Synthetic double-stranded RNA analogs recognized by Toll-like receptor 3 (TLR3) are an attractive adjuvant candidate for vaccines, especially against intracellular pathogens or tumors, because of their ability to enhance T cell and antibody responses. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been limited due to heterogeneous molecular size, inconsistent activity, poor stability, and toxicity. To overcome these limitations, we developed a novel dsRNA-based TLR3 agonist named NexaVant (NVT) by using PCR-coupled bidirectional in vitro transcription. Agarose gel electrophoresis and reverse phase-HPLC analysis demonstrated that NVT is a single 275-kDa homogeneous molecule. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without any signs of serious toxicity. NVT stimulated TLR3 and increased the expression of viral nucleic acid sensors TLR3, MDA-5, and RIG-1. When intramuscularly injected into C57BL/6 mice, ovalbumin (OVA) plus NVT highly increased the migration of dendritic cells (DCs), macrophages, and neutrophils into inguinal lymph node (iLN) compared with OVA alone. In addition, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-β production. Furthermore, NVT exhibited an appropriate adjuvanticity because it elevated OVA-specific IgG, in particular, higher levels of IgG2c (Th1-type) but lower IgG1 (Th2-type). Concomitantly, NVT increased the levels of Th1-type T cells such as IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ cells in response to OVA stimulation. Collectively, we suggest that NVT with appropriate safety and effectiveness is a novel and promising adjuvant for vaccines, especially those requiring T cell mediated immunity such as viral and cancer vaccines.

Nexavant was reported as an alternative to the TLR3 agonist of Poly(I:C) and its derivatives. The physicochemical properties, signaling pathways, anti-cancer effects, and mechanisms of Nexavant were investigated. The distinctive characteristics of Nexavant compared to that of Poly(I:C) were demonstrated by precise quantification, enhanced thermostability, and increased resistance to RNase A. Unlike Poly(I:C), which activates TLR3, RIG-I, and MDA5, Nexavant stimulates signaling through TLR3 and RIG-I but not through MDA5. Compared to Poly(I:C), an intratumoral Nexavant treatment led to a unique immune response, immune cell infiltration, and suppression of tumor growth in various animal cancer models. Nexavant therapy outperformed anti-PD-1 antibody treatment in all the tested models and showed a synergistic effect in combinational therapy, especially in well-defined cold tumor models. The effect was similar to that of nivolumab in a humanized mouse model. Intranasal instillation of Nexavant led to the recruitment of immune cells (NK, CD4+ T, and CD8+ T) to the lungs, suppressing lung metastasis and improving animal survival. Our study highlighted Nexavant’s defined nature for clinical use and unique signaling pathways and its potential as a standalone anti-cancer agent or in combination with anti-PD-1 antibodies.

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

Background: Safe, microbiome-based interventions for autoimmune hyperthyroidism are lacking. We isolated the lactic acid bacterium NES-428 from kimchi and previously demonstrated that it shares 99% 16S-rRNA identity with Lactobacillus brevis reference strains, confirming NES-428 as a novel strain. Here we evaluated its immunomodulatory and anti-thyroid activity in cellular and murine models. Methods: Jurkat T cells (5 × 10⁶) were incubated with heat-killed NES-428 for 24 h and subsequently stimulated with phorbol 12-myristate 13-acetate/ionomycin (50 ng mL⁻¹/1 µg mL⁻¹) for 6 h; cytokine transcripts were quantified by qRT-PCR. Hyperthyroidism was induced in female BALB/c mice by three intramuscular injections of adenovirus-encoding human TSH receptor (Ad-TSHR). Mice received a daily oral dose of NES-428 (1 × 10⁹ CFU) for 15 weeks. Serum thyroxine (T₄) levels, splenocyte cytokine secretion, and thyroid histopathology were assessed. Statistical analyses employed one-way ANOVA with Tukey post hoc or log-rank tests (α = 0.05). Results: NES-428 pre-conditioning of Jurkat cells significantly down-regulated IL-2 and IFN-γ transcripts (−48% and –43%, respectively; p < 0.01) compared with stimulated controls while modestly increasing IL-4 (+26%). In Ad-TSHR mice, daily NES-428 reduced mean serum T₄ from 11.2 ± 2.1 to 5.8 ± 1.4 µg dL⁻¹ (p < 0.001), restored body weight gain, and normalized follicular architecture relative to untreated hyperthyroid animals. NES-428 supplementation also lowered splenocyte IFN-γ secretion by 58% and raised IL-4 by 41% (p < 0.05). Conclusions: The kimchi-derived strain NES-428 attenuates Th1-skewed cytokine responses and ameliorates experimental hyperthyroidism in vivo. These findings support further investigation of NES-428 as a probiotic candidate for immune modulation in Graves’ disease.

