Joanna L. Richens

Quantitative Validation and Comparison of Multiplex Cytokine Kits

The focus of biomarker studies is shifting toward deciphering patterns of biomolecules as they provide a more comprehensive depiction of disease than individual biomarkers. Multiplexing technologies are crucial in deciphering such patterns, but it is essential that they are validated for reproducibility and precision to ensure accurate protein identification. Here the authors examine such properties in Cytokine Bead Array (CBA) and Luminex kits and compare concentration measurements to those obtained using enzyme-linked immunosorbent assay (ELISA). Luminex kits were found to be highly reproducible and reliable; however, CBA kits were not due to aberrant standards. Absolute cytokine concentrations were dependent on the detection kit, but correlations with ELISA were good for all technologies.

Layer-by-Layer Assembly of Supported Lipid Bilayer Poly-L-Lysine Multilayers.

Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.

The electrical interplay between proteins and lipids in membranes.

All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure. The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid-protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid-protein interactions.

Rational targeting of subclasses of intermolecular interactions: elimination of nonspecific binding for analyte sensing.

The ability to target and control intermolecular interactions is crucial in the development of several different technologies. Here we offer a tool to rationally design liquid media systems that can modulate specific intermolecular interactions. This has broad implications in deciphering the nature of intermolecular forces in complex solutions and offers insight into the forces that govern both specific and nonspecific binding in a given system. Nonspecific binding still continues to be a problem when dealing with analyte detection across a range of different detection technologies. Here, we exemplify the problem of nonspecific binding on model membrane systems and when dealing with low-abundance protein detection on commercially available SPR technology. A range of different soluble reagents that target specific subclasses of intermolecular interactions have been tested and optimized to virtually eliminate nonspecific binding while leaving specific interactions unperturbed. Thiocyanate ions are used to target nonpolar interactions, and small reagents such as glycylglycylglycine are used to modulate the dielectric constant, which targets charge-charge and dipole interactions. We show that with rational design and careful modulation these reagents offer a step forward in dissecting the intermolecular forces that govern binding, alongside offering nonspecific binding elimination in detection systems.

α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir’s affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers.

Reverse engineering of Alzheimer's disease based on biomarker pathways analysis

Alzheimers disease (AD) poses an increasingly profound problem to society, yet progress toward a genuine understanding of the disease remains worryingly slow. Perhaps, the most outstanding problem with the biology of AD is the question of its mechanistic origins, that is, it remains unclear wherein the molecular failures occur that underlie the disease. We demonstrate how molecular biomarkers could help define the nature of AD in terms of the early biochemical events that correlate with disease progression. We use a novel panel of biomolecules that appears in cerebrospinal fluid of AD patients. As changes in the relative abundance of these molecular markers are associated with progression to AD from mild cognitive impairment, we make the assumption that by tracking their origins we can identify the biochemical conditions that predispose their presence and consequently cause the onset of AD. We couple these protein markers with an analysis of a series of genetic factors and together this hypothesis essentially allows us to redefine AD in terms of the molecular pathways that underlie the disease.

Rationalising the role of Keratin 9 as a biomarker for Alzheimer's disease.

Keratin 9 was recently identified as an important component of a biomarker panel which demonstrated a high diagnostic accuracy (87%) for Alzheimer’s disease (AD). Understanding how a protein which is predominantly expressed in palmoplantar epidermis is implicated in AD may shed new light on the mechanisms underlying the disease. Here we use immunoassays to examine blood plasma expression patterns of Keratin 9 and its relationship to other AD-associated proteins. We correlate this with the use of an in silico analysis tool VisANT to elucidate possible pathways through which the involvement of Keratin 9 may take place. We identify possible links with Dickkopf-1, a negative regulator of the wnt pathway, and propose that the abnormal expression of Keratin 9 in AD blood and cerebrospinal fluid may be a result of blood brain barrier dysregulation and disruption of the ubiquitin proteasome system. Our findings suggest that dysregulated Keratin 9 expression is a consequence of AD pathology but, as it interacts with a broad range of proteins, it may have other, as yet uncharacterized, downstream effects which could contribute to AD onset and progression.

Highly sensitive multipoint real-time kinetic detection of Surface Plasmon bioanalytes with custom CMOS cameras

Phase sensitive Surface Plasmon Resonance (SPR) techniques are a popular means of characterizing biomolecular interactions. However, limitations due to the narrow dynamic range and difficulty in adapting the method for multi-point sensing have restricted its range of applications. This paper presents a compact phase sensitive SPR technology using a custom CMOS camera. The system is exceptionally versatile enabling one to trade dynamic range for sensitivity without altering the optical system. We present results showing sensitivity over the array of better than 10−6 Refractive Index Units (RIU) over a refractive index range of 2×10−2 RIU, with peak sensitivity of 3×10−7 RIU at the center of this range. We also explain how simply altering the settings of polarization components can give sensitivity on the order of 10−8 RIU albeit at the cost of lower dynamic range. The consistent response of the custom CMOS camera in the system also allowed us to demonstrate precise quantitative detection of two Fibrinogen antibody–protein binding sites. Moreover, we use the system to determine reaction kinetics and argue how the multipoint detection gives useful insight into the molecular binding mechanisms.

Surface-enhanced Raman scattering measurement from a lipid bilayer encapsulating a single decahedral nanoparticle mediated by an optical trap

We present a new technique for the study of model membranes on the length-scale of a single nano-sized liposome. Silver decahedral nanoparticles have been encapsulated by a model unilamellar lipid bilayer creating nano-sized lipid vesicles. The metal core has two roles (i) increasing the polarizability of vesicles, enabling a single vesicle to be isolated and confined in an optical trap, and (ii) enhancing Raman scattering from the bilayer, via the high surface-plasmon field at the sharp vertices of the decahedral particles. Combined this has allowed us to measure a Raman fingerprint from a single vesicle of 50 nm-diameter, containing just ∼104 lipid molecules in a bilayer membrane over a surface area of <0.01 μm2, equivalent to a volume of approximately 1 zepto-litre. Raman scattering is a weak and inefficient process and previous studies have required either a substantially larger bilayer area in order to obtain a detectable signal, or the tagging of lipid molecules with a chromophore to provide an indirect probe of the bilayer. Our approach is fully label-free and bio-compatible and, in the future, it will enable much more localized studies of the heterogeneous structure of lipid bilayers and of membrane-bound components than is currently possible.

The interactions of squalene, alkanes and other mineral oils with model membranes; effects on membrane heterogeneity and function

Droplet interface bilayers (DIBs) offer many favourable facets as an artificial membrane system but the influence of any residual oil that remains in the bilayer following preparation is ill-defined. In this study the fluorescent membrane probes di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (Di-8-ANEPPS) and Fluoresceinphosphatidylethanolamine (FPE) were used to help understand the nature of the phospholipid-oil interaction and to examine any structural and functional consequences of such interactions on membrane bilayer properties. Concentration-dependent modifications of the membrane dipole potential were found to occur in phospholipid vesicles exposed to a variety of different oils. Incorporation of oil into the lipid bilayer was shown to have no significant effect on the movement of fatty acids across the lipid bilayer. Changes in membrane heterogeneity were, however, demonstrated with increased microdomain formation being visible in the bilayer following exposure to mineral oil, pentadecane and squalene. As it is important that artificial systems provide an accurate representation of the membrane environment, careful consideration should be taken prior to the application of DIBs in studies of membrane structure and organisation.