Strategy to develop broadly effective multivalent COVID-19 vaccines against emerging variants based on Ad5/35 platform.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron strain has evolved into highly divergent variants with several sub-lineages. These newly emerging variants threaten the efficacy of available COVID-19 vaccines. To mitigate the occurrence of breakthrough infections and re-infections, and more importantly, to reduce the disease burden, it is essential to develop a strategy for producing updated multivalent vaccines that can provide broad neutralization against both currently circulating and emerging variants. We developed bivalent vaccine AdCLD-CoV19-1 BA.5/BA.2.75 and trivalent vaccines AdCLD-CoV19-1 XBB/BN.1/BQ.1.1 and AdCLD-CoV19-1 XBB.1.5/BN.1/BQ.1.1 using an Ad5/35 platform-based non-replicating recombinant adenoviral vector. We compared immune responses elicited by the monovalent and multivalent vaccines in mice and macaques. We found that the BA.5/BA.2.75 bivalent and the XBB/BN.1/BQ.1.1 and XBB.1.5/BN.1/BQ.1.1 trivalent vaccines exhibited improved cross-neutralization ability compared to their respective monovalent vaccines. These data suggest that the developed multivalent vaccines enhance immunity against circulating Omicron subvariants and effectively elicit neutralizing antibodies across a broad spectrum of SARS-CoV-2 variants.

Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1.

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells in vivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRß-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRß-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRß-sufficient Tfh cells. Loss of LXRß confers inactivation of GSK3ß induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/ß-catenin pathway, leading to elevated TCF-1 expression in CD4+ T cells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ T cells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3ß-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.

Functional Characterization of the GNAT Family Histone Acetyltransferase Elp3 and GcnE in Aspergillus fumigatus.

Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a pivotal role in the regulation of gene expression and diverse biological processes. However, the function of GNAT family HATs, especially Elp3, in the opportunistic human pathogenic fungus Aspergillus fumigatus is largely unknown. To investigate the roles of the GNAT family HATs Elp3 and GcnE in the A. fumigatus, we have generated and characterized individual null Δelp3 and ΔgcnE mutants. The radial growth of fungal colonies was significantly decreased by the loss of elp3 or gcnE, and the number of asexual spores (conidia) in the ΔgcnE mutant was significantly reduced. Moreover, the mRNA levels of the key asexual development regulators were also significantly low in the ΔgcnE mutant compared to wild type (WT). Whereas both the Δelp3 and ΔgcnE mutants were markedly impaired in the formation of adherent biofilms, the ΔgcnE mutant showed a complete loss of surface structure and of intercellular matrix. The ΔgcnE mutant responded differently to oxidative stressors and showed significant susceptibility to triazole antifungal agents. Furthermore, Elp3 and GcnE function oppositely in the production of secondary metabolites, and the ΔgcnE mutant showed attenuated virulence. In conclusion, Elp3 and GcnE are associated with diverse biological processes and can be potential targets for controlling the pathogenic fungus.

Waveguide-type Maxwellian near-eye display using a pin-mirror holographic optical element array.

We propose a novel, to the best of our knowledge, waveguide-type optical see-through Maxwellian near-eye display for augmented reality. A pin-mirror holographic optical element (HOE) array enables the Maxwellian view and eye-box replication. Virtual images with deep depth of field are presented by each pin-mirror HOE, alleviating the discrepancy between vergence and accommodation distance. The compact form factor is achieved by the thin waveguide and HOE couplers.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Highly Fluorescent Gold Cluster Assembly.

The photoluminescence (PL) of metal nanoclusters (NCs), originating from their molecule-like electronic structure, is one of the most intriguing properties of NCs. Although various strategies such as tailoring the size, structure, and chemical environment of NCs have shown to improve the PL, their quantum yields (QYs) are still lagging far behind those of conventional luminescent materials, including quantum dots and organic fluorophores. Herein, we report the synthesis of highly luminescent gold cluster assembly (GCA) from Zn2+-ion-mediated assembly of Au4(SRCOO-)4 clusters using mercaptocarboxylic acid as a protective ligand and reductant as well as a growth suppressor. The synergetic combination of unique aurophilic interactions among Au4 clusters and the rigidified chemical environment induced by metal ion chelation through carboxylate groups is responsible for the ultrabright greenish-blue fluorescence with a QY up to 90%. Furthermore, the unique flexibility of dis/reassembly and the aggregation-dependent strong fluorescence of GCA offer a great potential for applications in biodegradable and trackable drug delivery systems.