Graves’ disease is an autoimmune hyperthyroid disorder in which thyroid peroxidase (TPO) plays a central role in excessive thyroid hormone production. However, current TPO-targeting drugs, such as propylthiouracil, have limited selectivity and can cause serious adverse effects. In this study, we established an integrated discovery pipeline to identify and optimize novel small-molecule TPO inhibitors. The pipeline began with high-throughput screening of approximately 7000 compounds from the KRICT chemical library for peroxidase inhibition, followed by cytotoxicity filtering and iterative medicinal chemistry to generate NE-2 derivatives. Lead compounds (NE-2-6, NE-2-7, and NE-2-8) were evaluated using enzymatic assays, selectivity profiling against myeloperoxidase (MPO) and lactoperoxidase (LPO), molecular docking, plasma pharmacokinetic profiling, and in vivo antithyroid pharmacodynamic testing in an Ad-TSHR289-induced thyroid hyperfunction model. NE-2-6 exhibited potent TPO inhibitory activity and relative selectivity within the tested peroxidase panel. UV–visible spectral scanning and H₂O₂-dependent inhibition assays suggested that NE-2-6 perturbs the heme-associated catalytic environment of TPO; however, these data do not establish a definitive binding mode or kinetic inhibition mechanism. In the Ad-TSHR289-induced thyroid hyperfunction model, NE-2-6 reduced serum T4 levels, supporting antithyroid pharmacodynamic activity. Because autoimmune endpoints and dose-matched PK–PD relationships were not fully assessed, the findings should be interpreted as preliminary preclinical evidence supporting further evaluation of NE-2-6 as a TPO-targeting antithyroid candidate.

Background: Processed pseudogenes and retrogenes are defined by their RNA-mediated origin and, by virtue of this origin-based definition, are often interpreted as discrete genomic insertions. The completion of telomere-to-telomere (T2T) reference assemblies has substantially improved the resolution of segmental duplication architectures and centromeric satellite sequences that were previously inaccessible, allowing genomic structural contexts that were effectively invisible in earlier references to be directly examined. Results: Using the SEPTIN14P-CICP locus family as a case study, chain-based comparative analyses showed that a genomic window spanning the SEPTIN14 3′ terminal exon and the adjacent processed pseudogene CICP12 is dispersed into multiple segmental duplication-associated units across great apes, rather than being maintained as a single orthologous locus. Genome-wide analyses further indicated that annotated CICP loci preferentially localize within segmental duplication blocks and accumulate near pericentromeric or subtelomeric regions. Despite this duplication-associated dispersion, codon-based selection analyses revealed pervasive purifying selection acting on the full-length SEPTIN14 coding sequence and its 3′ terminal exon, arguing against a model in which the terminal exon was newly formed through segmental duplication. Together, these results show that when highly conserved, strongly constrained coding regions are embedded within segmental duplication-rich regions, co-dispersed processed pseudogene copies can be interpreted as distinct from independently generated LINE-1-mediated insertions and as reflecting secondary structural propagation. Conclusions: When considered in light of origin-based definitions of processed pseudogenes and retrogenes, and specifically within duplication-rich and structurally unstable genomic regions resolved by T2T-level assemblies, these results suggest that multiple annotated loci can arise through secondary propagation of a single RNA-derived insertion. Under such contexts, incorporation of selective constraint and cross-species conservation enables more reliable distinction between source insertions and their secondarily propagated copies. This case study highlights a limitation of current annotation frameworks and demonstrates the need for more precise annotation that incorporates evolutionary and structural context in the T2T era.