A Rational Design Approach to Developing Second Generation Fabry Disease Treatments

Fabry disease is an X-linked lysosomal storage disorder that affects approximately 1 in 40,000 males in its classical form and as many as 1:4,600 in its late-onset form [1]. The disease is caused by mutations in the gene encoding α- galactosidase (α-GAL), which results in deficient levels of α-GAL activity in the lysosomes of patients [2, 3]. This lack of enzymatic activity causes macromolecular substrates to accumulate in tissues, and can result in a wide range of symptoms such as impaired renal and cardiac function [4]. The severity of disease is linked to the amount of residual enzyme activity [5, 6]. Mutations resulting in little to no residual activity lead to the more severe classical form of the disease, whereas those that retain a fraction of wild-type enzyme levels lead to the less severe late-onset form of the disease. The FDA has approved both enzyme replacement therapy (ERT) and pharmacological chaperone therapy (PCT) for the treatment of Fabry disease. ERT involves the intravenous injection of recombinant α-GAL. The injected enzyme is delivered to the lysosome through the binding of extracellular mannose- 6-phosphate receptors. ERT has been shown to clear accumulated substrate in the majority of tissue types, and has shown to slow the impairment of organ function typically associated with the disease [7, 8]. Despite being broadly efficacious for the treatment of both forms of Fabry disease, additional treatment options were developed. PCT recently received approval for the treatment of a select number of Fabry patients. The treatment involves the oral administration of Galafold™, a small molecule also known as 1-deoxygalactonojirimycin (DGJ). DGJ is a potent competitive inhibitor of α-GAL, and acts by stabilizing mutant forms of α-GAL as they traffic to the lysosome [9, 10]. Only a portion of Fabry patients are approved to receive this treatment [11]. These patients possess one of the 348 genetic mutations for which this treatment has been found applicable. The majority of these patients have the late-onset form of the disease and possess some level of residual α-GAL activity. Despite the success of PCT and ERT at treating a broad number of patients there still exists a number of shortcomings associated with either treatment. One such shortcoming is the immunogenicity associated with the recombinant ERT enzymes. 88% of patients receiving ERT develop immune responses including both IgG and IgE based reactions [12-14]. It is our belief that the immune response is present because classical Fabry patients produce no correctly folded α-GAL, and when correctly folded α-GAL is presented to the immune system it is treated as foreign. This results in immune response and is likely to trigger the formation of neutralizing antibodies. In order to bypass this issue, we interconverted the active sites of α-GAL and a highly homologous human enzyme, α-NAGAL. The engineered enzymes possess lower catalytic efficiency than the wild-type counterparts, but have acquired the selectivity of their counterparts. We confirmed this through enzymatic characterization and through x-ray crystallography. Most importantly we showed that enzymes retain their native antigenicity. By engineering novel functionality into previously existing protein scaffolds we have highlighted a rational approach to engineering less immunogenic therapeutics. In addition to possessing undesirable immunogenic properties, the efficiency of ERT in the clearance of substrate in podocyte cells has come into question [15, 16]. These cells play a critical role in the function of the glomerular filtration barrier and ultimately kidney function. Due to the function of these cells, they form the third layer of filtration as well as performing vital role in the structure of glomerular capillaries, they are naturally a problematic cell type for serum-circulated ERT molecules to reach. Permeability of this barrier has been shown to be dependent on charge and molecular diameter, with permeability having an inverse relationship with molecular diameter. By engineering mutations that disrupt the dimer interface of α-GAL we have successfully expressed and purified a monomeric form of the enzyme, which possesses slightly lower than wild-type levels of activity and stability. The monomeric α-GAL may provide an excellent point of entry towards engineering a more potent ERT molecule for the treatment of renal variants of Fabry disease. While investigating the selectivity and affinity of α-Gal and α-NAGAL towards galactose and α-N-acetylgalactosamine analogs, we observed that at concentrations similar to those used in clinical trials DGJ significantly inhibits human β-GAL. This observation led us to further investigate the effect of α-linked functional groups attached to the position corresponding to C1 in a galactose scaffold. In DGJ and the 2-acetamido variant, DGJNAc, this position is occupied by a hydrogen. We utilized inhibition assays and x-ray crystallography to probe the effect of certain functional groups at this position on specificity and affinity towards α-GAL or α-NAGAL. We determined that these groups provided little to no change in affinity, but provided increased specificity towards α-selecting active sites. These findings coupled with previous studies performed by members of the Garman lab provide a clear set of guidelines towards developing ligands specific towards either α-GAL α-NAGAL

