Chloride Ligands on DNA-Stabilized Silver Nanoclusters.

DNA-stabilized silver nanoclusters (AgN-DNAs) are known to have one or two DNA oligomer ligands per nanocluster. Here, we present the first evidence that AgN-DNA species can possess additional chloride ligands that lead to increased stability in biologically relevant concentrations of chloride. Mass spectrometry of five chromatographically isolated near-infrared (NIR)-emissive AgN-DNA species with previously reported X-ray crystal structures determines their molecular formulas to be (DNA)2[Ag16Cl2]8+. Chloride ligands can be exchanged for bromides, which red-shift the optical spectra of these emitters. Density functional theory (DFT) calculations of the 6-electron nanocluster show that the two newly identified chloride ligands were previously assigned as low-occupancy silvers by X-ray crystallography. DFT also confirms the stability of chloride in the crystallographic structure, yields qualitative agreement between computed and measured UV-vis absorption spectra, and provides interpretation of the 35Cl-nuclear magnetic resonance spectrum of (DNA)2[Ag16Cl2]8+. A reanalysis of the X-ray crystal structure confirms that the two previously assigned low-occupancy silvers are, in fact, chlorides, yielding (DNA)2[Ag16Cl2]8+. Using the unusual stability of (DNA)2[Ag16Cl2]8+ in biologically relevant saline solutions as a possible indicator of other chloride-containing AgN-DNAs, we identified an additional AgN-DNA with a chloride ligand by high-throughput screening. Inclusion of chlorides on AgN-DNAs presents a promising new route to expand the diversity of AgN-DNA structure-property relationships and to imbue these emitters with favorable stability for biophotonics applications.

A Disulfide-Stabilized Aß that Forms Dimers but Does Not Form Fibrils.

Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide.

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

KCNQ5 activation is a unifying molecular mechanism shared by genetically and culturally diverse botanical hypotensive folk medicines.

Botanical folk medicines have been used throughout human history to treat common disorders such as hypertension, often with unknown underlying mechanisms. Here, we discovered that hypotensive folk medicines from a genetically diverse range of plant species each selectively activated the vascular-expressed KCNQ5 potassium channel, a feature lacking in the modern synthetic pharmacopeia, whereas nonhypotensive plant extracts did not. Analyzing constituents of the hypotensive Sophora flavescens root, we found that the quinolizidine alkaloid aloperine is a KCNQ-dependent vasorelaxant that potently and isoform-selectively activates KCNQ5 by binding near the foot of the channel voltage sensor. Our findings reveal that KCNQ5-selective activation is a defining molecular mechanistic signature of genetically diverse traditional botanical hypotensives, transcending plant genus and human cultural boundaries. Discovery of botanical KCNQ5-selective potassium channel openers may enable future targeted therapies for diseases including hypertension and KCNQ5 loss-of-function encephalopathy.

Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN).

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal ß-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a ß-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal ß-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.

TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA.

Siderophore nutrition tests with Caulobacter crescentus strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. C. crescentus did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [59Fe]apoferrichrome and 59Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd ], 1 nM; Michaelis-Menten constant [KM ], 0.1 nM; Vmax, 19 pMol/109 cells/min) were similar to those of Escherichia coli FhuA. Transport properties for 59Fe-citrate were similar to those of E. coli FecA (KM , 5.3 nM; Vmax, 29 pMol/109 cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028, 00138, 02277, and 03023 as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to Escherichia coli FepA and BtuB. A Δ02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for heme/hemoglobin utilization).IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus, identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by C. crescentus, its heme/hemoglobin transporter (HutA).

Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread.

UNLABELLED: Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1ß, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. IMPORTANCE: Extracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.

Formation and Nanomechanical Properties of Silver-Mediated Guanine DNA Duplexes in Aqueous Solution.

