Transplantation and Noninvasive Longitudinal Imaging of Parathyroid Cells: A Proof-of-Concept Study.

The parathyroid cell is a vital regulator of extracellular calcium levels, operating through the secretion of parathyroid hormone (PTH). Despite its importance, the regulation of PTH secretion remains complex and not fully understood, representing a unique interplay between extracellular and intracellular calcium, and hormone secretion. One significant challenge in parathyroid research has been the difficulty in maintaining cells for in-depth cellular investigations.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins.

Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry.

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins.

Investigating Daptomycin-Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs.

Daptomycin is a cyclic lipopeptide antibiotic that targets the lipid membrane of Gram-positive bacteria. Membrane fluidity and charge can affect daptomycin activity, but its mechanisms are poorly understood because it is challenging to study daptomycin interactions within lipid bilayers. Here, we combined native mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP) to study daptomycin-membrane interactions with different lipid bilayer nanodiscs.

Stability and Dissociation of Adeno-Associated Viral Capsids by Variable Temperature-Charge Detection-Mass Spectrometry.

Adeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray.

Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry.

Native mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nanoelectrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethylene-glycol.

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties.

Background: The ATP-sensitive K (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K channels, and the regulatory subunit sulfonylurea receptor (SURx).

Nanomechanical Properties of Artificial Lipid Bilayers Composed of Fluid and Polymerizable Lipids.

Polymerization enhances the stability of a planar supported lipid bilayer (PSLB) but it also changes its chemical and mechanical properties, attenuates lipid diffusion, and may affect the activity of integral membrane proteins. Mixed bilayers composed of fluid lipids and poly(lipids) may provide an appropriate combination of polymeric stability coupled with the fluidity and elasticity needed to maintain the bioactivity of reconstituted receptors. Previously (, , , 12483-12491) we showed that binary mixtures of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC form phase-segregated PSLBs composed of nanoscale fluid and poly(lipid) domains.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K channels.

ATP-sensitive K (K) channels couple cellular metabolism to electrical activity in many cell types. Wild-type K channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane.

Quantification of intracellular HNO delivery with capillary zone electrophoresis.

Redox signaling, wherein reactive and diffusible small molecules are channeled into specific messenger functions, is a critical component of signal transduction. A central principle of redox signaling is that the redox modulators are produced in a highly controlled fashion to specifically modify biotargets. Thiols serve as primary mediators of redox signaling as a function of the rich variety of adducts, which allows initiation of distinct cellular effects.

Direct interaction of the ATP-sensitive K channel by the tyrosine kinase inhibitors imatinib, sunitinib and nilotinib.

The ATP-regulated K channel (K) plays an essential role in the control of many physiological processes, and contains a ATP-binding site. Tyrosine kinase inhibitors (TKI) are commonly used drugs, that primarily target ATP-binding sites in tyrosine kinases. Herein, we used the patch-clamp technique to examine the effects of three clinically established TKIs on K channel activity in isolated membrane patches, using a pancreatic β-cell line as a K channel source.

Surface Modification of Glass/PDMS Microfluidic Valve Assemblies Enhances Valve Electrical Resistance.

Microfluidic instrumentation offers unique advantages in biotechnology applications including reduced sample and reagent consumption, rapid mixing and reaction times, and a high degree of process automation. As dimensions decrease, the ratio of surface area to volume within a fluidic architecture increases, which gives rise to some of the unique advantages inherent to microfluidics. Thus, manipulation of surface characteristics presents a promising approach to tailor the performance of microfluidic systems.

Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids.

Polymerization of synthetic phospholipid monomers has been widely used to enhance the stability of lipid membranes in applications such as membrane-based biosensing, where the inherent instability of fluid-phase lipid bilayers can be problematic. However, lipid polymerization typically decreases membrane fluidity, which may be required to maintain the activity of reconstituted integral proteins and peptides. Prior work has shown that a bilayer composed of binary mixtures of poly(lipid) and fluid lipid exhibits enhanced stability and supports the function of incorporated biomolecules.

Hybrid Nanoparticle Platform for Nanoscale Scintillation Proximity Assay.

