Cholesterol reduction by immunization with a PCSK9 mimic.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a plasma protein that controls cholesterol homeostasis. Here, we design a human PCSK9 mimic, named HIT01, with no consecutive 9-residue stretch in common with any human protein as a potential heart attack vaccine. Murine immunizations with HIT01 reduce low-density lipoprotein (LDL) and cholesterol levels by 40% and 30%, respectively. Immunization of cynomolgus macaques with HIT01-K21Q-R218E, a cleavage-resistant variant, elicits high-titer PCSK9-directed antibody responses and significantly reduces serum levels of cholesterol 2 weeks after each immunization. However, HIT01-K21Q-R218E immunizations also increase serum PCSK9 levels by up to 5-fold, likely due to PCSK9-binding antibodies altering the half-life of PCSK9. While vaccination with a PCSK9 mimic can induce antibodies that block interactions of PCSK9 with the LDL receptor, PCSK9-binding antibodies appear to alter homeostatic levels of PCSK9, thereby confounding its vaccine impact. Our results nevertheless suggest a mechanism for increasing the half-life of soluble regulatory factors by vaccination.

Development of chemical tools based on GSK-7975A to study store-operated calcium entry in cells.

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release-activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.

Enhancing Anti-SARS-CoV-2 Neutralizing Immunity by Genetic Delivery of Enveloped Virus-like Particles Displaying SARS-CoV-2 Spikes.

New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit-DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines.

B-cell epitope discovery: The first protein flexibility-based algorithm-Zika virus conserved epitope demonstration.

Antibody-antigen interaction-at antigenic local environments called B-cell epitopes-is a prominent mechanism for neutralization of infection. Effective mimicry, and display, of B-cell epitopes is key to vaccine design. Here, a physical approach is evaluated for the discovery of epitopes which evolve slowly over closely related pathogens (conserved epitopes). The approach is 1) protein flexibility-based and 2) demonstrated with clinically relevant enveloped viruses, simulated via molecular dynamics. The approach is validated against 1) seven structurally characterized enveloped virus epitopes which evolved the least (out of thirty-nine enveloped virus-antibody structures), 2) two structurally characterized non-enveloped virus epitopes which evolved slowly (out of eight non-enveloped virus-antibody structures), and 3) eight preexisting epitope and peptide discovery algorithms. Rationale for a new benchmarking scheme is presented. A data-driven epitope clustering algorithm is introduced. The prediction of five Zika virus epitopes (for future exploration on recombinant vaccine technologies) is demonstrated. For the first time, protein flexibility is shown to outperform solvent accessible surface area as an epitope discovery metric.

Assessment of Crosslinkers between Peptide Antigen and Carrier Protein for Fusion Peptide-Directed Vaccines against HIV-1.

Conjugate-vaccine immunogens require three components: a carrier protein, an antigen, and a crosslinker, capable of coupling antigen to carrier protein, while preserving both T-cell responses from carrier protein and B-cell responses from antigen. We previously showed that the N-terminal eight residues of the HIV-1 fusion peptide (FP8) as an antigen could prime for broad cross-clade neutralizing responses, that recombinant heavy chain of tetanus toxin (rTTHC) as a carrier protein provided optimal responses, and that choice of crosslinker could impact both antigenicity and immunogenicity. Here, we delve more deeply into the impact of varying the linker between FP8 and rTTHC. In specific, we assessed the physical properties, the antigenicity, and the immunogenicity of conjugates for crosslinkers ranging in spacer-arm length from 1.5 to 95.2 Å, with varying hydrophobicity and crosslinking-functional groups. Conjugates coupled with different degrees of multimerization and peptide-to-rTTHC stoichiometry, but all were well recognized by HIV-fusion-peptide-directed antibodies VRC34.01, VRC34.05, PGT151, and ACS202 except for the conjugate with the longest linker (24-PEGylated SMCC; SM(PEG)24), which had lower affinity for ACS202, as did the conjugate with the shortest linker (succinimidyl iodoacetate; SIA), which also had the lowest peptide-to-rTTHC stoichiometry. Murine immunizations testing seven FP8-rTTHC conjugates elicited fusion-peptide-directed antibody responses, with SIA- and SM(PEG)24-linked conjugates eliciting lower responses than the other five conjugates. After boosting with prefusion-closed envelope trimers from strains BG505 clade A and consensus clade C, trimer-directed antibody-binding responses were lower for the SIA-linked conjugate; elicited neutralizing responses were similar, however, though statistically lower for the SM(PEG)24-linked conjugate, when tested against a strain especially sensitive to fusion-peptide-directed responses. Overall, correlation analyses revealed the immunogenicity of FP8-rTTHC conjugates to be negatively impacted by hydrophilicity and extremes of length or low peptide-carrier stoichiometry, but robust to other linker parameters, with several commonly used crosslinkers yielding statistically indistinguishable serological results.

