Interfacing lipid bilayer nanodiscs and silicon photonic sensor arrays for multiplexed protein-lipid and protein-membrane protein interaction screening.

Soluble proteins are key mediators of many biochemical signaling pathways via direct interaction with the lipid bilayer and via membrane-bound receptors. Components of the cell membrane are involved in many important biological processes, including viral infection, blood clotting, and signal transduction, and as such, they are common targets of therapeutic agents. Therefore, the development of analytical approaches to study interactions at the cell membrane is of critical importance. Herein, we integrate two key technologies, silicon photonic microring resonator arrays and phospholipid bilayer nanodiscs, which together allow multiplexed screening of soluble protein interactions with lipid and membrane-embedded targets. Microring resonator arrays are an intrinsically multiplexable, label-free analysis platform that has previously been applied to studying protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. Nanodiscs are protein-stabilized lipid assemblies that represent a convenient construct to mimic the native phospholipid bilayer, investigate the effects of membrane composition, and solubilize membrane-embedded targets. Exploiting the natural affinity of nanodisc-supported lipid bilayers for oxide-passivated silicon, we assembled single and multiplex sensor arrays via direct physisorption, characterizing electrostatic effects on the nanodisc attachment. Using model systems, we demonstrate the applicability of this platform for the parallel screening of protein interactions with nanodisc-embedded lipids, glycolipids, and membrane proteins.

Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators.

Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.

Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells.

Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC alpha(1) subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells.

Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors.

Adeno-associated virus (AAV) vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. An inherent characteristic of the manufacturing process is production of empty capsids that lack the transgene and are therefore unable to provide the intended therapeutic benefit. The effect of empty capsids on clinical outcomes is not well understood, but there are immunogenicity and efficacy concerns, and these empty capsids are considered a product-related impurity. Therefore, empty capsids should be controlled during the manufacturing process and monitored through analytical testing, but there are limited techniques available that are capable of quantifying capsid content and even fewer that are amenable to validation and implementation as registered release tests in a regulated environment. In addition, there is currently not a widely accepted gold standard technique for quantifying capsid content, and the understanding of how the results compare between different orthogonal technologies is limited. The current study utilizes a comprehensive assessment to evaluate diverse analytical techniques for their ability to quantitate capsid content.

Role of soluble guanylyl cyclase-cyclic GMP signaling in tumor cell proliferation.

Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO-cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO-receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGC alpha(1), robust levels of sGC beta(1) were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine); sGC activator), NOC-18+BAY41-2272, IBMX (3-isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC-18+BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO-sGC-cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms.

Anti-inflammatory influence of P-selectin on human mononuclear cells.

The purpose of this study was to determine if P-selectin, an adhesion molecule involved in the transendothelial movement of leukocytes, might also have a direct influence on the function of cells that come into contact with it. Human peripheral blood mononuclear cells were incubated on immobilized P-selectin or a control substrate (bovine serum albumin, BSA) and stimulated with bacterial lipopolysaccharide (LPS). After 24 h, the concentrations of proinflammatory cytokines in the supernatants of LPS-stimulated cells incubated on P-selectin were <50% of those produced by cells incubated on BSA (interleukin-1beta: P=0.001, tumor necrosis factor-alpha: P=0.004, and interferon-gamma : P=0.026). In contrast, cells incubated on P-selectin produced 74% more of the anti-inflammatory cytokine interleukin-10 than cells incubated on BSA (P=0.013). Neither P-selectin nor BSA stimulated cytokine production in the absence of LPS. Thus, P-selectin modulated the cytokine secretion of peripheral blood mononuclear cells in a coordinated manner that reduced the inflammatory potential of the cells.

The permeation of dynorphin A 1-6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability.

Dynorphin A 1-17 (Dyn A 1-17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1-17 metabolism at the blood-brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1-6 is identified as a metabolite of Dyn A 1-17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC-MS/MS. The transport of Dyn A 1-6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1-6.

Analytical and biological methods for probing the blood-brain barrier.

The blood-brain barrier (BBB) is an important interface between the peripheral and central nervous systems. It protects the brain against the infiltration of harmful substances and regulates the permeation of beneficial endogenous substances from the blood into the extracellular fluid of the brain. It can also present a major obstacle in the development of drugs that are targeted for the central nervous system. Several methods have been developed to investigate the transport and metabolism of drugs, peptides, and endogenous compounds at the BBB. In vivo methods include intravenous injection, brain perfusion, positron emission tomography, and microdialysis sampling. Researchers have also developed in vitro cell-culture models that can be employed to investigate transport and metabolism at the BBB without the complication of systemic involvement. All these methods require sensitive and selective analytical methods to monitor the transport and metabolism of the compounds of interest at the BBB.