Waveguide-type see-through dual focus near-eye display with a polarization grating.

Waveguide-type near-eye displays have useful properties such as compact form factor, lightweight and see-through capability. Conventional systems, however, support only a single image plane fixed at a certain distance, which may induce eye fatigue due to the vergence-accommodation conflict. In this paper, we propose a waveguide-type near-eye display with two image planes using a polarization grating. Two images with orthogonal polarizations propagate within the waveguide with different total internal reflection angles and form virtual images at different distances. The use of the polarization grating and two pairs of holographic optical elements enables dual image plane formation by a single waveguide with high transparency for the real scene. Optical experiments confirm the principle of the proposed optical system.

Differentiation of c-Kit+ CD24+ natural killer cells into myeloid cells in a GATA-2-dependent manner.

Myeloid progenitor cells have generally been considered the predominant source of myeloid cells under steady-state conditions. Here we show that NK cells contributed to a myeloid cell lineage pool in naïve and tumor-bearing mice. Using fate tracing of NKp46+ cells, we found that myeloid cells could be derived from NK cells. Notably, among mature CD11b+ CD27+ NK cells, c-Kit+ CD24+ NK cells were capable of differentiating into a range of myeloid lineages in vitro and produced neutrophils and monocytes in vivo. The differentiation was completely inhibited by NK-stimulating cytokines. In addition to the potential for differentiation into myeloid cells, c-Kit+ CD24+ NK cells retained NK cell phenotypes and effector functions. Mechanistically, GATA-2 was necessary for the differentiation of c-Kit+ CD24+ NK cells. Therefore, we discovered that GATA-2-dependent differentiation of c-Kit+ CD24+ NK cells contributes to myeloid cell development and identified a novel pathway for myeloid lineage commitment under physiological conditions.

Characterization of the mbsA Gene Encoding a Putative APSES Transcription Factor in Aspergillus fumigatus.

The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ΔmbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ΔmbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ΔmbsA conidium. Comparative transcriptomic analyses revealed that the ΔmbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ΔmbsA mutant produced higher amount of GT, ΔmbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.

Deep Tumor Penetration of Drug-Loaded Nanoparticles by Click Reaction-Assisted Immune Cell Targeting Strategy.

Nanoparticles have been extensively used to deliver therapeutic drugs to tumor tissues through the extravasation of a leaky vessel via enhanced permeation and retention effect (EPR, passive targeting) or targeted interaction of tumor-specific ligands (active targeting). However, the therapeutic efficacy of drug-loaded nanoparticles is hampered by its heterogeneous distribution owing to limited penetration in tumor tissue. Inspired by the fact that cancer cells can recruit inflammatory immune cells to support their survival, we developed a click reaction-assisted immune cell targeting (CRAIT) strategy to deliver drug-loaded nanoparticles deep into the avascular regions of the tumor. Immune cell-targeting CD11b antibodies are modified with trans-cyclooctene to enable bioorthogonal click chemistry with mesoporous silica nanoparticles functionalized with tetrazines (MSNs-Tz). Sequential injection of modified antibodies and MSNs-Tz at intervals of 24 h results in targeted conjugation of the nanoparticles onto CD11b+ myeloid cells, which serve as active vectors into tumor interiors. We show that the CRAIT strategy allows the deep tumor penetration of drug-loaded nanoparticles, resulting in enhanced therapeutic efficacy in an orthotopic 4T1 breast tumor model. The CRAIT strategy does not require ex vivo manipulation of cells and can be applied to various types of cells and nanovehicles.

Molecular-Level Understanding of Continuous Growth from Iron-Oxo Clusters to Iron Oxide Nanoparticles.