Optimized photoluminescence quantum yield in upconversion composites considering the scattering, inner-filter effects, thickness, self-absorption, and temperature.

Optimizing upconversion (UC) composites is challenging as numerous effects influence their unique emission mechanism. Low scattering mediums increase the number of dopants excited, however, high scattering mediums increase the UC efficiency due to its non-linear power dependency. Scattering also leads to greater thermal effects and emission saturation at lower excitation power density (PD). In this work, a photoluminescence quantum yield (PLQY) increase of 270% was observed when hexagonal NaYF4:(18%)Yb3+,(2%)Er3+ phosphor is in air compared to a refractive index-matched medium. Furthermore, the primary inner-filter effect causes a 94% PLQY decrease when the excitation focal point is moved from the front of the phosphor to 8.4 mm deep. Increasing this effect limits the maximum excitation PD, reduces thermal effects, and leads to emission saturation at higher excitation PDs. Additionally, self-absorption decreases the PLQY as the phosphor's thickness increases from 1 to 9 mm. Finally, in comparison to a cuboid cuvette, a 27% PLQY increase occurs when characterizing the phosphor in a cylindrical cuvette due to a lensing effect of the curved glass, as supported by simulations. Overall, addressing the effects presented in this work is necessary to both maximize UC composite performance as well as report their PLQY more reliably.

Critical Power Density: A Metric To Compare the Excitation Power Density Dependence of Photon Upconversion in Different Inorganic Host Materials.

In photon upconversion (UC) based on triplet-triplet annihilation, the upconversion photoluminescent quantum yield (UC-PLQY) depends on the excitation power density in a way that can be described by a single figure of merit. This figure of merit, the threshold value, allows the excitation power density required for efficient UC-PLQY to be compared between different triplet-triplet annihilation systems. Here, we investigate the excitation power density dependence of two-photon UC processes in a series of four lanthanide-doped inorganic host materials (oxides, fluorides, and chlorides) all doped with 18 mol % Yb3+ sensitizer ions and 2 mol % Er3+ activator ions. We demonstrate that an analogous figure of merit, which we call the critical power density (CPD), accurately describes the UC power dependence of these samples. Better CPD values are obtained when the lifetime of the intermediate states is long. The UC-PLQY at the CPD is linked to the saturation UC-PLQY. Thus, a measurement of the UC-PLQY at this low power density can be used to estimate the theoretical saturation UC-PLQY in the absence of deleterious effects such as laser-induced heating. This is compared to another method to estimate the saturation based on the CPD model, namely, taking half of the level's PLQY under direct excitation. Our careful analysis of the upconversion spectra as a function of excitation power density gives several insights into the differing upconversion pathways in the hosts and proves to be a useful tool for their comparison.

New Insights in 4f(12)5d(1) Excited States of Tm(2+) through Excited State Excitation Spectroscopy.

Optical excitation of ions or molecules typically leads to an expansion of the equilibrium bond lengths in the excited electronic state. However, for 4f(n-1)5d(1) excited states in lanthanide ions both expansion and contraction relative to the 4f(n) ground state have been reported, depending on the crystal field and nature of the 5d state. To probe the equilibrium distance offset between different 4f(n-1)5d(1) excited states, we report excited state excitation (ESE) spectra for Tm(2+) doped in CsCaBr3 and CsCaCl3 using two-color excited state excitation spectroscopy. The ESE spectra reveal sharp lines at low energies, confirming a similar distance offset for 4f(n-1)5d(t2g)(1) states. At higher energies, broader bands are observed, which indicate the presence of excited states with a different offset. On the basis of ab initio embedded-cluster calculations, the broad bands are assigned to two-photon d-d absorption from the excited state. In this work, we demonstrate that ESE is a powerful spectroscopic tool, giving access to information which cannot be obtained through regular one-photon spectroscopy.

Plasmon enhanced upconversion luminescence near gold nanoparticles--simulation and analysis of the interactions: errata.

The procedure used in our previous publication [Opt. Express 20, 271, (2012)] to calculate how coupling to a spherical gold nanoparticle changes the upconversion luminescence of Er(3+) ions contained several errors. The errors are corrected here.

Plasmon enhanced upconversion luminescence near gold nanoparticles-simulation and analysis of the interactions.

