Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a dominant inherited kidney disorder characterized by lipoprotein thrombi in glomerular capillaries. Single-amino-acid mutations in apoE have been associated with the development of the disease, although the mechanism is unknown. In an effort to gain mechanistic insight linking the presence of such mutations and the development of LPG, we evaluated the effects of three of the most common apoE3 variants associated with this disease, namely R145P(Sendai), R147P(Chicago), and R158P(Osaka or Kurashiki), on the structural and conformational integrity of the protein. All three variants were found to have significantly reduced helical content, to expose a larger portion of hydrophobic surface to the solvent, and to be significantly thermodynamically destabilized, often lacking functionally relevant unfolding intermediates. Furthermore, all variants were aggregation prone and had enhanced sensitivity to protease digestion. Finally, although the variants were able to form discoidal lipoprotein particles, discrete subpopulations of poorly formed or aberrant particles were evident. Furthermore, these lipoprotein particles were thermodynamically destabilized and aggregation prone. Overall, our data suggest that these mutations induce a generalized unfolding of the N-terminal domain of apoE3 toward a molten-globule-like structure. ApoE3 N-terminal domain unfolding due to mutation may constitute a common mechanism underlying the protein's association with the pathogenesis of LPG.

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT.

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I(-/-) mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-ß-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I(-/-) × apoE(-/-) mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-ß-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1(-/-) or LCAT(-/-) mice. Coexpression of apoA-IV and LCAT in apoA-I(-/-) mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.

Role of the hydrophobic and charged residues in the 218-226 region of apoA-I in the biogenesis of HDL.

We investigated the significance of hydrophobic and charged residues 218-226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I⁻/⁻ mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-ß- and α4-HDL particles. In apoA-I⁻/⁻ × apoE⁻/⁻ mice, the same mutant formed few discoidal and pre-ß-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I⁻/⁻ mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218-222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218-222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-ß particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.

Erratum: Addition to "High On/Off Ratio Carbon Quantum Dot-Chitosan Biomemristors with Coplanar Nanogap Electrodes".

[This corrects the article DOI: 10.1021/acsaelm.2c00979.].

The influence of hydrogenation and oxygen vacancies on molybdenum oxides work function and gap states for application in organic optoelectronics.

Molybdenum oxide is used as a low-resistance anode interfacial layer in applications such as organic light emitting diodes and organic photovoltaics. However, little is known about the correlation between its stoichiometry and electronic properties, such as work function and occupied gap states. In addition, despite the fact that the knowledge of the exact oxide stoichiometry is of paramount importance, few studies have appeared in the literature discussing how this stoichiometry can be controlled to permit the desirable modification of the oxide's electronic structure. This work aims to investigate the beneficial role of hydrogenation (the incorporation of hydrogen within the oxide lattice) versus oxygen vacancy formation in tuning the electronic structure of molybdenum oxides while maintaining their high work function. A large improvement in the operational characteristics of both polymer light emitting devices and bulk heterojunction solar cells incorporating hydrogenated Mo oxides as hole injection/extraction layers was achieved as a result of favorable energy level alignment at the metal oxide/organic interface and enhanced charge transport through the formation of a large density of gap states near the Fermi level.

Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides.

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.

Radiofrequency Schottky Diodes Based on p-Doped Copper(I) Thiocyanate (CuSCN).

Schottky diodes based on inexpensive materials that can be processed using simple manufacturing methods are of particular importance for the next generation of flexible electronics. Although a number of high-frequency n-type diodes and rectifiers have been demonstrated, the progress with p-type diodes is lagging behind, mainly due to the intrinsically low conductivities of existing p-type semiconducting materials that are compatible with low-temperature, flexible, substrate-friendly processes. Herein, we report on CuSCN Schottky diodes, where the semiconductor is processed from solution, featuring coplanar Al-Au nanogap electrodes (<15 nm), patterned via adhesion lithography. The abundant CuSCN material is doped with the molecular p-type dopant fluorofullerene C60F48 to improve the diode's operating characteristics. Rectifier circuits fabricated with the doped CuSCN/C60F48 diodes exhibit a 30-fold increase in the cutoff frequency as compared to pristine CuSCN diodes (from 140 kHz to 4 MHz), while they are able to deliver output voltages of >100 mV for a VIN = ±5 V at the commercially relevant frequency of 13.56 MHz. The enhanced diode and circuit performance is attributed to the improved charge transport across CuSCN induced by C60F48. The ensuing diode technology can be used in flexible complementary circuits targeting low-energy-budget applications for the emerging internet of things device ecosystem.

