCSK9 is self-processed to a complex of its prodomain and catalytic domain like a typical protein convertase, but it does not develop normal proteolytic activity. Instead, its propeptide remains complexed with the catalytic domain, and the C-terminal Gln152 of the prodomain occupies the active site like a substrate for peptide synthesis.

Biophysical and biochemical approach to locating an inhibitor binding site on cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) transfers neutral lipids between different types of plasma lipoprotein. Inhibitors of CETP elevate the fraction of plasma cholesterol associated with high-density lipoproteins and are being developed as new agents for the prevention and treatment of cardiovascular disease. The molecular basis of their function is not yet fully understood.

Fluorescently labeled analogues of dofetilide as high-affinity fluorescence polarization ligands for the human ether-a-go-go-related gene (hERG) channel.

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide.

Nanoscale electrostatic actuators in liquid electrolytes.

Equilibrium and energy analyses were performed for an electrostatic actuator consisting of two plane parallel electrodes, operated using DC voltages, separated by a liquid electrolyte. One electrode is fixed, and the other electrode is connected to a spring and is free to move. The mechanical equilibrium includes the spring force, the van der Waals force, and the electrochemical force as given by the solution of the linearized Poisson-Boltzmann equation.

A semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues.

An in vitro semiquantitative reactive metabolite detection assay is described that incorporates NADPH-supplemented human liver microsomes, a novel quaternary ammonium glutathione analogue conjugating agent (QA-GSH), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection. The assay was developed to have high sample capacity and the potential for high sample throughput. MS/MS detection is selective and sensitive for the QA-GSH conjugating agent and semiquantitation of QA-GSH-reactive metabolite conjugates is performed using QA-GSH standards added to samples prior to analysis [i.

Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library.

The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent.

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure.

Cyclization of N-terminal S-carbamoylmethylcysteine causing loss of 17 Da from peptides and extra peaks in peptide maps.

Enzymatic digests of proteins S-alkylated with iodoacetamide may contain peptides with N-terminal S-carbamoylmethylcysteine. These can be partly converted to a form with 17 Da lower mass and increased HPLC retention. Proof by synthesis supported by MS/MS and NMR spectroscopy was used to show that N-terminal S-carbamoylmethyl-L-cysteine can cyclize, losing NH3 to form an N-terminal residue of (R)-5-oxoperhydro-1,4-thiazine-3-carboxylic acid.