We investigate plasmon resonances in gold nanoparticles to enhance the quantum yield of upconverting materials. For this purpose, we use a rate equation model that describes the upconversion of trivalent erbium based upconverters. Changes of the optical field acting on the upconverter and the changes to the transition probabilities of the upconverter in the proximity of a gold nanoparticle are calculated using Mie theory and exact electrodynamic theory respectively. With this data, the influence on the luminescence of the upconverter is determined using the rate equation model. The results show that upconversion luminescence can be increased in the proximity of a spherical gold nanoparticle due to the change in the optical field and the modification of the transition rates.

Generation of one light-induced metastable nitrosyl linkage isomer in [Pt(NH3)4Cl(NO)]Cl2 in the red spectral range.

One metastable linkage nitrosyl isomer can be generated in [Pt(NH(3))(4)Cl(NO)]Cl(2) by irradiation with light in the red spectral range. The potential energy barrier for the thermal relaxation of the metastable state to the ground state has an amount of E(A) = (0.27 +/- 0.03) eV. The decay follows the Arrhenius law and E(A) is independent of temperature. At room temperature the metastable state has a lifetime of tau = 3.8 x 10(-5) s after generation by pulsed laser illumination. Below T = 100 K about 30% linkage NO isomers can be generated in a powder sample by irradiation with lambda = 658 nm. DFT calculations demonstrate the rotation of the NO ligand from Pt-N-O to Pt-O-N as a unique linkage isomer. Consequently, only one new nu(NO) stretching vibration is detected with a shift from 1673 cm(-1) to 1793 cm(-1) by 120 cm(-1), to higher frequencies in good agreement with the DFT calculations. In the metastable state new electronic absorption bands are observed in the blue-green and near infrared spectral range. The metastable state can be optically accessed via a (5d + pi(NO)) -->pi*(NO) transition. [Pt(NH(3))(4)Cl(NO)]Cl(2) is diamagnetic with a Pt(5d(8)) configuration and thus represents the first {MNO}(8) complex with experimental evidence for a light-induced nitrosyl linkage isomer.

Long-lived light-induced metastable states in trans-[Ru(NH3)4(H2O)NOCl3 x H2O and related compounds.

The existence of two light-induced long-lived metastable states SI, SII in irradiated trans-[Ru(NH3)4(H2O)NO]Cl3 x H2O and trans-[Ru(NH3)4(OH)NO]Cl2 is revealed by differential scanning calorimetry measurements and calculations based on density functional theory. Irradiation with light in the blue spectral range leads to the population of SI, while SII can be obtained by transferring SI into SII with irradiation of light in the near infrared spectral range. The population and transfer of the metastable states is described by exponential functions and the thermal decays are evaluated according to Arrhenius' law, yielding activation energies of EA(SI) = 0.95(3) eV, EA(SII) = 0.69(3) eV and frequency factors of Z(SI) = 2 x 10(14) s(-1), Z(SII) = 3 x 10(13) s(-1) for trans-[Ru(NH3)4(H2O)NO]Cl3 x H2O, while EA(SI) = 0.91(3) eV, EA(SII) = 0.60(3) eV, Z(SI) = 6 x 10(14) s(-1), Z(SII) = 1 x 10(13) s(-1) for trans-[Ru(NH3)4(OH)NO]Cl2. The observations are compared with the ground state potential surface calculated by density functional theory, where the metastable states correspond to a side-on bonded (SII) and isonitrosyl (SI) configuration of the NO ligand. The calculations provide the energetic minima of the ground state and the metastable states SI and SII as well as the saddle points along the reaction coordinate Q, which corresponds roughly to a rotation of the NO ligand by about 90 degrees (SII) and 180 degrees (SI), and therefore allows for the comparison between observed and calculated activation energies.

Cooperative radiative and nonradiative effects in K2NaScF6 codoped with V3+ and Er3+.

K2NaScF6 crystals codoped with V3+ and Er3+ exhibit some novel cooperative near-IR to visible upconversion processes at cryogenic temperatures. V3+ mainly acts as a broadband sensitizer. The V3+ 3T1g --> 3T2g excitation between 13,500 and 15,500 cm(-1), after fast relaxation to V3+ 1T2g, can be transferred to Er3+ 4I(11/2), and then upconversion takes place. Four upconversion mechanisms are identified and characterized. For narrow-band laser excitation the overall efficiency of the upconversion processes is low. However, at 12 K for broadband excitation, such as in a lamp, between 12,000 and 14,500 cm(-1) the number of emitted visible photons is roughly doubled by codoping V3+ in addition to Er3+.

Increasing the crystallisation temperature to access new spin clusters: conversion of [Ni8(cit)6(OH)2(H2O)2]10- to [Ni8(cit)6(OH)2]10-.

The role of temperature in the formation of high nuclearity nickel(II) citrate spin clusters is explored, revealing how changes in structure and hence magnetic properties can be triggered through desolvation and ligand reorganisation.