Contribution of Noncanonical Antigens to Virulence and Adaptive Immunity in Human Infection with Enterotoxigenic E. coli.

Enterotoxigenic Escherichia coli (ETEC) contributes significantly to the substantial burden of infectious diarrhea among children living in low- and middle-income countries. In the absence of a vaccine for ETEC, children succumb to acute dehydration as well as nondiarrheal sequelae related to these infections, including malnutrition. The considerable diversity of ETEC genomes has complicated canonical vaccine development approaches defined by a subset of ETEC pathovar-specific antigens known as colonization factors (CFs). To identify additional conserved immunogens unique to this pathovar, we employed an "open-aperture" approach to capture all potential conserved ETEC surface antigens, in which we mined the genomic sequences of 89 ETEC isolates, bioinformatically selected potential surface-exposed pathovar-specific antigens conserved in more than 40% of the genomes (n = 118), and assembled the representative proteins onto microarrays, complemented with known or putative colonization factor subunit molecules (n = 52) and toxin subunits. These arrays were then used to interrogate samples from individuals with acute symptomatic ETEC infections. Surprisingly, in this approach, we found that immune responses were largely constrained to a small number of antigens, including individual colonization factor antigens and EtpA, an extracellular adhesin. In a Bangladeshi cohort of naturally infected children <2 years of age, both EtpA and a second antigen, EatA, elicited significant serologic responses that were associated with protection from symptomatic illness. In addition, children infected with ETEC isolates bearing either etpA or eatA genes were significantly more likely to develop symptomatic disease. These studies support a role for antigens not presently targeted by vaccines (noncanonical) in virulence and the development of adaptive immune responses during ETEC infections. These findings may inform vaccine design efforts to complement existing approaches.

Heparin acts as a structural component of ß-endorphin amyloid fibrils rather than a simple aggregation promoter.

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide ß-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in the presence of heparin leads to a loss of function. Applying correlative optical super-resolution microscopy methods, we show that heparin incorporates into emerging ß-endorphin fibrils forming an integral component and is essential for amyloid templating. This will have direct implications on ß-endorphin's normal physiological function and raises concerns on the biological relevance of heparin-promoted amyloid models.

Remote laser ablation electrospray ionization mass spectrometry for non-proximate analysis of biological tissues.

RATIONALE: We introduce remote laser ablation electrospray ionization (LAESI), a novel, non-proximate ambient sampling technique. Remote LAESI allows additional analytical instrumentation to be incorporated during sample analysis. This work demonstrates the utility of remote LAESI and, when combined with optical microscopy, allows for the microscopy-guided sampling of biological tissues. METHODS: Rapid prototyping using a 3D printer was applied to produce various ablation chamber geometries. A focused 5 ns, 2.94 µm laser pulse kept at 10 Hz ablated the sample within the chamber, remote to the mass spectrometer inlet. Ablated particulates were carried through a transfer tube by N2 gas, delivered to the electrospray plume and ionized. A long-distance microscope was used to capture images of tissues before, during and after ablation. RESULTS: Optimized remote LAESI was found to have a 27% transport efficiency compared with conventional LAESI, sufficient for many applications. A comparable molecular coverage was obtained with remote LAESI for the analysis of plant tissue. Proof-of-principle experiments using a pansy flower and a maple leaf indicated the functionality of this approach for selecting domains of interest for analysis by optical microscopy and obtaining chemical information from those selected regions by remote LAESI-MS. CONCLUSIONS: Remote LAESI is an ambient non-proximate sampling technique, proven to detect metabolites in biological tissues. When combined with optical microscopy, remote LAESI allows for the simultaneous acquisition of morphological and chemical information. This technique has important implications for histology, where chemical information for specific locations within a tissue is critical.

Why do the [PhSiO(1.5)](8,10,12) cages self-brominate primarily in the ortho position? Modeling reveals a strong cage influence on the mechanism.

(PhSiO1.5)8,10,12 cages are bulky, electron withdrawing like CF3; yet self-brominate (60 °C), favoring ortho substitution: PhT8 (≈85%), PhT10 (≈75%) and PhT12 (60%). First-principles calculations suggest bromination initiates when Br2 is "trapped" via H-bonding to ortho-H's, followed by polarization via strong interactions with cage faces, possibly cage LUMOs.

Nano building blocks via iodination of [PhSiO1.5]n, forming [p-I-C6H4SiO1.5]n (n = 8, 10, 12), and a new route to high-surface-area, thermally stable, microporous materials via thermal elimination of I2.