β-particle emitting radionuclides, such as H, C, P, P, and S, are important molecular labels due to their small size and the prevalence of these atoms in biomolecules but are challenging to selectively detect and quantify within aqueous biological samples and systems. Here, we present a core-shell nanoparticle-based scintillation proximity assay platform (nanoSPA) for the separation-free, selective detection of radiolabeled analytes. nanoSPA is prepared by incorporating scintillant fluorophores into polystyrene core particles and encapsulating the scintillant-doped cores within functionalized silica shells.

Enhanced Fluorescent Protein Activity in Polymer Scaffold-Stabilized Phospholipid Nanoshells Using Neutral Redox Initiator Polymerization Conditions.

Phospholipid nanoshells, for example, liposomes, provide a versatile enabling platform for the development of nanometer-sized biosensors and molecular delivery systems. Utilization of phospholipid nanoshells is limited by the inherent instability in complex biological environments, where the phospholipid nanoshell may disassemble and degrade, thus releasing the contents and destroying sensor function. Polymer scaffold stabilization (PSS), wherein the phospholipid nanoshells are prepared by partitioning reactive monomers into the lipid bilayer lamella followed by radical polymerization, has emerged to increase phospholipid nanoshell stability.

Rapid Formation of Polymer Frits in Fused Silica Capillaries Using Spatially defined Thermal Free-Radical Initiated Polymerization.

Column preparation in capillary chromatography commonly relies upon the generation of on-column porous frits. Here, we report a simple, robust and low-cost approach for preparing polymer frits on-column, in a rapid and spatially controlled manner using thermal free-radical initiated polymerization. In this approach, a simple, temperature-controlled heating apparatus is positioned adjacent to a 100 m i.

Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells.

The inwardly rectifying K (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.

Polystyrene-Core, Silica-Shell Scintillant Nanoparticles for Low-Energy Radionuclide Quantification in Aqueous Media.

β-particle emitting radionuclides are useful molecular labels due to their abundance in biomolecules. Detection of β-emission from H, S, and P, important biological isotopes, is challenging due to the low energies (E ≤ 300 keV) and short penetration depths (≤0.6 mm) in aqueous media.

Enhanced Temporal Resolution with Ion Channel-Functionalized Sensors Using a Conductance-Based Measurement Protocol.

The binding of a target analyte to an ion channel (IC), which is readily detected electrochemically in a label-free manner with single-molecule selectivity and specificity, has generated widespread interest in using natural and engineered ICs as transducers in biosensing platforms. To date, the majority of developments in IC-functionalized sensing have focused on IC selectivity or sensitivity or development of suitable membrane environments and aperture geometries. Comparatively little work has addressed analytical performance criteria, particularly criteria required for temporal measurements of dynamic processes.

Methacrylate Polymer Scaffolding Enhances the Stability of Suspended Lipid Bilayers for Ion Channel Recordings and Biosensor Development.

Black lipid membranes (BLMs) provide a synthetic environment that facilitates measurement of ion channel activity in diverse analytical platforms. The limited electrical, mechanical and temporal stabilities of BLMs pose a significant challenge to development of highly stable measurement platforms. Here, ethylene glycol dimethacrylate (EGDMA) and butyl methacrylate (BMA) were partitioned into BLMs and photopolymerized to create a cross-linked polymer scaffold in the bilayer lamella that dramatically improved BLM stability.

Fate of fluorescent core-shell silica nanoparticles during simulated secondary wastewater treatment.

Increasing use of silica nanoparticles (SiO2 NPs) in consumer products and industrial processes leads to SiO2 NP discharge into wastewater. Thus, there is a need to understand the fate of SiO2 NPs during wastewater treatment. However, the detection of SiO2 NPs in environmental systems is hindered by the elevated background levels of natural silicon.

Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography.

The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles.

Ion channel probes for scanning ion conductance microscopy.

The sensitivity and selectivity of ion channels provide an appealing opportunity for sensor development. Here, we describe ion channel probes (ICPs), which consist of multiple ion channels reconstituted into lipid bilayers suspended across the opening of perflourinated glass micropipets. When incorporated with a scanning ion conductance microscope (SICM), ICPs displayed a distance-dependent current response that depended on the number of ion channels in the membrane.

Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine.

A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present.