Genomics Research Center, Academia Sinica

The programmed death-ligand 1 (PD-L1) plays a critical role for promoting cancer immune evasion. However, the resistance to PD-L1-targeted immunotherapy greatly limits its application. α-lipoic acid (ALA) is an endogenous antioxidant, while whether ALA affects PD-L1 expression remains unknown. In IFN-γ-stimulated castration-resistant prostate cancer (CRPC)-mimicking PC3 and DU145 cells, the expression of PD-L1 and its regulatory genes was determined by Western blotting, RT-PCR, and immunofluorescence. The T-cell-mediated tumor-killing activity was evaluated in a co-culture system of cancer cells and Jurkat T cells. ALA significantly inhibits IFN-γ-induced PD-L1 protein and mRNA expression without affecting its degradation. The upstream genes accounting for PD-L1 induction, including JAK1/STAT1/IRF-1 cascade, c-Myc, HIF-1α, and GSK3β activity, were markedly suppressed by ALA. The decreased expression of PD-L1 and these regulators by ALA is also modulated by attenuation of mTOR/p70S6K/4EBP1-dependent protein translation and ROS production. In the co-culture system, ALA markedly increased T-cell-mediated tumor-killing activity compared to that of ALA treatment alone, suggesting that ALA may augment the antitumor immunity. Collectively, we demonstrated that ALA-mediated inhibition of PD-L1 expression is regulated by multiple mechanisms, which indicates that ALA may be a potential agent to enhance cancer immunotherapy, particularly in CRPC.

Described here is the synthesis and assembly of a new-generation mRNA-bifunctional lipid nanoparticle (mRNA-BLNP3) for selective delivery of mRNA to antigen-presenting cells (APCs). Compared to mRNA-BLNP1 and BLNP2 (BioRxiv,doi.org/10.1101/2023.12.26.572282), the mRNA-BLNP3, incorporating a glycolipid with the lipid moiety designed to target the CD1d receptor on dendritic cells (DCs) and the sugar head group targeting the mannose-binding receptors on APCs is more selective with stronger target-specific immune responses. It was shown that vaccination in mice with mRNA-BLNP1 elicited enhanced cytokine induction and antibody responses compared to traditional mRNA-LNPs, and the mRNA-BLNP2 vaccine, incorporating a mannose-glycolipid, further improved DC targeting. However, BLNP3, incorporating the glycolipid with an aryl-mannose head group targeting the mannose receptor on APCs and the same lipid moiety targeting the Cd1d receptor on DCs, showed superior lymph node targeting in vivo with reduced liver accumulation, enhanced mRNA expression in DCs and macrophages, and increased DC maturation. Immunization in mice with mRNA-BLNP3 elicited enhanced humoral and cellular immune responses compared to mRNA-BLNP1 and mRNA-BLNP2, with higher antigen-specific IgG titers and granzyme B–producing CD8+ T cells, demonstrating that BLNP3 is a promising bifunctional lipid nanoparticle for delivery of mRNA vaccines with improved efficacy and safety.