We describe the synthesis and characterization of the homologous p-iodophenylsilsesquioxanes (SQs) [p-I-C(6)H(4)SiO(1.5)](n) (n = 8, 10, 12) via ICl-promoted iodination (-40 to -60 degrees C) with overall yields of 80-90% and > 95% para selectivity following recrystallization. Characterization by NMR, FTIR, TGA, and single-crystal X-ray diffraction are reported and compared to data previously published for I(8)OPS. Coincidentally, we report a new synthesis of the elusive pentagonal decaphenyl SQ (dPS) [C(6)H(4)SiO(1.5)](10) and its characterization by NMR and single-crystal X-ray studies. These unique macromolecules possess equivalent chemical functionality but varying symmetries (cubic, pentagonal, and D(2d) dodecahedral), offering the potential to develop homologous series of functionalized star and dendrimer compounds with quite different core geometries and thereby providing the potential to greatly vary structure-property relationships in derivative compounds and nanocomposites made therefrom. We find that all three compounds decompose on heating to approximately 400 degrees C/N(2) with loss of I(2) to form robust, microporous materials with BET surface areas of 500-700 m(2)/g, pore volumes of 0.25-0.31 cm(3)/g, average pore widths of 8 A, and oxidative stabilities > or = 500 degrees C and with solid-phase morphologies varying from crystalline to mostly amorphous, as indicated by powder XRD and SEM studies. These latter findings point to important symmetry effects relating directly to packing in the crystalline phase prior to thermolysis.

Synthesis and photophysical properties of stilbeneoctasilsesquioxanes. Emission behavior coupled with theoretical modeling studies suggest a 3-D excited state involving the silica core.

A set of stilbene-substituted octasilicates [p-RStil(x)Ph(8-x)SiO(1.5)](8) (R = H, Me, MeO, Cl, NMe(2) and x = 5.3-8) and [o-MeStilSiO(1.5)](8) were prepared. Model compounds were also prepared including the corner and half cages: [p-MeStilSi(OEt)(3)], [p-Me(2)NStilSi(OSiMe(3))(3)], and [p-Me(2)NStilSi(O)(OSiMe)](4). These compounds were characterized by MALDI-TOF, TGA, FTIR, and (1)H NMR techniques. Their photophysical properties were characterized by UV-vis, two-photon absorption, and cathodoluminescence spectroscopy (on solid powders), including studies on the effects of solvent polarity and changes in concentration. These molecules are typically soluble, easily purified, and robust, showing T(d(5%)) > 400 degrees C in air. The full and partial cages all show UV-vis absorption spectra (in THF) identical to the spectrum of trans-stilbene, except for [o-MeStilSiO(1.5)](8), which exhibits an absorption spectrum blue-shifted from trans-stilbene. However, the partial cages show emissions that are red-shifted by approximately 20 nm, as found for stilbene-siloxane macrocycles, suggesting some interaction of the silicon center(s) with the stilbene pi* orbital in both the corner and half cages. In contrast, the emission spectra of the full cages show red-shifts of 60-100 nm. These large red-shifts are supported by density functional theoretical calculations and proposed to result from interactions of the stilbene pi* orbitals with a LUMO centered within the cage that has 4A(1) symmetry and involves contributions from all Si and oxygen atoms and the organic substituents. Given that this LUMO has 3-D symmetry, it appears that all of the stilbene units interact in the excited state, consistent with theoretical results, which show an increased red-shift with an increase in the functionalization of a single corner to functionalization of all eight corners with stilbene. In the case of the Me(2)N- derivatives, this interaction is primarily a charge-transfer interaction, as witnessed by the influence of solvent polarity on the emission behavior. More importantly, the two-photon absorption behavior is 2-3 times greater on a per p-Me(2)Nstilbene basis for the full cage than for the corner or half cages. Similar observations were made for p-NH(2)stilbenevinyl(8)OS cages, where the greater conjugation lengths led to even greater red-shifts (120 nm) and two-photon absorption cross sections. Cathodoluminescence studies done on [p-MeStilSiO(1.5)](8) or [p-MeStilOS](8) powders exhibit essentially the same emissions as seen in solution at high dilution. Given that only the emissions are greatly red-shifted in these molecules, whereas the ground-state UV-vis absorptions are not changed from trans-stilbene, except for the ortho derivative, which is blue-shifted 10 nm. It appears that the interactions are only in the excited state. Theoretical results show that the HOMO and LUMO states are always the pi and pi* states on the stilbene, which show very weak shifts with increasing degrees of functionalization, consistent with the small changes in the UV-vis spectra. The band gap between the lowest unoccupied 4a1 symmetry core state localized inside the silsesquioxane cage and the highest occupied state (pi state on stilbene), however, is markedly decreased as the number of stilbene functional groups is increased. This is consistent with the significant red-shifts in the emission spectra. The results suggest that the emission occurs from the 4a1 state localized on the cage. Moreover, for the compounds [p-RStil(6-7)Ph(2-1)OS](8), the emissions are blue-shifted compared to those of the fully substituted compounds, suggesting the molecular symmetry is reduced (from cubic), thereby reducing the potential for 3-D delocalization and raising the energy of the LUMO. The implications are that these octafunctional molecules exhibit some form of 3-D interaction in the excited state that might permit their use as molecular transistors as well as for energy collection and dispersion as molecular antennas, for example, and for nonlinear optical applications.

