Silica modified with polymeric amphiphilic nanoparticles as first dimension for multidimensional separation techniques.

This paper addresses the evaluation of a new amphiphilic nanoparticle supported on silica and its application as sorbent in on-line solid phase extraction. The investigated sorbent material is a copolymer composed by [2- (Acryloyloxy) ethyl] trimethylammonium chloride (block A) and butyl acrylate (block B) prepared by reversible addition-fragmentation chain transfer. After polymerization the nanoparticles were adsorbed into silica surface by electrostatic attraction providing to the sorbent both, apolar and polar characteristics. After the fundamental studies to understand the main features of the synthesized nanoparticles supported on silica, this sorbent material was packed into an extraction column. This column was connected on line with liquid chromatography-tandem mass spectrometry and utilized for automated online determination of several analytes as Ochratoxin A, azoxystrobin, cyproconazole and difenoconazole, in wine and water samples.

Sphingomyelin ability to act as chiral selector using nanodisc electrokinetic chromatography.

Understanding how stereochemistry affects interactions with cell membranes is important for effective drug development. Chirality has been shown to greatly effect pharmaceutical distribution and metabolism within the cell. However it has been thought that interactions with, and passive diffusion through, the membrane are not stereochemically selective. Various studies have produced conflicting results regarding whether interactions with lipid bilayers are or can be stereoselective. In the current work, stereoselective interactions between a pair of atropisomers, R-(+)/(S)-(-) 1,1'-Bi-2-naphthol, and sphingomyelin nanodisc bilayers, are demonstrated. This is accomplished using nanodisc electrokinetic chromatography, demonstrating that this approach is sensitive to subtle differences in affinity between small molecule probes and lipid bilayers. Using the same approach, no evidence of stereoselectivity was observed using enantiomer or diastereomer probes of varied chemistry and structure.

Effects of structure on the performance of latex nanoparticles as a pseudostationary phase in electrokinetic chromatography.

The fundamental relationships between the structure and chemistry of latex nanoparticles synthesized by reversible addition fragmentation chain transfer (RAFT) controlled living polymerization and their subsequent performance as pseudostationary phases (PSP) are reported in this paper. RAFT enables the rational optimization of latex nanoparticle pseudostationary phases and control of the behavior of the PSP. Nanoparticles comprised of amphiphilic diblock copolymers of 2-acrylamido-2-methylpropane sulfonic acid-derived ionic/hydrophilic blocks and butyl- ethyl- or methyl-acrylate-derived hydrophobic blocks were synthesized in two sizes. The mobility, methylene selectivity, and efficiency of each of the six pseudostationary phases are reported, as well as the relationship between monomer quantity and NP size. Linear solvation energy relationships are reported and compared to SDS micelles and previous nanoparticle pseudostationary phases. The solvation characteristics and selectivity of nanoparticle pseudostationary phases is shown to be affected primarily by the structure of the hydrophobic copolymer block. Butyl acrylate nanoparticles 17 nm in diameter are found to provide the best overall separation performance with over 500 thousand theoretical plates generated in 6 min separations.

Determination of lipid bilayer affinities and solvation characteristics by electrokinetic chromatography using polymer-bound lipid bilayer nanodiscs.

Styrene-maleic acid polymer-bound lipid bilayer nanodiscs have been investigated and characterized by electrokinetic chromatography. Linear solvation energy relationship analysis was employed to characterize the changes in solvation environment of nanodiscs of varied belt to lipid ratio, belt polymer chemistry and molecular weight, and lipid composition. Increases in the lipid to belt polymer ratio resulted in smaller, more cohesive nanodiscs with greater electrophoretic mobility. Nanodisc structures with belt polymers of different chemistry and molecular weight were compared and showed only minor changes in solvent characteristics and selectivity consistent with changes in structure of the lipid bilayer. Seven phospholipid and sphingomyelin nanodiscs of different lipid composition were characterized. Changes in lipid head group structure had a significant effect on bilayer-solute interactions. In most cases, changes in alkyl tail structure had no discernible effect on solvation environment aside from those explained by changes in the gel-liquid transition temperature. Comparison to vesicles of similar lipid composition show only minor differences in solvation environment, likely due to differences in lipid composition and bilayer curvature. Together these results provide evidence that the dominant solute-nanodisc interactions are with the lipid bilayer and that head group chemistry has a greater impact on bilayer-solute interactions than alkyl tail or belt polymer structure. Nanodisc electrokinetic chromatography is demonstrated to allow characterization of solute interactions with lipid bilayers of varied composition.