Exploring COVID-19 Impacts on Maine Tourism Using an Online Photo-Sharing Site

This article explores the impact of the COVID-19 pandemic on travel patterns and visitor spending within Maine using data from the social media site Flickr. The authors compared the geographic information attached to Flickr photo posts in Maine before and after March 2020 when pandemic travel restrictions began to be implemented. Maps show that Maine visitors shifted to more northern, rural, and inland areas. The direct economic impact of these patterns are indicated through the correlation of Flickr photo posts to sales tax data. Lastly, we examined divergent movement patterns and social media use of visitors coming from places with and without travel restrictions. Flickr photo analysis can be a safe, cost-effective, accessible way to help assess policy and develop tourism management strategies, especially useful during a time of rapid change and public health crisis such as the COVID-19 pandemic

Light-Induced Atomic Desorption for loading a Sodium Magneto-Optical Trap

We report studies of photon-stimulated desorption (PSD), also known as light-induced atomic desorption(LIAD), of sodium atoms from a vacuum cell glass surface used for loading a magneto-optical trap (MOT). Fluorescence detection was used to record the trapped atom number and the desorption rate. We observed a steep wavelength dependence of the desorption process above 2.6 eV photon energy, a result significant for estimations of sodium vapor density in the lunar atmosphere. Our data fit well to a simple model for the loading of the MOT dependent only on the sodium desorption rate and residual gas density. Up to 3.7x10^7 Na atoms were confined under ultra-high vacuum conditions, creating promising loading conditions for a vapor cell based atomic Bose-Einstein condensate of sodium.Comment: Sodium LIAD loaded MOT, 7 pages, 5 figures. Revised submitted manuscript with minor corrections, new data presented, Fig.5 change

Dynamic regulation of PU.1 expression in multipotent hematopoietic progenitors

PU.1 is an Ets family transcription factor that is essential for fetal liver hematopoiesis. We have generated a PU.1gfp reporter strain that allowed us to examine the expression of PU.1 in all hematopoietic cell lineages and their early progenitors. Within the bone marrow progenitor compartment, PU.1 is highly expressed in the hematopoietic stem cell, the common lymphoid progenitor, and a proportion of common myeloid progenitors (CMPs). Based on Flt3 and PU.1 expression, the CMP could be divided into three subpopulations, Flt3+ PU.1hi, Flt3− PU.1hi, and Flt3− PU.1lo CMPs. Colony-forming assays and in vivo lineage reconstitution demonstrated that the Flt3+ PU.1hi and Flt3− PU.1hi CMPs were efficient precursors for granulocyte/macrophage progenitors (GMPs), whereas the Flt3− PU.1lo CMPs were highly enriched for committed megakaryocyte/erythrocyte progenitors (MEPs). CMPs have been shown to rapidly differentiate into GMPs and MEPs in vitro. Interestingly, short-term culture revealed that the Flt3+ PU.1hi and Flt3− PU.1hi CMPs rapidly became CD16/32high (reminiscent of GMPs) in culture, whereas the Flt3− PU.1lo CMPs were the immediate precursors of the MEP. Thus, down-regulation of PU.1 expression in the CMP is the first molecularly identified event associated with the restriction of differentiation to erythroid and megakaryocyte lineages

Byzantine Agreement Given Partial Broadcast

This paper considers unconditionally secure protocols for reliable broadcast among a set of n players, where up to t of the players can be corrupted by a (Byzantine) adversary but the remaining h = n - t players remain honest. In the standard model with a complete, synchronous network of bilateral authenticated communication channels among the players, broadcast is achievable if and only if 2n/h < 3. We show that, by extending this model by the existence of partial broadcast channels among subsets of b players, global broadcast can be achieved if and only if the number h of honest players satisfies 2n/h < b + 1. Achievability is demonstrated by protocols with communication and computation complexities polynomial in the size of the network, i.e., in the number of partial broadcast channels. A respective characterization for the related consensus problem is also give

Excavating Awareness and Power in Data Science: A Manifesto for Trustworthy Pervasive Data Research