Fluoride rearrangement reactions of polyphenyl- and polyvinylsilsesquioxanes as a facile route to mixed functional phenyl, vinyl T10 and T12 silsesquioxanes.

Polyphenylsilsesquioxane [PhSiO(1.5)](n) (PPS) and polyvinylsilsesquioxane [vinylSiO(1.5)](n) (PVS) are polymeric byproducts of the syntheses of the related T(8) octamers [PhSiO(1.5)](8) and [vinylSiO(1.5)](8). Here we demonstrate that random-structured PPS and PVS rearrange in the presence of catalytic amounts of Bu(4)N(+)F(-) in THF to form mixed-functionality polyhedral T(10) and T(12) silsesquioxane (SQ) cages in 80-90% yields. Through control of the initial ratio of starting materials, we can statistically tailor the average values for x for the vinyl(x)Ph(10-x)T(10) and vinyl(x)Ph(12-x)T(12) products. Metathetical coupling of x approximately = 2 vinyl cages with 4-bromostyrene produces SQs with an average of two 4-bromostyrenyl substituents. These products can be reacted via Heck coupling with vinylSi(OEt)(3) to produce SQs with vinylSi(OEt)(3) end-caps. Alternately, Heck coupling with the originally produced x approximately = 2 vinyl SQs leads to "beads on a chain" SQ oligomers joined by conjugated organic tethers. The functionalized T(10) and T(12) cages, metathesis, and Heck compounds were characterized by standard analytical methods (MALDI-TOF MS, (1)H and (13)C NMR spectroscopy, TGA, and GPC). MALDI confirms the elaboration of the cages after each synthetic step, and GPC verifies the presence of higher molecular weight SQ oligomers. TGA shows that all of these compounds are thermally stable in air (>300 degrees C). The UV-vis absorption and emission behavior of the Heck oligomers reveals exceptional red-shifts (> or = 60 nm) compared to the vinylSi(OEt)(3) end-capped model compounds, suggesting electronic interactions through the SQ silica cores. Such phenomena may imply 3-D conjugation through the cores themselves.

High-throughput screening of nanoparticle catalysts made by flame spray pyrolysis as hydrocarbon/NO oxidation catalysts.

We describe here the use of liquid-feed flame spray pyrolysis (LF-FSP) to produce high surface area, nonporous, mixed-metal oxide nanopowders that were subsequently subjected to high-throughput screening to assess a set of materials for deNO(x) catalysis and hydrocarbon combustion. We were able to easily screen some 40 LF-FSP produced materials. LF-FSP produces nanopowders that very often consist of kinetic rather than thermodynamic phases. Such materials are difficult to access or are completely inaccessible via traditional catalyst preparation methods. Indeed, our studies identified a set of Ce(1-x)Zr(x)O(2) and Al(2)O(3)-Ce(1-x)Zr(x)O(2) nanopowders that offer surprisingly good activities for both NO(x) reduction and propane/propene oxidation both in high-throughput screening and in continuous flow catalytic studies. All of these catalysts offer activities comparable to traditional Pt/Al(2)O(3) catalysts but without Pt. Thus, although Pt-free, they are quite active for several extremely important emission control reactions, especially considering that these are only first generation materials. Indeed, efforts to dope the active catalysts with Pt actually led to lower catalytic activities. Thus the potential exists to completely change the materials used in emission control devices, especially for high-temperature reactions as these materials have already been exposed to 1500 degrees C; however, much research must be done before this potential is verified.

