Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD.

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.

Lyophilization of Adjuvanted Vaccines: Methods for Formulation of a Thermostable Freeze-Dried Product.

Lyophilization of vaccines is advantageous for the distribution and storage of thermally labile products, particularly in regions where cold chain management is difficult. To date, current lyophilized vaccines do not contain an adjuvant. Instead, adjuvanted vaccines may be presented as a two vial system, that require bedside-mixing prior to immunization. Here we present an example of a lyophilization cycle that we have used to successfully freeze-dry an adjuvanted protein formulation in a single vial.

Particle Sizing of Nanoparticle Adjuvant Formulations by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA).

Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are two orthogonal and complementary methods of measuring size of particles in a sample. These technologies use the theory of Brownian motion by analyzing the random changes of light intensity scattered by particles in solution. Both techniques can be used to characterize particle size distribution of proteins and formulations in the nanometer to low micron range.Each method has benefits over the other. DLS is a quick and simple measurement that is ideal for monodisperse particles and can also analyze a distribution of particles over a wide range of sizes. NTA provides a size distribution that is less susceptible to the influence of a few large particles, and has the added benefit of being able to measure particle concentration. Here we describe methods for measuring the particle size and concentration of an oil-in-water nanoemulsion.

Staining and Transfer Techniques for SDS-PAGE Gels to Minimize Oil-in-Water Emulsion Adjuvant Interference.

Adjuvants in modern vaccines boost and shape immune responses and allow for antigen dose-sparing. Analysis of protein antigens in the presence of adjuvants can prove challenging, especially if the adjuvant interferes with visualization of the protein band on an SDS-PAGE gel. In this chapter, a variety of different techniques are presented to mitigate the interference of a nanoemulsion adjuvant, GLA-SE, with different recombinant proteins of varying molecular weight by addressing sample preparation and staining methods.

Interactions Between Antigens and Nanoemulsion Adjuvants: Separation and Characterization Techniques.

Determining the association of vaccine components in a formulation is of interest for designing and optimizing well characterized vaccines. Three methods are described to assess interactions between protein antigens and oil-in-water nanoemulsion adjuvants. The methods include (1) ultracentrifugation to measure free versus adjuvant-associated protein, (2) size exclusion chromatography (SEC) to qualitatively assess existing interactions, and (3) Native PAGE as a means to visualize the formulation run in its native state on a polyacrylamide gel. As with many techniques, the methods alone are not definitive, but data from multiple orthogonal assays can provide a more complete picture of protein-adjuvant interactions.

Ethnic differences in physical pain sensitivity: role of acculturation.

Although research suggests that Asian Americans are more reactive to physical pain than European Americans, some evidence suggests that the observed differences in ethnicity may actually reflect Asian Americans' differing levels of acculturation. Two studies were conducted to test this hypothesis. In Study 1, first- and second-generation Asian Americans and European Americans took part in a cold pressor task. Evidence of heightened pain responses was found only among first-generation Asian Americans. Study 2 further controlled for ethnicity and replicated this pattern in finding heightened pain reactions among mainland Chinese students in Hong Kong relative to Hong Kong Chinese students. These findings suggest a role for acculturation in accounting for ethnic differences in physical pain sensitivity.

Regenerable DNA-functionalized hydrogels for ultrasensitive, instrument-free mercury(II) detection and removal in water.

Mercury is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, new materials are required to not only detect but also effectively remove mercury from environmental sources such as water. We herein describe a polyacrylamide hydrogel-based sensor functionalized with a thymine-rich DNA that can simultaneously detect and remove mercury from water. Detection is achieved by selective binding of Hg(2+) between two thymine bases, inducing a hairpin structure where, upon addition of SYBR Green I dye, green fluorescence is observed. In the absence of Hg(2+), however, addition of the dye results in yellow fluorescence. Using the naked eye, the detection limit in a 50 mL water sample is 10 nM Hg(2+). This sensor can be regenerated using a simple acid treatment and can remove Hg(2+) from water at a rate of approximately 1 h(-1). This sensor was also used to detect and remove Hg(2+) from samples of Lake Ontario water spiked with mercury. In addition, these hydrogel-based sensors are resistant to nuclease and can be rehydrated from dried gels for storage and DNA protection. Similar methods can be used to functionalize hydrogels with other nucleic acids, proteins, and small molecules for environmental and biomedical applications.