Silver cations can mediate base pairing of guanine (G) DNA oligomers, yielding linear parallel G-Ag+-G duplexes with enhanced stabilities compared to those of canonical DNA duplexes. To enable their use in programmable DNA nanotechnologies, it is critical to understand solution-state formation and the nanomechanical stiffness of G-Ag+-G duplexes. Using temperature-controlled circular dichroism (CD) spectroscopy, we find that heating mixtures of G oligomers and silver salt above 50 °C fully destabilizes G-quadruplex structures and converts oligomers to G-Ag+-G duplexes. Electrospray ionization mass spectrometry supports that G-Ag+-G duplexes form at stoichiometries of 1 Ag+ per base pair, and CD spectroscopy suggests that as the Ag+/base stoichiometry increases further, G-Ag+-G duplexes undergo additional morphological changes. Using liquid-phase atomic force microscopy, we find that this excess Ag+ enables assembly of long fiberlike structures with ∼2.5 nm heights equivalent to a single DNA duplex but with lengths that far exceed a single duplex. Finally, using the conditions established to form single G-Ag+-G duplexes, we use a surface forces apparatus (SFA) to compare the solution-phase stiffness of single G-Ag+-G duplexes with dG-dC Watson-Crick-Franklin duplexes. SFA shows that G-Ag+-G duplexes are 1.3 times stiffer than dG-dC duplexes, confirming gas-phase ion mobility spectrometry measurements and computational predictions. These findings may guide the development of structural DNA nanotechnologies that rely on silver-mediated base pairing.

Synthesis of site-specific Fab-drug conjugates using ADP-ribosyl cyclases.

Targeted delivery of small-molecule drugs via covalent attachments to monoclonal antibodies has proved successful in clinic. For this purpose, full-length antibodies are mainly used as drug-carrying vehicles. Despite their flexible conjugation sites and versatile biological activities, intact immunoglobulins with conjugated drugs, which feature relatively large molecular weights, tend to have restricted tissue distribution and penetration and low fractions of payloads. Linking small-molecule therapeutics to other formats of antibody may lead to conjugates with optimal properties. Here, we designed and synthesized ADP-ribosyl cyclase-enabled fragment antigen-binding (Fab) drug conjugates (ARC-FDCs) by utilizing CD38 catalytic activity. Through rapidly forming a stable covalent bond with a nicotinamide adenine dinucleotide (NAD+ )-based drug linker at its active site, CD38 genetically fused with Fab mediates robust site-specific drug conjugations via enzymatic reactions. Generated ARC-FDCs with defined drug-to-Fab ratios display potent and antigen-dependent cytotoxicity against breast cancer cells. This work demonstrates a new strategy for developing site-specific FDCs. It may be applicable to different antibody scaffolds for therapeutic conjugations, leading to novel targeted agents.

Branched oligopeptides form nanocapsules with lipid vesicle characteristics.

In a recent article (Gudlur et al. PLOS ONE, 2012, 7 (9) e45374), we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These polycationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the perinuclear region where they persist without any apparent degradation. In this report, we examine the lipidlike properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4 °C. Having the ability to prepare stable uniform nanoscale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.

Electron count and ligand composition influence the optical and chiroptical signatures of far-red and NIR-emissive DNA-stabilized silver nanoclusters.

Near-infrared (NIR) emissive DNA-stabilized silver nanoclusters (AgN-DNAs) are promising fluorophores in the biological tissue transparency windows. Hundreds of NIR-emissive AgN-DNAs have recently been discovered, but their structure-property relationships remain poorly understood. Here, we investigate 19 different far-red and NIR emissive AgN-DNA species stabilized by 10-base DNA templates, including well-studied emitters whose compositions and chiroptical properties have never been reported before. The molecular formula of each purified species is determined by high-resolution mass spectrometry and correlated to its optical absorbance, emission, and circular dichroism (CD) spectra. We find that there are four distinct compositions for AgN-DNAs emissive at the far red/NIR spectral border. These emitters are either 8-electron clusters stabilized by two DNA oligomer copies or 6-electron clusters with one of three different ligand compositions: two oligomer copies, three oligomer copies, or two oligomer copies with additional chlorido ligands. Distinct optical and chiroptical signatures of 6-electron AgN-DNAs correlate with each ligand composition. AgN-DNAs with three oligomer ligands exhibit shorter Stokes shifts than AgN-DNAs with two oligomers, and AgN-DNAs with chlorido ligands have increased Stokes shifts and significantly suppressed visible CD transitions. Nanocluster electron count also significantly influences electronic structure and optical properties, with 6-electron and 8-electron AgN-DNAs exhibiting distinct absorbance and CD spectral features. This study shows that the optical and chiroptical properties of NIR-emissive AgN-DNAs are highly sensitive to nanocluster composition and illustrates the diversity of structure-property relationships for NIR-emissive AgN-DNAs, which could be harnessed to precisely tune these emitters for bioimaging applications.