Nano-alpha-Al2O3 by liquid-feed flame spray pyrolysis.

Nanometre-sized particles of transition (t)-aluminas are important for the fabrication of high-quality alumina ceramics. Multiple tons are produced each year using a variety of gas-phase processes. The nanoparticles produced by these methods consist mainly of the undesired delta phase with some gamma- and theta-Al(2)O(3). Nano-t-aluminas should provide access to dense nano/submicrometre-grained alpha-Al(2)O(3) shapes offering significant advantages over micrometre-grained shapes. Unfortunately, polymorphism coupled with the high activation energy for nucleating alpha-Al(2)O(3) greatly impedes efforts to process dense alpha-Al(2)O(3) with controlled grain sizes, especially for submicrometre materials. Typically alpha-Al(2)O(3) nucleation within t-aluminas is sporadic rather than uniform, leading to exaggerated grain growth and vermicular microstructures without full densification (5). Thus, production of quantities of nano-alpha-Al(2)O(3) from multiple nano-t-aluminas for seeding or direct processing of alpha-Al(2)O(3) monoliths could greatly change how alpha-Al(2)O(3) components are processed. We report here that liquid-feed flame spray pyrolysis of nano-t-aluminas converts them to dispersible 30-80 nm alpha-Al(2)O(3) powders (50-85% phase transformed). Surprisingly, the powder surfaces are fully dehydrated. These powders pressureless sinter to more than 99.5% dense alpha-Al(2)O(3) with final grain sizes < or =500 nm without sintering aids.

Continuous-wave ultraviolet laser action in strongly scattering Nd-doped alumina.

We report electrically pumped, cw laser action near 405 nm from Nd(3+) -doped delta -alumina nanopowders. To our knowledge, this is the first report of stimulated emission from the high-lying F(2) -excited states, achieved through feedback from strong elastic scattering of light over transport path lengths shorter than half a wavelength.

Metabolism of tresperimus by rat aorta semicarbazide-sensitive amine oxidase (SSAO).

Tresperimus (Cellimis), a new immunosuppressive agent, is mainly eliminated in the rat through metabolism, in which the oxidative deamination of the primary amine of the drug plays a major role. We have previously demonstrated in vivo the significant involvement of semicarbazide-sensitive amine oxidase (SSAO) in this reaction. Rat aorta, a tissue with one of the highest specific SSAO activities, was tested as a new in vitro model to elucidate tresperimus metabolism, using a combination of liquid chromatography/mass spectrometry (LC/MS) and high-performance liquid chromatography (HPLC) analyses. The metabolites resulting from the main metabolic pathway of the drug were formed in rat aorta homogenates. The use of various SSAO, lysyl oxidase and monoamine oxidase inhibitors confirmed that SSAO is predominantly involved in the main site of tresperimus metabolism but also in every metabolic pathway of the drug, including deamination of tresperimus metabolites M3 (desaminopropyl derivative of tresperimus) and M6 (guanidinohexylamine). A microsomal fraction of the rat aorta was used to characterize tresperimus deamination. The moderate affinity of membrane-bound SSAO for tresperimus, with a Km value of 66 microM, was counterbalanced by a catalytic efficiency superior to that of certain physiological substrates of SSAO, such as methylamine. The rat aorta provided an interesting model with which to study tresperimus metabolism, highlighting the important role that SSAO could play as a phase I oxidative enzyme in the metabolism of certain exogenous amines at the vascular level.

Organic/inorganic hybrid composites from cubic silsesquioxanes.

A new class of epoxy nanocomposites with completely defined organic/inorganic phases was prepared by reacting octakis(glycidyldimethylsiloxy)octasilsesquioxane [(glydicylMe(2)SiOSiO(1.5))(8)] (OG) with diaminodiphenylmethane (DDM) at various compositional ratios. The effects of reaction curing conditions on nanostructural organization and mechanical properties were explored. A commercial epoxy resin based on the diglycidyl ether of bisphenol A (DGEBA) was used as a reference material throughout these studies. FTIR was used to follow the curing process and to demonstrate that the silsesquioxane structure is preserved during processing. OG/DDM composites possess comparable tensile moduli (E) and fracture toughness (K(IC)) to, and better thermal stabilities than, DGEBA/DDM cured under similar conditions. Dynamic mechanical analysis and model reaction studies suggest that the maximum cross-link density is obtained at N = 0.5 (NH(2):epoxy groups = 0.5) whereas the mechanical properties are maximized at N = 1.0. Digestion of the inorganic core with HF followed by GPC analysis of the resulting organic tether fragments when combined with the model reaction studies confirms that, at N = 0.5, each organic tether connects four cubes, while, at N = 1.0, linear tethers connecting two cubes dominate the network structure. Thus, well-defined nanocomposites with controlled variation of the organic tether architecture can be made and their properties assessed.