Few genome-wide association studies have examined genetic variants associated with hepatocellular carcinoma (HCC) risk in individuals seronegative for HBsAg and anti-HCV, and the long-term impact of these variants remains uncertain. This multi-stage study analyzed adults >30 years old who were seronegative for HBsAg and anti-HCV. In the genome-wide association study discovery phase, 765 HCC cases and 9,949 controls were analyzed for 308,693 SNPs, with significant SNPs confirmed in community-based (171 HCC cases, 684 controls) and hospital-based (470 HCC cases, 5,460 controls) validation sets. A cohort of 67,909 participants, followed from 2012 to 2021, was used to evaluate the long-term HCC risk associated with these variants. Ten SNPs in PNPLA3/SAMM50 were significantly associated with HCC risk (p <1.62 × 10-7) and were in high linkage disequilibrium. Three SNPs (rs738409, rs2281135, rs2235776) were replicated and demonstrated strong associations with HCC, independent of steatosis. Over 267,238 person-years of follow-up, 32 new HCC cases occurred, with elevated risks observed in individuals homozygous for the risk genotypes: GG (rs738409), AA (rs2281135), and TT (rs2235776). The adjusted hazard ratios (95% CI) were 3.37 (1.32-8.63), 2.80 (1.06-7.37), and 2.64 (0.91-7.66), respectively. In allelic models, carriers of the risk allele had higher HCC risk, with adjusted hazard ratios (95% CI) of 1.88 (1.14-3.10) for rs738409, 1.68 (1.02-2.78) for rs2281135, and 1.61 (0.98-2.67) for rs2235776. This study identified PNPLA3/SAMM50 variants significantly associated with long-term HCC risk in individuals without viral hepatitis. These findings highlight their potential utility as biomarkers for HCC risk stratification and underscore the need for further research into underlying mechanisms. Variants in the PNPLA3/SAMM50 locus were significantly associated with hepatocellular carcinoma (HCC) risk in individuals seronegative for HBsAg and anti-HCV. The stepwise increase in HCC risk with higher numbers of risk alleles suggests their potential utility as biomarkers to identify high-risk individuals without a history of chronic hepatitis B or C virus infection. These findings provide a foundation for genetic risk stratification and precision prevention strategies in populations where non-viral etiologies of HCC are becoming increasingly important.

Secondary growth is a key characteristic evolved from seed plants and generates secondary xylem-the most abundant tissue on Earth. Recent studies have uncovered xylem developmental lineages in eudicots and magnoliids of angiosperms. However, xylem development in gymnosperms, the other representative clade of seed plants, remained elusive. We performed single-cell transcriptomics for xylem cells of conifers (Cunninghamia lanceolata), the major clade in gymnosperms. Using Seurat and scVI-based cross-species integration, we reconstructed the xylem differentiation trajectories and revealed that the radial system is conserved across seed plants, while the axial system in C. lanceolata exhibits a composite lineage architecture resembling both eudicots and magnoliids. To validate these trajectories, we established a multi-modal spatial framework incorporating spatial transcriptomics, spatial proteomics, and spatial metabolomics. These three spatial layers provided orthogonal evidence confirming cell-type identities and trajectory inference. Additionally, we identified a xylem cell population unique to gymnosperms, suggesting a lineage-specific specialization. Together, our findings uncover a more complex ancestral xylem architecture in gymnosperms and propose a progressive simplification of axial developmental programs from gymnosperms to angiosperms, highlighting a trajectory of reductive evolution in seed plant vascular development.

Phage shock protein A (PspA), a recently identified member of the endosomal sorting complex required for transport III (ESCRT-III) superfamily, is proposed to be critical in stress-induced membrane remodeling in bacteria; however, the precise function and mechanism remain largely unexplored. In this study, we employed various lipid membrane models, including giant unilamellar vesicles, small unilamellar vesicles, and supported lipid bilayers, to study membrane-related activities of PspA. Through cell-free protein synthesis of PspA and biophysical characterizations, we demonstrated its capacity to self-assemble and uncovered the decisive role of the α1 region in facilitating this self-assembly. Notably, assays using lipid bilayer-based systems revealed a range of membrane-associated activities, including binding, disruption, and remodeling, encompassing membrane tubulation, elongation, and the formation of double-membrane vesicles. We discovered that the K55 residue within the α1 region is a key determinant for lipid binding and observed its effects on overall membrane remodeling activities, while the R44 residue is crucial for forming stable PspA rods. Together, these findings highlight the importance of the α1 domain in mediating membrane remodeling activity and suggest this may serve as a viable mechanism for other ESCRT-III proteins.