Spatially resolved detection of small molecules from press-dried plant tissue using MALDI imaging.

Premise: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a chemical imaging method that can visualize spatial distributions of particular molecules. Plant tissue imaging has so far mostly used cryosectioning, which can be impractical for the preparation of large-area imaging samples, such as full flower petals. Imaging unsectioned plant tissue presents its own difficulties in extracting metabolites to the surface due to the waxy cuticle. Methods: We address this by using established delipidation techniques combined with a solvent vapor extraction prior to applying the matrix with many low-concentration sprays. Results: Using this procedure, we imaged tissue from three different plant species (two flowers and one carnivorous plant leaf). Material factorization analysis of the resulting data reveals a wide range of plant-specific small molecules with varying degrees of localization to specific portions of the tissue samples, while facilitating detection and removal of signal from background sources. Conclusions: This work demonstrates applicability of MALDI-MSI to press-dried plant samples without freezing or cryosectioning, setting the stage for spatially resolved molecule identification. Increased mass resolution and inclusion of tandem mass spectrometry are necessary next steps to allow more specific and reliable compound identification.

Premisa: Matrix­assisted laser desorption/ionization mass spectrometry imaging (MALDI­MSI) es un método de imagen química que puede visualizar distribuciones espaciales de moléculas particulares. Hasta ahora, las imágenes de tejido vegetal han utilizado principalmente la criosección, lo cual puede ser poco práctico para la preparación de muestras de imágenes con áreas grandes, tales como los pétalos completos de una flor. La obtención de imágenes de tejido vegetal no seccionado presenta sus propias dificultades durante la extracción de metabolitos a la superficie, debido a la cutícula cerosa de la planta. Métodos: Abordamos esto usando técnicas establecidas de deslipidación combinados con una extracción de vapor por solvente antes de aplicar por aspersión la matriz en bajas concentraciones. Resultados: Usando este procedimiento, obtuvimos imágenes de tejido de tres especies de plantas diferentes (dos flores y una hoja de planta carnívora). Análisis de factorización material de los datos obtenidos revelaron una amplia gama de pequeñas moléculas específicas en plantas con diversos grados de localización en porciones específicas de las muestras de tejido, al igual que facilitó la detección y remoción de las señales de fondo. Conclusión: Nuestro trabajo demuestra la aplicabilidad de MALDI­MSI hacía muestras de plantas secadas a presión sin congelación o criosección, creando el marco para la identificación de moléculas resueltas espacialmente. Aumento de la resolución de masas e inclusión de la espectrometría de masas en tándem son pasos necesarios para obtener identificación de compuestos más específica y confiable.

DNA Stabilizes Eight-Electron Superatom Silver Nanoclusters with Broadband Downconversion and Microsecond-Lived Luminescence.

DNA oligomers are known to serve as stabilizing ligands for silver nanoclusters (AgN-DNAs) with rod-like nanocluster geometries and nanosecond-lived fluorescence. Here, we report two AgN-DNAs that possess distinctly different structural properties and are the first to exhibit only microsecond-lived luminescence. These emitters are characterized by significant broadband downconversion from the ultraviolet/visible to the near-infrared region. Circular dichroism spectroscopy shows that the structures of these two AgN-DNAs differ significantly from previously reported AgN-DNAs. We find that these nanoclusters contain eight valence electrons, making them the first reported DNA-stabilized luminescent quasi-spherical superatoms. This work demonstrates the important role that nanocluster composition and geometry play in dictating luminescence properties of AgN-DNAs and significantly expands the space of structure-property relations that can be achieved for AgN-DNAs.