Evaluation of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) cationic polymer capillary coating for capillary electrophoresis and electrokinetic chromatography separations.

Capillary electrophoresis and electrokinetic chromatography are typically carried out in unmodified fused-silica capillaries under conditions that result in a strong negative zeta potential at the capillary wall and a robust cathodic electroosmotic flow. Modification of the capillary wall to reverse the zeta potential and mask silanol sites can improve separation performance by reducing or eliminating analyte adsorption, and is essential when conducting electrokinetic chromatography separations with cationic latex nanoparticle pseudo-stationary phases. Semipermanent modification of the capillary walls by coating with cationic polymers has proven to be facile and effective. In this study, poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymers were synthesized by reversible addition-fragmentation chain transfer polymerization and used as physically adsorbed semipermanent coatings for capillary electrophoresis and electrokinetic chromatography separations. An initial synthesis of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymer coating produced strong and stable anodic electroosmotic flow of -5.7 to -5.4 × 10-4 cm2 /V⋅s over the pH range of 4-7. Significant differences in the magnitude of the electroosmotic flow and effectiveness were observed between synthetic batches, however. For electrokinetic chromatography separations, the best performing batches of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymer performed as well as the commercially available cationic polymer polyethyleneimine, whereas polydiallylammonium chloride and hexadimethrine bromide did not perform well.

Performance and selectivity of cationic nanoparticle pseudo-stationary phases in electrokinetic chromatography.

Electrokinetic chromatography (EKC) is a powerful analytical technique that uses an ionic pseudo-stationary phase (PSP) to separate neutral compounds. Although anionic surfactants are the most common choice for PSP, cationic latex nanoparticles are an attractive alternative. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize several types of diblock copolymers that self-assemble into latex nanoparticles, which were characterized by a variety of techniques including diffusion NMR. The performance of each nanoparticle as a PSP was studied by using a homologous series of ketones and linear solvation energy relationships (LSER) analysis. A cationic homopolymer coating was found to be necessary to prevent band broadening caused by analyte interactions with nanoparticles adsorbed to the capillary surface. No significant difference in methylene selectivity or LSER parameters was observed between nanoparticles with different cationic shells, but differences were observed between nanoparticles with different hydrophobic cores. Cationic latex nanoparticles behaved more like anionic latex nanoparticles than like cationic surfactants, suggesting that selectivity is primarily driven by the hydrophobic portion of a PSP. Cationic latex nanoparticles in combination with a homopolymer cationic capillary coating are an excellent choice for EKC analyses where an anodic electroosmotic flow is required.

Phospholipid bilayer affinities and solvation characteristics by electrokinetic chromatography with a nanodisc pseudostationary phase.

Phospholipid bilayer nanodiscs composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and synthetic maleic acid-styrene copolymer belts have been introduced as a pseudostationary phase (PSP) in electrokinetic chromatography and demonstrated good performance. The nanodiscs provide a suitable migration range and high theoretical plate counts. Using this nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was determined and characterized. Good correlation is observed between retention factors and octanol water partition coefficients for particular categories of solutes, but the general correlation is weak primarily because the nanodiscs show stronger affinity than octanol for hydrogen bond donors. This suggests that a more appropriate application of this technology is to measure and characterize interactions between solutes and lipid bilayers directly. Linear solvation energy relationship analysis of the nanodisc-solute interactions in this study demonstrates that the nanodiscs provide a solvation environment with low cohesivity and weak hydrogen bond donating ability, and provide relatively strong hydrogen bond acceptor strength.

Diminished amygdala activation and behavioral threat response following traumatic brain injury.