Green algae, commonly used as a source of food nutrients,containulvan, which is a water-soluble sulfated heteropolysaccharide isolatedfrom Ulva. Ulvan exhibits a range of biological properties,including antitumor, antiviral, anticoagulant, and immunomodulatoryactivities. Its sugar backbone consists of 1→4-linked disacchariderepeating units, which are categorized as ulvanobiuronic acids (typesA3′S and B3′S) and ulvanobioses(types U3′S and U2S3′S). Despiteits promising therapeutic potential, the detailed structure–activityrelationship of ulvan remains unclear. In this study, an efficientstrategy was developed, utilizing orthogonally protected α1→4-linked l-Rha-d-Glc, l-Rha-l-Ido, and l-Rha-d-Xyl derivatives as disaccharide building blocksto synthesize di-, tetra-, hexa-, and octasaccharides of types A3′S, B3′S, and U3′S. The immunomodulatory effects of these compounds were evaluatedthrough cytokine induction experiments. The results revealed thatonly the B3′S and U3′S octasaccharideshad a significant influence on the gene expression of IL-1β,IL-6, IL-10, GM-CSF, and G-CSF. This chemical glycobiology approachoffers valuable insights into the biomedical applications of marineulvan oligosaccharides.

Gene therapy offers a promising treatment for Leber's hereditary optic neuropathy (LHON), a disease of retinal ganglion cell (RGC) degeneration with severe vision loss caused by mitochondria-NADH dehydrogenase 4 (MT-ND4) mutations. However, optimizing mitochondria-targeted gene delivery to promote RGC regeneration and visual-photoreception recovery remains challenging in LHON. Here, mitochondria-targeted wireless charging gold nanoparticles (WCGs), doubling as a wireless charging-mediated gene-delivery platform and electric stimulus-restored phototransduction, are developed for LHON treatment. Upon high-frequency magnetic field (HFMF) irradiation, WCGs enhanced MT-ND4 transfection efficiency, restored complex I activity and mitochondrial homeostasis in vitro, and further promoted RGC neurite outgrowth in LHON patient-derived iPSC-differentiated retinal organoids. Wireless charging combined with electric stimulation also facilitated in vivo gene delivery, effectively promoting neuronal recovery, preventing RGC degeneration, suppressing inflammation, and enhancing retinal electrophysiological function in the damaged retinas of LHON mice. Furthermore, single-cell RNA sequencing and spatiotemporal transcriptomic analysis revealed that HFMF-treatment reprograms Müller glia to enhance dendritic development, restore mitochondrial function, and express phototransduction genes to support photoreceptor function in vivo. Finally, functional retinal-optic-electrophysiological findings with spatiotemporal transcriptomic analysis at the single-cell level support that HFMF synergized with WCG/hND4 therapy, promoting retinal repairment with RGC neurite regeneration and recovering visual phototransduction in LHON mouse models.

Abstract The integration of symbionts into host cells during endosymbiosis significantly alters gene expression and cell physiology. Though alternative splicing facilitates cellular adaptation through rapid modulation of gene expression and protein isoform diversity, its regulatory role during endosymbiosis remains poorly understood. Paramecium bursaria, which harbors hundreds of Chlorella variabilis algae within its cytoplasm, offers a powerful model to study splicing during endosymbiosis, especially given its exceptionally short introns (median ∼24 nt). Using time-course RNA sequencing of symbiotic and aposymbiotic cells, we found that splicing, especially of 5’ proximal introns, enhances gene expression. Moreover, we identified 883 genes with differentially spliced introns, particularly enriched in transmembrane transporters essential for establishing nutrient exchange between a host cell and algal symbionts. Splicing regulation correlated with expression changes in conserved spliceosome components, implicating that these factors act as splicing enhancers or repressors during symbiosis. By exploring intron orthology across ciliates, we found that conserved introns exhibited more efficient splicing, characterized by lower GC content and uniform length, suggesting that intron evolution favors features that optimize expression. Our study reveals how splicing contributes to host adaptation during endosymbiosis and highlights the evolutionary dynamics of short introns in eukaryotes. Key points Paramecium retains conserved core spliceosome genes but employs unique splicing regulation. Alternative splicing regulates metabolism during endosymbiosis, especially for transporter genes. Older introns evolve toward ∼24 nt length and low GC for efficient splicing and expression.