Frequent public uproar over forms of data science that rely on information about people demonstrates the challenges of defining and demonstrating trustworthy digital data research practices. This paper reviews problems of trustworthiness in what we term pervasive data research: scholarship that relies on the rich information generated about people through digital interaction. We highlight the entwined problems of participant unawareness of such research and the relationship of pervasive data research to corporate datafication and surveillance. We suggest a way forward by drawing from the history of a different methodological approach in which researchers have struggled with trustworthy practice: ethnography. To grapple with the colonial legacy of their methods, ethnographers have developed analytic lenses and researcher practices that foreground relations of awareness and power. These lenses are inspiring but also challenging for pervasive data research, given the flattening of contexts inherent in digital data collection. We propose ways that pervasive data researchers can incorporate reflection on awareness and power within their research to support the development of trustworthy data science

Observed aerosol effects on marine cloud nucleation and supersaturation

Aerosol particles in the marine boundary layer include primary organic and salt particles from sea spray and combustion-derived particles from ships and coastal cities. These particle types serve as nuclei for marine cloud droplet activation, although the particles that activate depend on the particle size and composition as well as the supersaturation that results from cloud updraft velocities. The Eastern Pacific Emitted Aerosol Cloud Experiment (EPEACE) 2011 was a targeted aircraft campaign to assess how different particle types nucleate cloud droplets. As part of E-PEACE 2011, we studied the role of marine particles as cloud droplet nuclei and used emitted particle sources to separate particle-induced feedbacks from dynamical variability. The emitted particle sources included shipboard smoke-generated particles with 0.05-1 μm diameters (which produced tracks measured by satellite and had drop composition characteristic of organic smoke) and combustion particles from container ships with 0.05-0.2 μm diameters (which were measured in a variety of conditions with droplets containing both organic and sulfate components) [1]. Three central aspects of the collaborative E-PEACE results are: (1) the size and chemical composition of the emitted smoke particles compared to ship-track-forming cargo ship emissions as well as background marine particles, with particular attention to the role of organic particles, (2) the characteristics of cloud track formation for smoke and cargo ships, as well as the role of multi-layered low clouds, and (3) the implications of these findings for quantifying aerosol indirect effects. For comparison with the E-PEACE results, the preliminary results of the Stratocumulus Observations of Los-Angeles Emissions Derived Aerosol-Droplets (SOLEDAD) 2012 provided evidence of the cloud-nucleating roles of both marine organic particles and coastal urban pollution, with simultaneous measurements of the effective supersaturations of the clouds in the California coastal region

Secondary Organic Aerosol Coating Formation and Evaporation: Chamber Studies Using Black Carbon Seed Aerosol and the Single-Particle Soot Photometer

We report a protocol for using black carbon (BC) aerosol as the seed for secondary organic aerosol (SOA) formation in an environmental chamber. We employ a single-particle soot photometer (SP2) to probe single-particle SOA coating growth dynamics and find that SOA growth on nonspherical BC aerosol is diffusion-limited. Aerosol composition measurements with an Aerodyne high resolution time-of-flight aerosol mass spectrometer (AMS) confirm that the presence of BC seed does not alter the composition of SOA as compared to self-nucleated SOA or condensed SOA on ammonium sulfate seed. We employ a 3-wavelength photoacoustic soot spectrometer (PASS-3) to measure optical properties of the systems studied, including fullerene soot as the surrogate BC seed, nucleated naphthalene SOA from high-NO_x photooxidation, and nucleated α-pinene SOA from low-NO_x photooxidation. A core-and-shell Mie scattering model of the light absorption enhancement is in good agreement with measured enhancements for both the low- and high-NO_x α-pinene photooxidation systems, reinforcing the assumption of a core-shell morphology for coated BC particles. A discrepancy between measured and modeled absorption enhancement factors in the naphthalene photooxidation system is attributed to the wavelength-dependence of refractive index of the naphthalene SOA. The coating of high-NO_x α-pinene SOA decreases after reaching a peak thickness during irradiation, reflecting a volatility change in the aerosol, as confirmed by the relative magnitudes of f_(43) and f_(44) in the AMS spectra. The protocol described here provides a framework by which future studies of SOA optical properties and single-particle growth dynamics may be explored in environmental chambers