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution.

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.

Synthesis of site-specific antibody-drug conjugates by ADP-ribosyl cyclases.

Most of the current antibody-drug conjugates (ADCs) in clinic are heterogeneous mixtures. To produce homogeneous ADCs, established procedures often require multiple steps or long reaction times. The introduced mutations or foreign sequences may cause high immunogenicity. Here, we explore a new concept of transforming CD38 enzymatic activity into a facile approach for generating site-specific ADCs. This was achieved through coupling bifunctional antibody-CD38 fusion proteins with designer dinucleotide-based covalent inhibitors with stably attached payloads. The resulting adenosine diphosphate-ribosyl cyclase-enabled ADC (ARC-ADC) with a drug-to-antibody ratio of 2 could be rapidly generated through single-step conjugation. The generated ARC-ADC targeting human epidermal growth factor receptor 2 (HER2) displays excellent stability and potency against HER2-positive breast cancer both in vitro and in vivo. This proof-of-concept study demonstrates a new strategy for production of site-specific ADCs. It may provide a general approach for the development of a novel class of ADCs with potentially enhanced properties.

Salivary Cystatin SN Binds to Phytic Acid In Vitro and Is a Predictor of Nonheme Iron Bioavailability with Phytic Acid Supplementation in a Proof of Concept Pilot Study.

BACKGROUND: Acute phytic acid intake has been found to decrease iron bioavailability; however, repeated phytic acid consumption leads to iron absorption adaptation. Salivary proline-rich proteins (PRPs) have been shown to inhibit iron chelation to tannins and may mediate similar iron absorption adaptation with phytic acid intake. OBJECTIVES: The objectives of this study were to determine whether salivary proteins bind to phytic acid in vitro, and to explore a proof of concept in a pilot study that examined the impact of 4-wk, daily phytic acid supplementation on individuals' iron status, bioavailability, and salivary PRP concentrations. METHODS: High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization-time of flight were used to characterize in vitro salivary protein-phytic acid interactions. Nonanemic women (n = 7) consumed 350 mg phytic acid supplements 3 times daily for 4 wk, and meal challenges were employed to determine iron bioavailability, iron status, and salivary protein concentrations before and after supplementation periods. Enzyme-linked immunosorbent assay (ELISA) analysis of purified protein fractions and participant saliva identified proteins bound to phytic acid. RESULTS: In vitro salivary protein-phytic acid interaction identified cystatin SN, a non-proline rich salivary protein, as the specific bound protein to phytic acid. Iron bioavailability (P = 0.32), hemoglobin (P = 0.72), and serum ferritin (P = 0.08) concentrations were not reduced from week 0 to week 4 after phytic acid supplementation. Basic PRPs and cystatin SN concentrations were positively correlated with iron bioavailability at week 4. CONCLUSIONS: Overall, results suggest that phytic acid binds to the non-PRP cystatin SN and that salivary protein production may improve iron bioavailability with phytic acid consumption.

Linked Toll-Like Receptor Triagonists Stimulate Distinct, Combination-Dependent Innate Immune Responses.

Traditional vaccination strategies have failed to generate effective vaccines for many infections like tuberculosis and HIV. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like receptors (TLRs). Vaccines with multiple TLRs as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization-a critical feature of whole-cell vaccines. Second, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo. The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-κB, ISGs), cytokine profiles (IL12-p70, TNF-α, IL-6, IL-10, CCL2, IFN-α, IFN-ß, IFN-γ), and in vivo cytokine serum levels (IL-6, TNF-α, IL12-p40, IFN-α, IFN-ß). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more Th1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward TH2 biasing profiles despite the presence of TLR9. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development.

Parallel electrophoretic depletion, fractionation, concentration, and desalting of 96 complex biological samples for mass spectrometry.

The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.

The structural basis for inhibition of the classical and lectin complement pathways by S. aureus extracellular adherence protein.

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro-convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low-affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero-length crosslinking approach to map the Eap binding site to both the α'- and γ-chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand-binding modes.