The formation of inorganic nanoparticles has been understood based on the classical crystallization theory described by a burst of nucleation, where surface energy is known to play a critical role, and a diffusion-controlled growth process. However, this nucleation and growth model may not be universally applicable to the entire nanoparticle systems because different precursors and surface ligands are used during their synthesis. Their intrinsic chemical reactivity can lead to a formation pathway that deviates from a classical nucleation and growth model. The formation of metal oxide nanoparticles is one such case because of several distinct chemical aspects during their synthesis. Typical carboxylate surface ligands, which are often employed in the synthesis of oxide nanoparticles, tend to continuously remain on the surface of the nanoparticles throughout the growth process. They can also act as an oxygen source during the growth of metal oxide nanoparticles. Carboxylates are prone to chemical reactions with different chemical species in the synthesis such as alcohol or amine. Such reactions can frequently leave reactive hydroxyl groups on the surface. Herein, we track the entire growth process of iron oxide nanoparticles synthesized from conventional iron precursors, iron-oleate complexes, with strongly chelating carboxylate moieties. Mass spectrometry studies reveal that the iron-oleate precursor is a cluster comprising a tri-iron-oxo core and carboxylate ligands rather than a mononuclear complex. A combinatorial analysis shows that the entire growth, regulated by organic reactions of chelating ligands, is continuous without a discrete nucleation step.

RgsA Attenuates the PKA Signaling, Stress Response, and Virulence in the Human Opportunistic Pathogen Aspergillus fumigatus.

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA, brlA, and wetA are elevated in the ΔrgsA mutant. Moreover, ΔrgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by ΔrgsA. In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA, and the ΔrgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the ΔrgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by ΔrgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation-likely by attenuating PKA signaling.

General and Facile Coating of Single Cells via Mild Reduction.

Cell surface modification has been extensively studied to enhance the efficacy of cell therapy. Still, general accessibility and versatility are remaining challenges to meet the increasing demand for cell-based therapy. Herein, we present a facile and universal cell surface modification method that involves mild reduction of disulfide bonds in cell membrane protein to thiol groups. The reduced cells are successfully coated with biomolecules, polymers, and nanoparticles for an assortment of applications, including rapid cell assembly, in vivo cell monitoring, and localized cell-based drug delivery. No adverse effect on cellular morphology, viability, proliferation, and metabolism is observed. Furthermore, simultaneous coating with polyethylene glycol and dexamethasone-loaded nanoparticles facilitates enhanced cellular activities in mice, overcoming immune rejection.

Characterization of the rax1 gene encoding a putative regulator of G protein signaling in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is the major cause of life threatening invasive aspergillosis, and its small hydrophobic asexual spores (conidia) are the major infection agent. To better understand biology of A. fumigatus, we have characterized the rax1 gene encoding a putative regulator of G protein signaling (RGS). The deletion (Δ) of rax1 results in restricted colony growth and highly reduced number of conidia in A. fumigatus. Transcript levels of the three central activators of asexual development abaA, brlA, and wetA are significantly reduced in the Δrax1 mutant. However, the Δrax1 conidia, but not vegetative cells, are specifically resistant against H2O2 stress. The Δrax1 conidia accumulate higher mRNA levels of sakA encoding a key MAP kinase for stress response. Moreover, the Δrax1 conidia contain over five-fold amount of trehalose, an osmolyte and protein/membrane protectant. Transmission electron microscopy analyses indicate that the Δrax1 conidia have the thicker melanized-outermost cell wall layer compared to those of wild-type. In summary, Rax1 positively controls growth and development, and modulates intracellular trehalose amount, cell wall melanin levels in conidia, and spore resistance to H2O2.

Occlusion handling using angular spectrum convolution in fully analytical mesh based computer generated hologram.

Occlusion handling in computer-generated holography is of vast importance as it enhances depth information by presenting correct motion parallax of the 3D scene within the viewing angle. In this paper, we propose a computationally efficient occlusion handling technique based on a fully analytic mesh based computer generated holography. The proposed technique uses angular spectrum convolution that renders exact occlusion while preserving all other aspects of the fully analytic mesh based computer generated holography. The proposed method is computationally efficient as only a single convolution operation is required for each mesh without numerical propagation between the meshes. The proposed method is also exact as it performs the occlusion processing in the tilted mesh plane, being free from artifacts coming from orthographic spatial masking. The proposed method can be applied to the self and the mutual occlusions between the objects in the 3D scene. The computer simulated results show the feasibility of the proposed method.

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus.

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus.

High-resolution three-photon biomedical imaging using doped ZnS nanocrystals.

