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In the nervous system, a myelin sheath that originates from oligodendrocytes or Schwann cells wraps around axons to facilitate electrical signal transduction. The interface between an axon and myelin is maintained by a number of biomolecular interactions. Among the interactions are those between GD1a and GT1b gangliosides on the axon and myelin-associated glycoprotein (MAG) on myelin. Interestingly, these interactions can also inhibit neuronal outgrowth. Ganglioside–MAG interactions are often studied in cellular or animal models where their relative concentrations are not easily controlled or in assays where the gangliosides and MAG are not presented as part of fluid lipid bilayers. Here, we present an approach to characterize MAG–ganglioside interactions in real time, where MAG, GD1a, and GT1b contents are controlled and they are in their in vivo orientation within fluid lipid bilayers. Using a quartz crystal microbalance with dissipation monitoring (QCM-D) biosensor functionalized with a supported lipid bilayer (SLB) and MAG, we detect vesicular GD1a and GT1b binding and determine the interaction kinetics as a function of vesicular ganglioside content. MAG-bound vesicles are deformed similarly, regardless of the ganglioside or its mole fraction. We further demonstrate how MAG–ganglioside interactions can be disrupted by antiganglioside antibodies that override MAG-based neuron growth inhibition.

Bacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.

identified and analyzed by making meas urements on a population of single enti ties, for example, single liposomes [4][5][6][7][8][9] or EVs. [10,11] Traditional assays that treat a population as an ensemble obscure the distributions of particle properties, and they are blind to the asynchronous events that are revealed at the single cell, particle or molecule level.Single entity measurements can be obtained using microscopy, [12][13][14][15] spec troscopy, [16,17] flow cytometry, [10,18] mass spectrometry, [19,20] electrical, [21] electro chemical, [22] and separation methods, [23] all of which can reveal particle heteroge neities. However, many of these methods have weaknesses that limit their appli cability. For example, electron micro scopy has the requisite spatial resolution to observe individual small particles, but because of the required sample processing and fixing, temporal dynamics among particles cannot be determined. Flow cytometry is unable to characterize nanoscale (50-500 nm) objects, such as EVs, without special instrument modifications. Additionally, flow cytometry, capillary electrophoresis, and elec trochemical cytometry have highthroughput capabilities, but they are unable to analyze a single isolated particle over long periods of time, and specific individual particles are gener ally not recoverable for further analysis. Also, some of these methods destroy the particles as part of the measurement pro cess, which prevents multiplex analysis of individual particles. To overcome these drawbacks, new approaches are necessary that leverage the strengths of optical microscopy and enable multiplex highthroughput measurements on single biological particles to reveal their physical, chemical, and physiological heterogeneities.Chemicallyspecific capture of individual particles on sur faces can enable single particle analysis. [24,25] Furthermore, this approach can be improved by patterning the capture molecules in densely packed, highly ordered arrays, resulting in densely packed, highly ordered arrays of particles. [7] Pat terning the capture molecules on a surface results in the cap ture of thousands of particles in ordered arrays within a single image frame. Patterning capture molecules into highly ordered arrays ensures that neighboring particles are optically resolv able, while at the same time particle surface density is maxi mized. Additionally, capture molecule nanodots that are similar Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access ...

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in fluorescence contrast. Here we show that membrane microdomains in giant unilamellar vesicles (GUVs) can be visualized with europium luminescence using a complex of europium (III) and tetracycline (EuTc). EuTc is unlike typical organic lipid probes in that it has a unique excitation/emission wavelength combination (396/617 nm), a very large Stokes shift (221 nm), and a very narrow emission bandwidth (8 nm). The probe preferentially interacts with liquid disordered domains in GUVs, which results in intensity contrast across the surface of phase-separated GUVs. Interestingly, EuTc also disrupts GM1 partitioning. After labeling phase-separated GUVs with EuTc, CTxB, which binds GM1, labels disordered domains. We hypothesize that this is due to the ability of EuTc to increase lipid membrane order, which we demonstrate by showing that EuTc significantly reduces lipid diffusion coefficients. We conclude that EuTc, and potentially other tetracycline derivatives complexed with metal ions, disrupt membrane organization of lipid bilayer membranes, which may have implications on the antibacterial activity of the tetracycline family of antibiotics.

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