Each year, approximately 3.8 million people suffer mild to moderate traumatic brain injuries (mTBI) that result in an array of neuropsychological symptoms and disorders. Despite these alarming statistics, the neurological bases of these persistent, debilitating neuropsychological symptoms are currently poorly understood. In this study we examined the effects of mTBI on the amygdala, a brain structure known to be critically involved in the processing of emotional stimuli. Seven days after lateral fluid percussion injury (LFPI), mice underwent a series of physiological and behavioral experiments to assess amygdala function. Brain-injured mice exhibited a decreased threat response in a cued fear conditioning paradigm, congruent with a decrease in amygdala excitability determined with basolateral amygdala (BLA) field excitatory post-synaptic potentials together with voltage-sensitive dye imaging (VSD). Furthermore, beyond exposing a general decrease in the excitability of the primary input of the amygdala, the lateral amygdala (LA), VSD also revealed a decrease in the relative strength or activation of internuclear amygdala circuit projections after LFPI. Thus, not only does activation of the LA require increased stimulation, but the proportion of this activation that is propagated to the primary output of the amygdala, the central amygdala, is also diminished following LFPI. Intracellular recordings revealed no changes in the intrinsic properties of BLA pyramidal neurons after LFPI. This data suggests that mild to moderate TBI has prominent effects on amygdala function and provides a potential neurological substrate for many of the neuropsychological symptoms suffered by TBI patients.

Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells.

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour.

RAFT polymerized nanoparticles: influences of shell and core chemistries on performance for electrokinetic chromatography.

The performance and solvation characteristics of two novel latex nanoparticle (NP) pseudo-stationary phases (PSPs) for EKC are determined and compared to those of previously reported micellar, polymeric, and NP materials. The new NPs have shells composed of strongly acidic poly(AMPS) as opposed to the poly(acrylic acid) shell of the prior NP, and have varied hydrophobic core chemistry of either poly(butyl acrylate) or poly(ethyl acrylate). The NPs poly(AMPS) shell shows only minor changes in mobility and selectivity between pH 4.9 and 9.4, allowing adjustment of pH to influence and optimize separation performance. All of the NP phases have significantly different solvation characteristics and selectivity relative to SDS micelles. The selectivity and solvent character are similar for NPs with poly(butyl acrylate) cores and different shells, but vary significantly between NPs with poly(butyl acrylate) versus poly(ethyl acrylate) cores. NPs with poly(butyl acrylate) cores are among the least cohesive PSPs reported to date, while the NP with poly(ethyl acrylate) core is among the most cohesive. The results demonstrate that PSPs with unique selectivity can be generated by altering the chemistry of the hydrophobic core.

Angiotensin II potentiates adrenergic and muscarinic modulation of guinea pig intracardiac neurons.

The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG II-induced modulation of firing was blocked by the angiotensin type 2 (AT(2)) receptor inhibitor PD 123319 and was mimicked by the AT(2) receptor agonist CGP-42112A. AT(1) receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT(2) receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.

Retention behavior and selectivity of a latex nanoparticle pseudostationary phase for electrokinetic chromatography.

The retention characteristics and separation selectivity of a novel latex nanoparticle (NP) pseudostationary phase (PSP) for electrokinetic chromatography have been characterized. The anionic NPs have very low or no affinity for cationic solutes, but show significant interactions and retention based on hydrophobic interactions. Retention factors of alkyl-phenyl ketones increase linearly with the concentration of the NPs and have zero or near zero y-intercepts as expected for electrokinetic chromatography with non-micellar PSPs. The retention factors of these solutes and representative pharmaceuticals decrease logarithmically with increases in the concentration of ACN in the background electrolyte, as expected for reversed-phase retention. Linear solvation energy relationship analysis indicates that the NPs are less cohesive than would be expected for polymeric PSPs with similar structure but that the overall separation selectivity can be expected to be similar to polymer PSPs with similar backbone chemistry. The results indicate that the hydrophobic core of the NPs is non-cohesive and is highly accessible to solutes, whereas the ionic head groups are not as accessible and do not contribute substantially to retention or selectivity.

Fine particulate matter source apportionment following a large woodstove changeout program in Libby, Montana.