Three-photon excitation is a process that occurs when three photons are simultaneously absorbed within a luminophore for photo-excitation through virtual states. Although the imaging application of this process was proposed decades ago, three-photon biomedical imaging has not been realized yet owing to its intrinsic low quantum efficiency. We herein report on high-resolution in vitro and in vivo imaging by combining three-photon excitation of ZnS nanocrystals and visible emission from Mn(2+) dopants. The large three-photon cross-section of the nanocrystals enabled targeted cellular imaging under high spatial resolution, approaching the theoretical limit of three-photon excitation. Owing to the enhanced Stokes shift achieved through nanocrystal doping, the three-photon process was successfully applied to high-resolution in vivo tumour-targeted imaging. Furthermore, the biocompatibility of ZnS nanocrystals offers great potential for clinical applications of three-photon imaging.

Self-assembled Fe3O4 nanoparticle clusters as high-performance anodes for lithium ion batteries via geometric confinement.

Although different kinds of metal oxide nanoparticles continue to be proposed as anode materials for lithium ion batteries (LIBs), their cycle life and power density are still not suitable for commercial applications. Metal oxide nanoparticles have a large storage capacity, but they suffer from the excessive generation of solid-electrolyte interphase (SEI) on the surface, low electrical conductivity, and mechanical degradation and pulverization resulted from severe volume expansion during cycling. Herein we present the preparation of mesoporous iron oxide nanoparticle clusters (MIONCs) by a bottom-up self-assembly approach and demonstrate that they exhibit excellent cyclic stability and rate capability derived from their three-dimensional mesoporous nanostructure. By controlling the geometric configuration, we can achieve stable interfaces between the electrolyte and active materials, resulting in SEI formation confined on the outer surface of the MIONCs.

Consumer preferences for telemedicine devices and services in South Korea.

OBJECTIVE: The scope of healthcare has been expanding from caring for sick people to keeping people from becoming sick, and telemedicine will play a significant role in this new healthcare paradigm. This study investigated consumer preferences and willingness to pay for attributes of telemedicine services in South Korea. A market simulation was conducted to examine the market shares of alternative services and their relationships to the perceived usefulness of service types and preferred device types. MATERIALS AND METHODS: Using a conjoint survey, we collected data on consumer preferences for six telemedicine service attributes. Data analysis used the Bayesian mixed logit model. The market simulation estimated the probabilities of a specific service alternative being chosen using estimated model coefficients. RESULTS: Wearable devices were the most preferred, followed by smart-home and smartphone devices. Consumers perceived managing blood glucose to be the most useful telemedicine service, followed by monitoring oxygen saturation and blood pressure. The market simulation indicated that consumer preferences for device types were associated with the types of chronic diseases for which management through telemedicine services is perceived to be useful. CONCLUSIONS: As the focus of healthcare moves from treating patients to keeping individuals healthy, a key factor for the successful deployment of telemedicine services is understanding consumer perceptions and attitudes. The results of this study revealed the dynamics of consumer preferences with regard to service attributes.

Modifying the Backbone Chemistry of PEG-Based Bottlebrush Block Copolymers for the Formation of Long-Circulating Nanoparticles.

Nanoparticle physicochemical properties have received great attention in optimizing the performance of nanoparticles for biomedical applications. For example, surface functionalization with small molecules or linear hydrophilic polymers is commonly used to tune the interaction of nanoparticles with proteins and cells. However, it is challenging to control the location of functional groups within the shell for conventional nanoparticles. Nanoparticle surfaces composed of shape-persistent bottlebrush polymers allow hierarchical control over the nanoparticle shell but the effect of the bottlebrush backbone on biological interactions is still unknown. The synthesis is reported of novel heterobifunctional poly(ethylene glycol) (PEG)-norbornene macromonomers modified with various small molecules to form bottlebrush polymers with different backbone chemistries. It is demonstrated that micellar nanoparticles composed of poly(lactic acid) (PLA)-PEG bottlebrush block copolymer (BBCP) with neutral and cationic backbone modifications exhibit significantly reduced cellular uptake compared to conventional unmodified BBCPs. Furthermore, the nanoparticles display long blood circulation half-lives of ≈22 hours and enhanced tumor accumulation in mice. Overall, this work sheds light on the importance of the bottlebrush polymer backbone and provides a strategy to improve the performance of nanoparticles in biomedical applications.