Subunit vaccines offer safety and precision but require adjuvants to overcome weak immunogenicity, particularly for the induction of cellular immunity. Herein, we develop a manganese pyrophosphate mineralized DNA bi-adjuvant-based subunit vaccine platform via biomineralization, which demonstrated the capability to elicit robust antigen-specific cellular immune responses. The Mn/CpG bi-adjuvant modulated the balance between Th1 and Th2 immune responses. More importantly, the synergistic activation of Toll-like receptor 9-like (TLR9) signaling pathways by CpG and cGAS-STING pathways mediated by Mn(II) ions robustly enhanced cellular immunity, which was a magnitude of enhancement over commercial aluminum-based adjuvants both in vitro and in vivo. Furthermore, the mechanistic studies revealed that the Mn(II)-based nanoadjuvant effectively promoted Th1-biased cellular immune response, as evidenced by an elevated IgG2a/IgG1 ratio and enhanced Th1-associated cytokine secretion, alongside a potential reduction in regulatory T cell activity. These insights established critical design principles essential for the development of next-generation manganese-derived adjuvant systems, thereby advancing the frontiers of vaccinology and immunology.

The widespread application of lipid nanoparticles (LNPs) as mRNA delivery vectors is constrained by the intrinsic trade-off between delivery efficacy and inflammatory reactogenicity. To address this limitation, we engineered a safe and potent LNP-mRNA delivery system by leveraging the dual activities of α-tocopherol (TP). Capitalizing on its well-documented anti-inflammatory and immunomodulatory properties, we hypothesized that incorporating TP or its derivatives into LNPs would concurrently mitigate carrier-induced inflammation and enhance antigen-specific immunogenicity. Our results demonstrated that TP succinate (TPS)-modified LNPs significantly improved in vitro mRNA delivery, achieving up to a 9.5-fold increase in protein expression alongside enhanced cellular uptake, without compromising biocompatibility. Following immunization, TPS-LNPs markedly reduced acute inflammatory reactogenicity, as evidenced by a threefold lower serum IL-6 level at 6 h compared to the standard formulation in blank-LNP groups, and by significantly attenuated cytokine levels in mRNA-loaded groups. In vivo, TPS-LNPs elicited robust and balanced immune responses, characterized by potent humoral immunity and enhanced antigen-specific T cell activation. Mechanistically, the enhanced immunogenicity was associated with upregulated CD40 expression on antigen-presenting cells. Importantly, the anti-inflammatory attributes of TP derivatives conferred an excellent safety profile, with no evidence of significant tissue damage or systemic toxicity. Our findings advocate for a functionalization paradigm in LNP design, wherein α-tocopherol derivatives serve as intrinsic modulators to recalibrate innate immunity, thereby proposing a new design benchmark for simultaneously safe and potent mRNA delivery.

Small interfering RNAs (siRNAs), bound by Argonaute, efficiently cleave target mRNAs through full complementary base pairing, achieving potent gene silencing. While chemical modifications are widely employed to improve siRNA stability and binding affinity, modifying the 5′-end nucleotide of the guide strand remains a primary strategy for enhancing potency. In this study, we performed a structural analysis of the 5′-end binding pocket of the RISC complex to design a novel series of uridine-derived modifications and screen a library including 001U-018U and vs30U as candidates. We further tested the 001 003 μm-modified siRNA binding activity via in vitro RNA-induced silencing complex (RISC) loading in HepG2 cells and in vivo RISC loading in mice. These function valiation results indicated a significant improvement in gene silencing activity for the modified siRNAs compared to unmodified control. Additionally, preliminarily safety evaluations demonstrated the modified nucleoside carry no detectable cytotoxicity or genomic insertion risks. Our findings expand the chemical space of siRNA modification and provide new insights into the design of high-potency siRNA drugs.