Before a community-wide woodstove changeout program, a chemical mass balance (CMB) source apportionment study was conducted in Libby, MT, during the winter of 2003-2004 to identify the sources of fine particulate matter (PM2.5) within the valley. Results from this study showed that residential woodstoves were the major source, contributing approximately 80% of the ambient PM2.5 throughout the winter months. In an effort to lower the ambient PM2.5, a large woodstove changeout program was conducted in Libby from 2005 to 2007 in which nearly 1200 old woodstoves were changed out with cleaner burning models. During the winter of 2007-2008, a follow-up CMB source apportionment study was conducted to evaluate the effectiveness of the changeout. Results from this study showed that average winter PM2.5 mass was reduced by 20%, and woodsmoke-related PM2.5 (as identified by the CMB model) was reduced by 28% when compared with the pre-changeout winter of 2003-2004. These results suggest that a woodstove changeout can be an effective tool in reducing ambient levels of PM2.5 in woodstove-impacted communities.

Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells.

The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2-C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole-cell patch-clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non-malignant human tissues.

Electrokinetic chromatography and mass spectrometric detection using latex nanoparticles as a pseudostationary phase.

The utility of novel latex nanoparticles as pseudostationary phases for electrokinetic chromatography with UV and mass spectrometric detection is demonstrated. The nanoparticles are synthesized using ab initio RAFT (reversible addition-fragmentation chain transfer) in emulsion polymerization, which yields small (63 nm) particles with a narrow size distribution, a hydrophobic core, and an ionic shell. The nanoparticles are shown to provide efficient and selective separations, with retention and separation selectivity dominated by hydrophobic interactions. The nanoparticles are highly retentive, such that they are effective at relatively low concentrations. Addition of the nanoparticles to the background electrolyte at these concentrations has a minor effect on the noise with UV detection, no measurable effect on the separation current, and minor effects on analyte ionization efficiency during electrospray ionization. The nanoparticles do not cause fouling or degradation of the electrospray-mass spectrometer interface even after several weeks of use. The combination of online sample preconcentration via sweeping and selective mass spectrometric detection yields low detection limits (10-16 ppb), particularly for more hydrophobic compounds.

Urinary levoglucosan as a biomarker of wood smoke: results of human exposure studies.

Urinary levoglucosan was investigated as a potential biomarker of wood smoke exposure in two different controlled experimental settings. Nine subjects were exposed to smoke from a campfire in a controlled setting, and four were exposed to smoke from an older-model wood stove. All subjects were asked to provide urine samples before and after exposure, and to wear personal particulate matter with a diameter of < or =2.5 microm (PM(2.5)) monitors during exposure. Urinary levoglucosan measurements from both studies showed no consistent response to the smoke exposure. A third experiment was conducted to assess the contribution of dietary factors to urinary levoglucosan levels. Nine subjects were asked to consume caramel and provide urine samples before and after consumption. Urinary levoglucosan levels increased within 2 h of caramel consumption and returned to pre-exposure levels within 24 h. These studies suggest that diet is a major factor in determining urinary levoglucosan levels and that recent dietary history needs to be taken into account for future work involving levoglucosan as a biomarker of wood smoke exposure.

Community woodstove changeout and impact on ambient concentrations of polycyclic aromatic hydrocarbons and phenolics.

A large woodstove changeout program was carried out in Libby, Montana, with the goal of reducing ambient levels of PM2.5. This provided researchers the opportunity to measure ambient concentrations of phenolic and polycyclic aromatic hydrocarbons (PAHs) before, during, and after the changeout of nearly 1200 stoves to evaluate the effectiveness of the intervention. Starting in the heating season of 2004/2005 and ending in the heating season of 2007/2008, 19 compounds were measured every three days using a high-volume polyurethane foam (PUF) sampler followed by gas chromatography and mass spectrometry analysis. Some of the organic species with the highest measured concentrations were also signature chemical markers for wood combustion. When comparing the measurements conducted during the heating season of 2004/2005 (prechangeout) to those of the heating season of 2007/2008 (postchangeout), there was a 64% average reduction in the measured concentrations of phenolics and PAHs, while the PM2.5 mass dropped by only 20% over the same time period. The results of this four year sampling program suggest that the Libby woodstove changeout program was successful in reducing overall concentrations of the measured phenolic and PAH compounds.

