A biophysical approach to the identification of novel ApoE chemical probes

Alzheimer’s disease (AD) is the most common type of dementia and, after age, the greatest risk factor for developing AD is the allelic variation of apolipoprotein E (ApoE), with homozygote carriers of the ApoE4 allele having an up to 12-fold greater risk of developing AD than noncarriers. Apolipoprotein E exists as three isoforms that differ in only two amino acid sites, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). These amino acid substitutions are assumed to alter ApoE structure and function, and be responsible for the detrimental effects of ApoE4 via a mechanism that remains unclear. The hypothesis that a structural difference between ApoE4 and ApoE3 (and ApoE2) is the cause of the ApoE4-associated increased risk for AD forms the basis of a therapeutic approach to modulate ApoE4 structure, and we were therefore interested in screening to identify new chemical probes for ApoE4. In this regard, a high-yield protocol was developed for the expression and purification of recombinant full-length ApoE, and three diverse biophysical screening assays were established and characterized; an optical label-free assay (Corning Epic) for hit identification and microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) as orthogonal assays for hit confirmation. The 707 compounds in the National Institute of Health clinical collection were screened for binding to ApoE4, from which six confirmed hits, as well as one analogue, were identified. Although the compounds did not differentiate between ApoE isoforms, these data nevertheless demonstrate the feasibility of using a biophysical approach to identifying compounds that bind to ApoE and that, with further optimization, might differentiate between isoforms to produce a molecule that selectively alters the function of ApoE4

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state.

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism

Additive manufacturing of cellular materials with tailored properties

The ability to pattern complex materials with high-speed and low-cost three-dimensional (3D) printing techniques is highly desirable. Here, we present progress on developing siloxane-based feedstock formulations, known as “inks,” for a unique 3D printing approach called Direct Ink Writing (DIW). DIW is a low-cost, mask-less printing route that enables rapid design and patterning of planar and three-dimensional (3D) microstructures. In this filamentary printing approach, a concentrated ink with tailored viscoelastic properties is deposited through a micro-nozzle that is translated using a multi-axis positioning stage. The ink rapidly solidifies as it is extruded so that 3D structures with fine features may be built up in a layer-by-layer fashion. We introduce the concept of tailoring the macro-scale mechanical properties by designing the 3D micro-architecture of the printed cellular silicone materials. We show the ability to obtain highly uniform or graded properties by simply adjusting the pattern design. Moreover, by understanding the materials-structure-processing property relationships, we have created a modeling-design-fabrication approach to achieve tailored mechanical properties. For example, we have created porous architectures that, in one case, are well suited for pure compression and, in a separate case, are better suited for shear environments. We expect that the ability to deterministically program mechanical performance from part-to-part and within a part will prove useful for many applications

One-step volumetric additive manufacturing of complex polymer structures.

Two limitations of additive manufacturing methods that arise from layer-based fabrication are slow speed and geometric constraints (which include poor surface quality). Both limitations are overcome in the work reported here, introducing a new volumetric additive fabrication paradigm that produces photopolymer structures with complex nonperiodic three-dimensional geometries on a time scale of seconds. We implement this approach using holographic patterning of light fields, demonstrate the fabrication of a variety of structures, and study the properties of the light patterns and photosensitive resins required for this fabrication approach. The results indicate that low-absorbing resins containing ~0.1% photoinitiator, illuminated at modest powers (~10 to 100 mW), may be successfully used to build full structures in ~1 to 10 s

MODELING APPROACHES IN ELECTROPHORETIC DEPOSITION

Electrophoretic deposition (EPD) occurs when electric-field-driven colloidal particles suspended in a fluid migrate toward an electrode or liquid−liquid interface where they assemble into a deposit. The deposition rate depends on many parameters, such as the applied field strength, volume fraction of colloids, and electrophoretic mobility. Enhanced control of the shape, composition, and performance of functional materials fabricated via EPD can benefit from computationally feasible models that predict transient formation and resulting morphology of colloidal depositions. Here we discuss a broad range of EPD modeling approaches, their applicability and predictive capabilities. The majority of available models provide continuum level descriptions of EPD [1] that inherently neglect inter-colloidal interactions. Such models predict mass deposition rates that depend (at least) on the electrophoretic velocity, electrode surface area, and fraction of colloids that stick to the deposit. Film thickness models suggest nonuniform deposits occur when the colloidal particle permittivity exceeds that of the suspension or near electrode edges where electric field singularities locally enhance deposition rates. Alternative particle level modeling of EPD is still nascent, but promises to offer more detailed predictive information about deposit formation and packing morphology than traditional approaches. To this end, we also present and evaluate a particle-based model of colloidal suspensions that undergo electrophoretic motion and deposition [2] using an extensive set of mesoscale simulations that characterize experimentally relevant colloidal suspensions. Since the model explicitly computes inter-colloidal interactions, it is uniquely poised to elucidate how deposition conditions influence defect structures and particle rearrangement within EPD colloidal crystals. We use the model to investigate how empirical parameters, such as electric field strength and electrolyte concentration, can be tuned in order to control the degree of colloidal ordering versus non-ordering that occurs during EPD. It is straightforward to configure the model to study how various preparations of the interface, e.g. a bare surface, a lattice of particles, an amorphous monolayer, etc., and also annealing schemes influence the deposit microstructure. [1] Ferrari, B.; Moreno, R. EPD Kinetics: A Review. Journal of European Ceramic Society. 2010 (5), pp 1069-1078. [2] Giera, B.; Zepeda-Ruiz, L. A.; Pascall, A. J.; Weisgraber, T. H. Mesoscale Particle-Based Model of Electrophoretic Deposition. Langmuir. 10.1021/acs.langmuir.6b04010 This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA2734

Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan.