Respiratory syncytial virus (RSV) is one of the primary contributors to lower respiratory tract infections, especially in infants, children, and the elderly. The quest for a clinically acceptable and potent RSV vaccine constitutes a long-standing hurdle in clinical practice. Herein, we propose an orthogonal nanoadjuvant-based nanovaccine platform that addresses the long-standing challenge of insufficient cellular immunity in RSV F subunit vaccination. The vaccine is constructed based on Mn/Al-layered double hydroxide (Mn/Al-LDH) nanosheet functionalized with RSV F antigen and poly-CpG motifs. In this system, Al acts as a humoral immunity adjuvant promoting potent antibody responses, while Mn and CpG can enhance cellular immunity through activation of the cGAS-STING pathway and Toll-like receptor (TLR), respectively. The orchestrated activation of multiple immune pathways synergistically produces a "high-entropy" immunostimulatory effect, which facilitates the recruitment and subsequent activation of potent CD4+ and CD8+ T cells, thus leading to more rapid antigen-specific immune activation and fostering the establishment of robust long-term T cell memory. Furthermore, an expanded CD134-positive subset within central memory T cells (TCM) is observed, demonstrating that the immune system is primed for robust clonal proliferation and differentiation into effector and memory cell lineages. This immunological setup ensures that any subsequent encounter with the specific antigen will trigger an immediate and vigorous secondary response. In murine challenge models, the nanovaccine outperforms traditional aluminum (Al)-based vaccines by significantly reducing viral load and mitigating lung pathology. This study establishes a new paradigm for developing efficacious RSV vaccines, and the underlying strategy is broadly applicable to vaccine development against other intractable pathogens.

Dysfunction of RNA-binding proteins, including TDP-43 and FUS, has been associated with amyotrophic lateral sclerosis (ALS); however, the underlying mechanisms are largely unknown. Here, we reported that a neuronal upregulation of TRIM72 (Tripartite Motif Containing 72) in FUS mutation knockin ALS models slows disease progression. TRIM72 interacts with Commander, a protein complex for recycling of membrane proteins, facilitating membrane repair and antioxidation. Exosomal TRIM72 is detected in ALS patient cerebrospinal fluid (CSF) and extracellular application of exosomal TRIM72 protects cell from membrane damage. In a sporadic ALS cohort, CSF TRIM72 level associates ALS disease progression. AAV-mediated neuronal expression of TRIM72 slows down the disease progressions in ALS models and in an ALS patient without adverse effects over a year treatment course. Taken together, our results suggest a universal neuronal protection of a TRIM family protein in cell-autonomous and non-cell-autonomous manners in ALS. ### Competing Interest Statement Y.J. is a scientific advisor at Beijing SineuGene Therapeutics Co., Ltd. W.G. and L.P. are full-time employees of Beijing SineuGene Therapeutics Co., Ltd. X.Z., and Y.J. have patent applications related to this work.

[This corrects the article on p. 312 in vol. 9, PMID: 30906631.].

BACKGROUND:Eps15 homology domain (EHD) proteins, including EHD1 to EHD4, play vital roles in tumor progression. In this study, we aimed to investigate which specific EHD proteins, if any, are implicated in tumor immune evasion and immunotherapy response. METHODS:The immunotherapy responses of lung adenocarcinoma (LUAD) patients were predicted using tumor immune dysfunction and exclusion (TIDE) analysis. The T cell killing assay was performed by co-culturing activated T cells with LUAD cells. The function of EHD1 as a regulator of programmed death-ligand 1 (PD-L1) endocytic recycling was determined by receptor internalization assays. Methylated RNA immunoprecipitation (MeRIP) was performed to investigate N6-methyladenosine (m6A) modification of EHD1 mRNA. The protein-protein interaction was revealed by the molecular docking analysis and validated by immunofluorescence (IF) and immunoprecipitation (IP) assays. RNA immunoprecipitation (RIP) was used to examine the interaction between YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) and EHD1 mRNA. The regulatory mechanism of YTHDF1 on EHD1 was investigated through the application of m6A-binding site mutation analysis. The murine LUAD cells were employed to establish subcutaneous xenograft models within immunocompetent C57BL/6 mice to assess the immunomodulatory impact of EHD1 in vivo. RESULTS:TIDE algorithms and survival analysis identified that EHD1 promoted LUAD immune escape. EHD1 knockdown enhanced T cell cytotoxicity in killing LUAD cells across all effector-to-target (E/T) ratios. EHD1 overexpression exerted the opposite effect. The molecular docking analysis revealed an interaction between EHD1 and the PD-L1 protein, verified by IF and IP. Furthermore, EHD1 knockdown inhibited PD-L1 recycling, thereby promoting its lysosomal degradation. Disruption of the EHD1/PD-L1 interaction impaired the regulatory function of EHD1 in tumor immune evasion. In an immune-competent mouse model, we found that EHD1 silencing impeded tumor immune evasion and enhanced the efficacy of anti‑PD‑1 therapy. MeRIP-qPCR confirmed obvious m6A modification of EHD1. Further, the EHD1 mRNA was found to bind to the YTHDF1 protein, an m6A reader. YTHDF1 overexpression up-regulated EHD1 expression by enhancing its mRNA stability in an m6A-dependent manner. CONCLUSION:Our study illuminates the role of m6A-modified EHD1 in tumor immune evasion and immunotherapy responses, thereby offering a novel avenue to potentially enhance immunotherapeutic sensitivity and improve the prognosis for patients with LUAD.

