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.

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.

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.

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.

A Microfluidic Platform for High-Throughput Screening of Small Mutant Libraries.

The screening and isolation of target microorganisms from mutated recombinant libraries are crucial for the advancement of synthetic biology and metabolic engineering. However, conventional screening tools present several limitations in throughput, cost, and labor. Herein, we describe a novel microfluidic high-throughput screening (HTS) platform with several advantages. The platform utilizes a fluid array to compartmentalize bacterial cells in well-ordered separated microwells and allows long-term cell culture with high throughput. The platform enables the extraction of selected target cells from the fluid array for additional culture and postanalysis by using a capillary-driven sample relocation method. To confirm the feasibility of the platform, we demonstrated two different types of HTS methods based on the levels of reporter gene expression and cellular growth rate difference. For the reporter gene-based HTS, a spike recovery approach was taken to demonstrate that target cells are successfully screened out from a mixture containing nontarget cells by repeating the culture and extraction processes. Additionally, the same platform allowed us to screen and sort target cells according to their cellular growth rate difference, which seems hard in conventional screening methods. Hence, the platform could be used for various microbiological assays, including the detection of cell-excreted metabolites, microbial biosensors, and other HTS systems.

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.

Boosting with variant-matched adenovirus-based vaccines promotes neutralizing antibody responses against SARS-CoV-2 Omicron sublineages in mice.

Global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron subvariants, such as BA.4, BA.5 and XBB.1.5, has been leading the recent wave of coronavirus disease 2019 (COVID-19). Unique mutations in the spike proteins of these emerging Omicron subvariants caused immune evasion from the pre-existing protective immunity induced by vaccination or natural infection. Previously, we developed AdCLD-CoV19-1, a non-replicating recombinant adenoviral vector that encodes the receptor binding domain of the spike protein of the ancestral SARS-CoV-2 strain. Based on the same recombinant adenoviral vector platform, updated vaccines that cover unique mutations found in each Omicron subvariant, including BA.1, BA.2, BA.4.1 and BA.5, were constructed. Preclinical studies revealed that each updated vaccine as a booster shot following primary vaccination targeting the ancestral strain improved neutralizing antibody responses against the pseudovirus of its respective strain most effectively. Of note, boosting with a vaccine targeting the BA.1 or BA.2 Omicron subvariant was most effective in neutralization against the pseudovirus of the BA.2.75 strain, whereas BA.4.1/5-adapted booster shots were most effective in neutralization against the BQ.1, BQ1.1 and BF.7 strains. Therefore, it is imperative to develop a vaccination strategy that can cover the unique spike mutations of currently circulating Omicron subvariants in order to prevent the next wave of COVID-19.

Amorphization induced by focused ion beam milling in metallic and electronic materials.

Focused ion beam (FIB) milling using high-energy gallium ions is widely used in the preparation of specimens for transmission electron microscopy (TEM). However, the energetic ion beam induces amorphization on the edge of specimens during milling, resulting in a mischievous influence on the clearness of high-quality transmission electron micrographs. In this work, the amorphization induced by the FIB milling was investigated by TEM for three kinds of materials, metallic materials in bulk shape, and semiconductive and electronic ceramic materials as a substrate for the deposition of thin films.

The MYST Family Histone Acetyltransferase SasC Governs Diverse Biological Processes in Aspergillus fumigatus.

The conserved MYST proteins form the largest family of histone acetyltransferases (HATs) that acetylate lysines within the N-terminal tails of histone, enabling active gene transcription. Here, we have investigated the biological and regulatory functions of the MYST family HAT SasC in the opportunistic human pathogenic fungus Aspergillus fumigatus using a series of genetic, biochemical, pathogenic, and transcriptomic analyses. The deletion (Δ) of sasC results in a drastically reduced colony growth, asexual development, spore germination, response to stresses, and the fungal virulence. Genome-wide expression analyses have revealed that the ΔsasC mutant showed 2402 significant differentially expressed genes: 1147 upregulated and 1255 downregulated. The representative upregulated gene resulting from ΔsasC is hacA, predicted to encode a bZIP transcription factor, whereas the UV-endonuclease UVE-1 was significantly downregulated by ΔsasC. Furthermore, our Western blot analyses suggest that SasC likely catalyzes the acetylation of H3K9, K3K14, and H3K29 in A. fumigatus. In conclusion, SasC is associated with diverse biological processes and can be a potential target for controlling pathogenic fungi.

Reassessment of the status of Streptomyces setonii and reclassification of Streptomyces fimicarius as a later synonym of Streptomyces setonii and Streptomyces albovinaceus as a later synonym of Streptomyces globisporus based on combined 16S rRNA/gyrB gene sequence analysis.

