Lisa A. Holland

Obesity is associated with impaired immune response to influenza vaccination in humans

Background:Obesity is an independent risk factor for morbidity and mortality from pandemic influenza H1N1. Influenza is a significant public health threat, killing an estimated 250 000–500 000 people worldwide each year. More than one in ten of the worlds adult population is obese and more than two-thirds of the US adult population is overweight or obese. No studies have compared humoral or cellular immune responses to influenza vaccination in healthy weight, overweight and obese populations despite clear public health importance.Objective:The study employed a convenience sample to determine the antibody response to the 2009–2010 inactivated trivalent influenza vaccine (TIV) in healthy weight, overweight and obese participants at 1 and 12 months post vaccination. In addition, activation of CD8+ T cells and expression of interferon-γ and granzyme B were measured in influenza-stimulated peripheral blood mononuclear cell (PBMC) cultures.Results:Body mass index (BMI) correlated positively with higher initial fold increase in IgG antibodies detected by enzyme-linked immunosorbent assay to TIV, confirmed by HAI antibody in a subset study. However, 12 months post vaccination, higher BMI was associated with a greater decline in influenza antibody titers. PBMCs challenged ex vivo with vaccine strain virus, demonstrated that obese individuals had decreased CD8+ T-cell activation and decreased expression of functional proteins compared with healthy weight individuals.Conclusion:These results suggest obesity may impair the ability to mount a protective immune response to influenza virus.

Nanoparticle–Protein Interactions: A Thermodynamic and Kinetic Study of the Adsorption of Bovine Serum Albumin to Gold Nanoparticle Surfaces

Investigating the adsorption process of proteins on nanoparticle surfaces is essential to understand how to control the biological interactions of functionalized nanoparticles. In this work, a library of spherical and rod-shaped gold nanoparticles (GNPs) was used to evaluate the process of protein adsorption to their surfaces. The binding of a model protein (bovine serum albumin, BSA) to GNPs as a function of particle shape, size, and surface charge was investigated. Two independent comparative analytical methods were used to evaluate the adsorption process: steady-state fluorescence quenching titration and affinity capillary electrophoresis (ACE). Although under favorable electrostatic conditions kinetic analysis showed a faster adsorption of BSA to the surface of cationic GNPs, equilibrium binding constant determinations indicated that BSA has a comparable binding affinity to all of the GNPs tested, regardless of surface charge. BSA was even found to adsorb strongly to GNPs with a pegylated/neutral surface. However, these fluorescence titrations suffer from significant interference from the strong light absorption of the GNPs. The BSA-GNP equilibrium binding constants, as determined by the ACE method, were 10(5) times lower than values determined using spectroscopic titrations. While both analytical methods could be suitable to determine the binding constants for protein adsorption to NP surfaces, both methods have limitations that complicate the determination of protein-GNP binding constants. The optical properties of GNPs interfere with Ka determinations by static fluorescence quenching analysis. ACE, in contrast, suffers from material compatibility issues, as positively charged GNPs adhere to the walls of the capillary during analysis. Researchers seeking to determine equilibrium binding constants for protein-GNP interactions should therefore utilize as many orthogonal techniques as possible to study a protein-GNP system.

Amperometric and voltammetric detection for capillary electrophoresis

The focus of this article is amperometric and voltammetric detection coupled with capillary electrophoresis. Fundamental concepts and progress in the field of capillary electrophoresis with electrochemical detection (CEEC) that have occurred within the past three years, including new methodologies and unique applications, are highlighted. This review contains 95 references.

Capillary electrophoresis in pharmaceutical analysis.

Capillary electrophoresis (CE) is a separation technique particularly suited to the analysis of pharmaceutical compounds. This review offers a detailed discussion of the four common modes of detection coupled to CE—UV absorption, fluorescence, electrochemical, and mass spectrometry—and gives examples of the use of these methods in pharmaceutical analyses. Sample preparation and pretreatment techniques used for CE separations are described, as well as methods of preconcentration including hydrophobic retention, affinity concentration, sample stacking, and isotachophoresis. The use of affinity CE, chiral CE, and capillary gel electrophoresis for analysis of pharmaceuticals is covered in detail, and recent advances in capillary electrochromatography and CE on a chip are also discussed.