Polycystic kidney disease channel and synaptotagmin homologues play roles in schizosaccharomyces pombe cell wall synthesis/repair and membrane protein trafficking.

Eukaryotic cells can sense a wide variety of environmental stresses, including changes in temperature, pH, osmolarity and nutrient availability. They respond to these changes through a variety of signal-transduction mechanisms, including activation of Ca(2+)-dependent signaling pathways. This research has discovered important implications in the function(s) of polycystic kidney disease (PKD) channels and the mechanisms through which they act in the control of cell growth and cell polarity in Schizosaccharomyces pombe by ion channel-mediated Ca(2+) signaling. Pkd2 was expressed maximally during the exponential growth phase. At the cell surface pkd2 was localized at the cell tip during the G(2) phase of the cell cycle, although following cell wall damage, the cell surface-expressed protein relocalized to the whole plasma membrane. Pkd2 depletion affected Golgi trafficking, resulting in a buildup of vesicles at the cell poles, and strongly affected plasma membrane protein delivery. Surface-localized pkd2 was present in the plasma membrane for a very short time and was rapidly internalized. Internalization was dependent on Ca(2+), enhanced by amphipaths and inhibited by gadolinium. The pkd2 protein was in a complex with a yeast synaptotagmin homologue and myosin V. Depletion of pkd2 severely affected the localization of glucan synthase. A role for pkd2 in a cell polarity and cell wall synthesis signaling complex with a synaptotagmin homologue, myosin V and glucan synthase is proposed.

Covalent modification of fused silica capillaries with quaternized polyamines to achieve robust and stable anodic electroosmotic flow.

Synthetic chemistry originally developed for the manufacture of chemically stable silica polyamine composites was adapted for the modification of fused silica capillaries for application in CE. Polyethyleneimine (PEI) and polyallylamine (PAA) were covalently bonded to the interior surface of fused silica capillaries utilizing 3-chloropropyltrichlorosilane (CPTCS) or 3-glycidoxypropyl-trimethoxysilane (GPTMS) to anchor the polymers to the surface. The surface-bound polymers were subsequently quaternized using methyl iodide (MeI). The resulting modified capillaries were studied using CE, and were shown to provide reproducible, stable, and robust anodic EOF throughout the pH range of 2-10. Surface modifications utilizing CPTCS could be rinsed with up to 6 M HCL or 1 M NaOH without significant loss of surface modifier. The utility of the modified capillaries for the separation of simple anions and cations was demonstrated.

Urinary levoglucosan as a biomarker of wood smoke exposure: observations in a mouse model and in children.

BACKGROUND: Biomass smoke is an important source of particulate matter (PM), and much remains to be discovered with respect to the human health effects associated with this specific PM source. Exposure to biomass smoke can occur in one of two main categories: short-term exposures consist of periodic, seasonal exposures typified by communities near forest fires or intentional agricultural burning, and long-term exposures are chronic and typified by the use of biomass materials for cooking or heating. Levoglucosan (LG), a sugar anhydride released by combustion of cellulose-containing materials, is an attractive candidate as a biomarker of wood smoke exposure. OBJECTIVES: In the present study, Balb/c mice and children were assessed for LG in urine to determine its feasibility as a biomarker. METHODS: We performed urinary detection of LG by gas chromatography/mass spectrometry after intranasal instillations of LG or concentrated PM (mice) or biomass exposure (mice or humans). RESULTS: After instillation, we recovered most of the LG within the first 4 hr. Experiments using glucose instillation proved the specificity of our system, and instillation of concentrated PM from wood smoke, ambient air, and diesel exhaust supported a connection between wood smoke and LG. In addition, LG was detected in the urine of mice exposed to wood smoke. Finally, a pilot human study proved our ability to detect LG in urine of children. CONCLUSIONS: These results demonstrate that LG in the lungs is detectable in the urine of both mice and humans and that it is a good candidate as a biomarker of exposure to biomass smoke.