The 16S rRNA and gyrB genes of 22 Streptomyces strains belonging to the Streptomyces griseus cluster were sequenced, and their taxonomic positions were re-evaluated. For correct analysis, all of the publicly available sequences of the species were collected and compared with those obtained in this study. Species for which no consensus sequence could be identified were excluded from the phylogenetic analysis. The levels of 16S rRNA gene sequence similarity within the cluster ranged from 98.6 to 100% with a mean value of 99.6 ± 0.3%, and those of the gyrB gene ranged from 93.6 to 99.9% with a mean value of 96.3 ± 1.5%. The observed average nucleotide substitution rate of the gyrB gene was ten times higher than that of the 16S rRNA gene, showing a far higher degree of variation. Strains sharing 99.3% or more gyrB sequence similarity (corresponding to an evolutionary distance of 0.0073) always formed monophyletic groups in both trees. Through the combined analysis of the two genes, clear cases of synonymy could be identified and, according to the priority rule, the assertion of the status of Streptomyces setonii as a distinct species and the reclassification of Streptomyces fimicarius as a later synonym of S. setonii and Streptomyces albovinaceus as a later synonym of Streptomyces globisporus are proposed. Emended descriptions of S. setonii and S. globisporus are provided.

The Lysine Demethylases KdmA and KdmB Differently Regulate Asexual Development, Stress Response, and Virulence in Aspergillus fumigatus.

Histone demethylases govern diverse cellular processes, including growth, development, and secondary metabolism. In the present study, we investigated the functions of two lysine demethylases, KdmA and KdmB, in the opportunistic human pathogenic fungus Aspergillus fumigatus. Experiments with mutants harboring deletions of genes encoding KdmA (ΔkdmA) and KdmB (ΔkdmB) showed that KdmA is necessary for normal growth and proper conidiation, whereas KdmB negatively regulates vegetative growth and conidiation. In both mutant strains, tolerance to H2O2 was significantly decreased, and the activities of both conidia-specific catalase (CatA) and mycelia-specific catalase (Cat1) were decreased. Both mutants had significantly increased sensitivity to the guanine nucleotide synthesis inhibitor 6-azauracil (6AU). The ΔkdmA mutant produced more gliotoxin (GT), but the virulence was not changed significantly in immunocompromised mice. In contrast, the production of GT and virulence were markedly reduced by the loss of kdmB. Comparative transcriptomic analyses revealed that the expression levels of developmental process-related genes and antioxidant activity-related genes were downregulated in both mutants. Taken together, we concluded that KdmA and KdmB have opposite roles in vegetative growth, asexual sporulation, and GT production. However, the two proteins were equally important for the development of resistance to 6AU.

Bacillus subtilis-Fermented Amomum xanthioides Ameliorates Metabolic-Syndrome-Like Pathological Conditions in Long-Term HFHFD-Fed Mice.

In modern society, numerous metabolic disorders are widespread globally. The present study aimed to demonstrate whether Bacillus subtilis-fermented Amomum xanthioides (BSAX) exerts anti-metabolic disturbance effects compared with the ethyl acetate fraction of Amomum xanthioides (EFAX), a previously verified functional fraction. Mice fed with a high-fat, high-fructose diet (HFHFD) for 10 wk presented a typical model of metabolic dysfunction, and BSAX significantly attenuated a string of metabolic-syndrome-related pathological parameters, such as body, fat, organ mass, lipid markers (TGs, TC, free fatty acids), and glucose metabolism (glucose, insulin), without influencing appetite. Further, BSAX markedly lowered malondialdehyde (MDA) and ROS in the blood and restored antioxidative parameters (SOD, GSH, and CAT in liver tissue, and total bilirubin in serum) by elevating Nrf2 and HO-1. Moreover, BSAX noticeably restored gut microbiota diversity and normalized lipid-metabolism-associated proteins, including SREBP-1, p-AMPK, and PPAR-α. Generally, most metabolic parameters were improved by BSAX to a greater extent than EFAX, except for liver weight and hepatic TC. In conclusion, BSAX alleviates metabolic dysfunction by enhancing lipid metabolism and antioxidative capacity and is more effective than EFAX. Therefore, the application of high-yield, effective BSAX might be a promising approach for curing and preventing metabolic disorders.

Combinatorial Metabolic Engineering Strategies for the Enhanced Production of Free Fatty Acids in Escherichia coli.

In this study, we evaluated the effects of several metabolic engineering strategies in a systematic and combinatorial manner to enhance the free fatty acid (FFA) production in Escherichia coli. The strategies included (i) overexpression of mutant thioesterase I ('TesAR64C) to efficiently release the FFAs from fatty acyl-ACP; (ii) coexpression of global regulatory protein FadR; (iii) heterologous expression of methylmalonyl-CoA carboxyltransferase and phosphoenolpyruvate carboxylase to synthesize fatty acid precursor molecule malonyl-CoA; and (iv) disruption of genes associated with membrane proteins (GusC, MdlA, and EnvR) to improve the cellular state and export the FFAs outside the cell. The synergistic effects of these genetic modifications in strain SBF50 yielded 7.2 ± 0.11 g/L FFAs at the shake flask level. In fed-batch cultivation under nitrogen-limiting conditions, strain SBF50 produced 33.6 ± 0.02 g/L FFAs with a productivity of 0.7 g/L/h from glucose, which is the maximum titer reported in E. coli to date. Combinatorial metabolic engineering approaches can prove to be highly useful for the large-scale production of FA-derived chemicals and fuels.