Characterization of an Integrated On-Capillary Dual Electrode for Capillary Electrophoresis-Electrochemistry

An integrated on-capillary dual electrode electrochemical detector for capillary electrophoresis consisting of two platinum wires is described. The electrodes are permanently fixed so that no alignment is necessary. The detector was evaluated for direct amperometric detection in the series configuration. Collection efficiencies were calculated for a series of phenolic acids exhibiting different degrees of chemical reversibility. The linearity and limits of detection at both electrodes were determined. The selectivity of the system was demonstrated by the detection of ferulic acid in beer.

Microscale exoglycosidase processing and lectin capture of glycans with phospholipid assisted capillary electrophoresis separations.

Capillary electrophoresis separations of glycans labeled with 1-aminopyrene-3,6,8-trisulfonic acid were achieved with separation efficiencies ranging from 480,000 to 640,000 theoretical plates in a 60.2 cm, 25 μm inner diameter fused silica capillary. Under these separation conditions, the coefficient of variation in peak area is 10%, and if labeling efficiency is estimated at 100%, the limit of detection is 15 fM. The capillary electrophoresis method incorporated phospholipid additives to enhance the separation of glycans with slight differences in hydrodynamic volume. In addition, the phospholipid additives supported the integration of the lectin concanavalin A as well as the enzymes α1-2,3 mannosidase or β1-4 galactosidase to provide structural and compositional information about the glycans subject to separation. The use of in-capillary cleavage of terminal glycan residues with exoglycosidases offers a number of advantages over benchtop enzymatic sequencing, including reduced consumption of analyte, as well as enzyme. These methods were used to evaluate glycans derived from the glycoproteins α1-acid glycoprotein, fetuin, and ribonuclease B, as well as from glycoproteins collected from MCF7 cells.

Transformable Capillary Electrophoresis for Oligosaccharide Separations Using Phospholipid Additives

Phospholipids are used as an additive in capillary electrophoresis to enhance the separation of glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B. The properties of phospholipid preparations are dependent upon composition, hydration, and temperature. Separation performance is evaluated as a function of these variables. A preparation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), with [DMPC]/[DHPC] = 2.5, in 10% lipid/aqueous buffer at 25 degrees C provides the best separation efficiency at an electric field strength of 400 V/cm. Resolution is enhanced with the additive. Concanavalin A, a lectin selective for high mannose and mannose branching glycans, and alpha1-2,3 mannosidase, an enzyme that cleaves 1-2 and 1-3 mannopyranosyl residues, are incorporated in the separation to provide additional selectivity and to expand the application of phospholipid additives for glycan separation.

Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009–2014)

This review of capillary electrophoresis methods for DNA analyses covers critical advances from 2009 to 2014, referencing 184 citations. Separation mechanisms based on free-zone capillary electrophoresis, Ogston sieving, and reptation are described. Two prevalent gel matrices for gel-facilitated sieving, which are linear polyacrylamide and polydimethylacrylamide, are compared in terms of performance, cost, viscosity, and passivation of electroosmotic flow. The role of capillary electrophoresis in the discovery, design, and characterization of DNA aptamers for molecular recognition is discussed. Expanding and emerging techniques in the field are also highlighted.

In vitro selection of a single-stranded DNA molecular recognition element against atrazine.

Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid‐assisted capillary electrophoresis

CE separations of glycans taken from the cancer drug, Trastuzumab (Herceptin®), were accomplished using phospholipid additives. Glycans were labeled with 1‐aminopyrene‐3,6,8‐trisulfonic acid and were separated with efficiencies as high as 510 000 theoretical plates in a 60.2 cm 25 μm id fused‐silica capillary. The thermally tunable phospholipid was loaded into the capillary when it possessed a viscosity similar to that of water. The temperature was increased, and the separations were performed when the material exhibited higher viscosity. Enzymes were integrated into the separation with the phospholipid additive. Neuraminidase, β1‐4 galactosidase, and β‐N‐acetylglucosaminidase were injected into the capillary without covalent modification and used for enzyme hydrolysis. Exoglycosidase enzymes cleaved the terminal glycan residues. The glycan sequence could be verified based on enzyme specificity. Neuraminidase was used to determine total glycan content of the low‐abundance glycans containing sialic acid. β1‐4 Galactosidase and β‐N‐acetylglucosaminidase were used sequentially in‐capillary, to determine the structure of the high‐abundance glycans.