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate.

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.

Evaluation of vetiver oil and seven insect-active essential oils against the Formosan subterranean termite.

Repellency and toxicity of 8 essential oils (vetiver grass, cassia leaf, clove bud, cedarwood, Eucalyptus globules, Eucalyptus citrodora, lemongrass and geranium) were evaluated against the Formosan subterranean termite, Coptotermes formosanus Shiraki. Vetiver oil proved the most effective repellent because of its long-lasting activity. Clove bud was the most toxic, killing 100% of termites in 2 days at 50 micrograms/cm2. The tunneling response of termites to vetiver oil also was examined. Vetiver oil decreased termite tunneling activity at concentrations as low as 5 micrograms/g sand. Tunneling and paper consumption were not observed when vetiver oil concentrations were higher than 25 micrograms/g sand. Bioactivity of the 8 oils against termites and chemical volatility were inversely associated. Listed in decreasing order of volatility, the major constituents of the 8 oils were: eucalyptol, citronellal, citral, citronellol, cinnamaldehyde, eugenol, thujopsene, and both alpha- and beta-vetivone. Vetivor oil is a promising novel termiticide with reduced environmental impact for use against subterranean termites.

Nootkatone is a repellent for Formosan subterranean termite (Coptotermes formosanus).

We examined the behavior of Formosan subterranean termites toward one of the components of vetiver grass oil, the roots of which manufacture insect repellents. We found nootkatone, a sesquiterpene ketone, isolated from vetiver oil is a strong repellent and toxicant to Formosan subterranean termites. The lowest effective concentration tested was 10 micrograms/g substrate. This is the first report of nootkatone being a repellent to insects.

Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP1A1.

Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.

Involvement of semicarbazide-sensitive amine oxidase in tresperimus metabolism in human and in rat.

The metabolism of tresperimus, a new immunosuppressive agent, was investigated in vivo and in vitro in rat and in human. Two metabolic pathways were identified at each side of the molecule with two deamination reactions on the spermidine moiety and hydrolysis of the amide bond leading to the liberation of guanidinohexylamine. As the major metabolic pathway of the drug seemed to be the oxidative deamination, the capacity of different amine oxidases to metabolize tresperimus was then tested using in vivo experiments in rat and in vitro studies in rat and human plasma. The increase of tresperimus plasma levels induced by the administration of hydralazine, an irreversible in vivo inhibitor of semicarbazide-sensitive amine oxidase (SSAO), reflected the major involvement of this enzyme in tresperimus metabolism. This result was confirmed in vitro in rat and human plasma by the use of semicarbazide, a specific SSAO inhibitor. As opposed to rat plasma, human plasma may be an interesting in vitro model to study the metabolism of a drug extensively metabolized by SSAO such as tresperimus. Indeed, SSAO activity was significantly higher in human plasma than in rat plasma. The second metabolic pathway of the drug, which only occurred in rat plasma, appeared thus as the major route of tresperimus metabolism in this biological matrix.

Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins.

Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants. This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells. In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes. The cellular localization and activity of the putative drug efflux system were evaluated. In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins. The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model. Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

Efficacy of vetiver oil and nootkatone as soil barriers against Formosan subterranean termite (Isoptera: Rhinotermitidae).

Vetiver oil and its components nootkatone and cedrene were assessed as sand treatments for their efficacy to disrupt food recruitment by Coptotermes formosanus Shiraki. Termites were required to tunnel through sand treated with vetiver oil, nootkatone, cedrene, or untreated sand to reach a food source. Results showed that sand treated with vetiver oil or nootkatone disrupted termite tunneling behavior. As a consequence, after 21 d, wood consumption and termite survival were significantly lower compared with cedrene-treated or untreated sand treatments. Sand treated with vetiver oil or nootkatone at 100 microg/g substrate were effective barriers to termites.