Abstract Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitrowith the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined thein vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE·DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs

Impact of the apolipoprotein E (epsilon) genotype on cardiometabolic risk markers and responsiveness to acute and chronic dietary fat manipulation

Apolipoprotein (APO) E (ε) genotype is considered to play an important role in lipid responses to dietary fat manipulation but the impact on novel cardiometabolic risk markers is unclear. To address this knowledge gap, we investigated the relationship between the APOE genotype and cardiometabolic risk markers in response to acute and chronic dietary fat intakes. Associations with fasting (baseline) outcome measures (n = 218) were determined using data from the chronic DIVAS (n = 191/195 adults at moderate cardiovascular disease risk) and acute DIVAS-2 (n = 27/32 postmenopausal women) studies examining the effects of diets/meals varying in saturated, polyunsaturated and monounsaturated (MUFA) fatty acid composition. Participants were retrospectively genotyped for APOE (rs429358, rs7412). For baseline cardiometabolic outcomes, E4 carriers had higher fasting total and low-density lipoprotein-cholesterol (LDL-C), total cholesterol: high-density lipoprotein-cholesterol (HDL-C) and LDL-C: HDL-C ratios, but lower C-reactive protein (CRP) than E3/E3 and E2 carriers (p ≤ 0.003). Digital volume pulse stiffness index was higher in E2 carriers than the E3/E3 group (p = 0.011). Following chronic dietary fat intake, the significant diet × genotype interaction was found for fasting triacylglycerol (p = 0.010), with indication of a differential responsiveness to MUFA intake between the E3/E3 and E4 carriers (p = 0.006). Test fat × genotype interactions were observed for the incremental area under the curve for the postprandial apolipoprotein B (apoB; p = 0.022) and digital volume pulse reflection index (DVP-RI; p = 0.030) responses after the MUFA-rich meals, with a reduction in E4 carriers and increase in the E3/E3 group for the apoB response, but an increase in E4 carriers and decrease in the E3/E3 group for the DVP-RI response. In conclusion, baseline associations between the APOE genotype and fasting lipids and CRP confirm previous findings, although a novel interaction with digital volume pulse arterial stiffness was observed in the fasted state and differential postprandial apoB and DVP-RI responses after the MUFA-rich meals. The reported differential impact of the APOE genotype on cardiometabolic markers in the acute and chronic state requires confirmation

Quantifying the potential exposure hazard due to energetic releases of CO2 from a failed sequestration well

AbstractWells are designed to bring fluids from depth to the earth’s surface quickly. As such they are the most likely pathway for CO2 to return to the surface in large quantities and present a hazard without adequate management. We surveyed oil industry experience of CO2 well failures, and separately, calculated the maximal CO2 flow rate from a 5000 ft depth supercritical CO2 reservoir. The calculated maximum of 20,000 tonne/day was set by the sound speed and the seven-inch well casing diameter, and was greater than any observed event. We used this flux to simulate atmospheric releases and the associated hazard utilizing the National Atmospheric Release Advisory Center (NARAC) tools and real meteorology at a representative location in the High Plains of the United States. Three cases representing a maximum hazard day (quiet winds <1 m s−1 near the wellhead) and medium and minimal hazard days (average winds 3 m s−1 and 7 m s−1) were assessed. As expected for such large releases, there is a near-well hazard when there is little or no wind. In all three cases the hazardous Temporary Emergency Exposure Levels (TEEL) 2 or 3 only occurred within the first few hundreds of meters. Because the preliminary 3-D model runs may not have been run at high enough resolution to accurately simulate very small distances, we also used a simple Gaussian plume model to provide an upper bound on the distance at which hazardous conditions might exist. This extremely conservative model, which ignores inhomogeneity in the mean wind and turbulence fields, also predicts possible hazardous concentrations up to several hundred meters downwind from a maximal release

Apolipoprotein E associated with reconstituted high-density lipoprotein-like particles is protected from aggregation.

Apolipoprotein E (APOE) genotype determines Alzheimer\u27s disease (AD) susceptibility, with the APOE ε4 allele being an established risk factor for late-onset AD. The ApoE lipidation status has been reported to impact amyloid-beta (Aβ) peptide metabolism. The details of how lipidation affects ApoE behavior remain to be elucidated. In this study, we prepared lipid-free and lipid-bound ApoE particles, mimicking the high-density lipoprotein particles found in vivo, for all three isoforms (ApoE2, ApoE3, and ApoE4) and biophysically characterized them. We find that lipid-free ApoE in solution has the tendency to aggregate in vitro in an isoform-dependent manner under near-physiological conditions and that aggregation is impeded by lipidation of ApoE