Rapid and up-scalable manufacturing of gigahertz nanogap diodes.

The massive deployment of fifth generation and internet of things technologies requires precise and high-throughput fabrication techniques for the mass production of radio frequency electronics. We use printable indium-gallium-zinc-oxide semiconductor in spontaneously formed self-aligned <10 nm nanogaps and flash-lamp annealing to demonstrate rapid manufacturing of nanogap Schottky diodes over arbitrary size substrates operating in 5 G frequencies. These diodes combine low junction capacitance with low turn-on voltage while exhibiting cut-off frequencies (intrinsic) of >100 GHz. Rectifier circuits constructed with these co-planar diodes can operate at ~47 GHz (extrinsic), making them the fastest large-area electronic devices demonstrated to date.

Emissive Charge-Transfer States at Hybrid Inorganic/Organic Heterojunctions Enable Low Non-Radiative Recombination and High-Performance Photodetectors.

Hybrid devices based on a heterojunction between inorganic and organic semiconductors have offered a means to combine the advantages of both classes of materials in optoelectronic devices, but, in practice, the performance of such devices has often been disappointing. Here, it is demonstrated that charge generation in hybrid inorganic-organic heterojunctions consisting of copper thiocyanate (CuSCN) and a variety of molecular acceptors (ITIC, IT-4F, Y6, PC70 BM, C70 , C60 ) proceeds via emissive charge-transfer (CT) states analogous to those found at all-organic heterojunctions. Importantly, contrary to what has been observed at previous organic-inorganic heterojunctions, the dissociation of the CT-exciton and subsequent charge separation is efficient, allowing the fabrication of planar photovoltaic devices with very low non-radiative voltage losses (0.21 ±  0.02 V). It is shown that such low non-radiative recombination enables the fabrication of simple and cost-effective near-IR (NIR) detectors with extremely low dark current (4 pA cm-2 ) and noise spectral density (3 fA Hz-1/2 ) at no external bias, leading to specific detectivities at NIR wavelengths of just under 1013 Jones, close to the performance of commercial silicon photodetectors. It is believed that this work demonstrates the possibility for hybrid heterojunctions to exploit the unique properties of both inorganic and organic semiconductors for high-performance opto-electronic devices.

Deep Ultraviolet Copper(I) Thiocyanate (CuSCN) Photodetectors Based on Coplanar Nanogap Electrodes Fabricated via Adhesion Lithography.

Adhesion lithography (a-Lith) is a versatile fabrication technique used to produce asymmetric coplanar electrodes separated by a <15 nm nanogap. Here, we use a-Lith to fabricate deep ultraviolet (DUV) photodetectors by combining coplanar asymmetric nanogap electrode architectures (Au/Al) with solution-processable wide-band-gap (3.5-3.9 eV) p-type semiconductor copper(I) thiocyanate (CuSCN). Because of the device's unique architecture, the detectors exhibit high responsivity (≈79 A W-1) and photosensitivity (≈720) when illuminated with a DUV-range (λpeak = 280 nm) light-emitting diode at 220 µW cm-2. Interestingly, the photosensitivity of the photodetectors remains fairly high (≈7) even at illuminating intensities down to 0.2 µW cm-2. The scalability of the a-Lith process combined with the unique properties of CuSCN paves the way to new forms of inexpensive, yet high-performance, photodetectors that can be manufactured on arbitrary substrate materials including plastic.

Hydrogen and nitrogen codoping of anatase TiO2 for efficiency enhancement in organic solar cells.

TiO2 has high chemical stability, strong catalytic activity and is an electron transport material in organic solar cells. However, the presence of trap states near the band edges of TiO2 arising from defects at grain boundaries significantly affects the efficiency of organic solar cells. To become an efficient electron transport material for organic photovoltaics and related devices, such as perovskite solar cells and photocatalytic devices, it is important to tailor its band edges via doping. Nitrogen p-type doping has attracted considerable attention in enhancing the photocatalytic efficiency of TiO2 under visible light irradiation while hydrogen n-type doping increases its electron conductivity. DFT calculations in TiO2 provide evidence that nitrogen and hydrogen can be incorporated in interstitial sites and possibly form NiHi, NiHO and NTiHi defects. The experimental results indicate that NiHi defects are most likely formed and these defects do not introduce deep level states. Furthermore, we show that the efficiency of P3HT:IC60BA-based organic photovoltaic devices is enhanced when using hydrogen-doping and nitrogen/hydrogen codoping of TiO2, both boosting the material n-type conductivity, with maximum power conversion efficiency reaching values of 6.51% and 6.58%, respectively, which are much higher than those of the cells with the as-deposited (4.87%) and nitrogen-doped TiO2 (4.46%).

Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination.

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A(2) (PLA(2)) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C(12)-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA(2). Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA(2). Mass spectrometry showed that only C(12)-NBD-FA, the PLA(2 )hydrolysis product, was detected in the reaction mixture, indicating that PLA(2) recognizes PVA-SbQ/C(12)-NBD-PtdCho as a surface to perform catalysis.

Allele-dependent thermodynamic and structural perturbations in ApoE variants associated with the correction of dyslipidemia and formation of spherical ApoE-containing HDL particles.

Overexpression of ApoE4[Leu261Ala/Trp264Ala/Phe265Ala] mutant (ApoE4mutC) prevents hypertriglyceridemia and promotes formation of spherical ApoE-containing HDL in ApoE(-/-) or ApoA-I(-/-) mice. Although, a similar phenotype was observed with ApoE2[Leu261Ala/Trp264Ala/Phe265Ala] (ApoE2mutC), small differences in cholesterol distribution to IDL/LDL, HDL2 and HDL3 fractions and ApoE distribution to HDL2 and HDL3 fractions suggested that ApoE allelic background can influence mutant ApoE properties. To understand the structural basis behind the properties of ApoE2mutC and ApoE4mutC variants we analyzed their structural and thermodynamic integrity in comparison to their wild-type counterparts. Circular dichroism spectroscopy revealed a significantly reduced helical content for both mutants compared to wild-type. The presence of mutation only marginally affected the thermal stability of ApoE4 but greatly affected the thermal stability profile of ApoE2 leading to a previously uncharacterized intermediate stage. Both ApoE4mutC and ApoE2mutC were slightly stabilized against chemical denaturation compared to their wild-type counterparts. ApoE2mutC, in contrast to ApoE4mutC, exposed a larger hydrophobic surface to the solvent as determined by a fluorescent probe. Both mutants remodeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles with identical kinetics to the wild-type proteins. Given the known conformational differences between ApoE2 and ApoE4, our findings suggest that the 261-265 region may be involved in inter-domain interactions within the ApoE molecule. Overall, we show that substitution of Leu261, Trp264 and Phe265 with Ala in ApoE2 leads to more pronounced perturbations of thermodynamic stability and structure than in ApoE4. The minimal perturbations in ApoE4mutC may make it a more suitable candidate for therapeutic applications for the correction of remnant removal disorders and atheroprotection.

All-organic sulfonium salts acting as efficient solution processed electron injection layer for PLEDs.

Herein we introduce the all-organic triphenylsulfonium (TPS) salts cathode interfacial layers (CILs), deposited from their methanolic solution, as a new simple strategy for circumventing the use of unstable low work function metals and obtaining charge balance and high electroluminescence efficiency in polymer light-emitting diodes (PLEDs). In particular, we show that the incorporation of TPS-triflate or TPS-nonaflate at the polymer/Al interface improved substantially the luminous efficiency of the device (from 2.4 to 7.9 cd/A) and reduced the turn-on and operating voltage, whereas an up to 4-fold increase in brightness (∼11 250 cd/m(2) for TPS-triflate and ∼14 682 cd/m(2) for TPS-nonaflate compared to ∼3221 cd/m(2) for the reference device) was observed in poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-2,1',3-thiadiazole)] (F8BT)-based PLEDs. This was mainly attributed to the favorable decrease of the electron injection barrier, as derived from the open-circuit voltage (Voc) measurements, which was also assisted by the conduction of electrons through the triphenylsulfonium salt sites. Density functional theory calculations indicated that the total energy of the anionic (reduced) form of the salt, that is, upon placing an electron to its lowest unoccupied molecular orbital, is lower than its neutral state, rendering the TPS-salts stable upon electron transfer in the solid state. Finally, the morphology optimization of the TPS-salt interlayer through controlling the processing parameters was found to be critical for achieving efficient electron injection and transport at the respective interfaces.

Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

BACKGROUND: Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent. CONCLUSIONS/SIGNIFICANCE: Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.