Background and Aims: Pyrrolizidine alkaloids (PAs), widely distributed in plants, are known to induce liver failure. Hepatic platelet accumulation has been reported during the progression of PA-induced liver injury (PA-ILI). This study aimed to investigate the mechanisms underlying platelet accumulation in PA-ILI. Methods: Cases of PA-ILI, non-PA-ILI, and control subjects were collected from patients hospitalized at Zhongshan Hospital, Fudan University (Shanghai, China) between 2012 and 2019. A mouse model of PA-ILI was established using monocrotaline administration. Liver RNA sequencing was performed, and gene interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins online database. Low-molecular-weight heparin and recombinant a disintegrin and metalloproteinase with a thrombospondin type I motif member 13 (ADAMTS13) were applied. The necrotic liver area, hepatic platelet accumulation, and von Willebrand factor (VWF) deposition were examined using hematoxylin and eosin staining and immunofluorescence assay. Results: Hepatic platelet accumulation, necrotic area expansion, and increased VWF expression were observed in both PA-ILI patients and mice. The Search Tool for the Retrieval of Interacting Genes/Proteins database indicated that ADAMTS13 regulates VWF expression and was differentially expressed in the livers of PA-ILI mice. Plasma and hepatic ADAMTS13 levels were significantly downregulated in both PA-ILI patients and mice. Systemic administration of recombinant ADAMTS13 decreased hepatic platelet accumulation, downregulated VWF expression, and mitigated mouse hepatic necrosis. Conclusions: Hepatic platelet accumulation in PA-ILI was confirmed in both patients and mice. Deficiency of ADAMTS13 plays a critical role in platelet accumulation in PA-ILI, suggesting that ADAMTS13 could be a potential therapeutic target for this condition.

Aluminum-containing adjuvants have been widely used in human vaccines, such as anti-COVID-19 vaccine, to enhance antigen-specific immune responses. However, a major limitation of aluminum adjuvants in clinical application is that they typically enhance humoral immune responses but have weaker effects on cellular immune responses. Therefore, there has been a continuous and active pursuit of novel adjuvants that can address the shortcomings of traditional adjuvants and improve vaccine efficacy. In this paper, we proposed a novel vaccine-adjuvant system utilizing the convenient physical mixing of natural proanthocyanidins (PCs) and its derivative with a recombinant RBD subunit vaccine, which induced comparable humoral immune responses compared to aluminum adjuvant. In the context of cellular immunity, PCs modified with 4-bromomethyl phenylboronic acid not only enhanced the maturation and migration of dendritic cells but also improved the antigen uptake and presentation capacity of monocytes, leading to robust activation of immune cells, particularly involving CD4(+) and CD8(+) T cells, thereby demonstrating a clear Th1/CTL response bias. In general, PCs andtheir derivatives could be potential innovative natural adjuvants which effectively balance the Th1/Th2 immune response to recompense for the deficiency of aluminum adjuvant, and our study also offers an approach to discovering safe and efficient natural adjuvants.

Implementation of adjuvants is a requirement for inducing antigen-specific immune responses against infectious diseases. The conventional aluminum adjuvants used in clinical trials suffer from insufficient cellular immune response. Herein, we develop a bi-adjuvant nanovaccine containing receptor-binding domain (RBD) as antigen and DNA programmed Mg/Al layered double hydroxide (Mg/Al-LDH) as adjuvants, and thus achieve co-activation of potent humoral and cellular immune responses. The Mg/Al-LDH consisting of positively charged layers is firstly employed as scaffold to adsorb antigen via electrostatic interaction, and then CpG and dendritic cell (DC)-targeting aptamers co-encoded ultra-long DNA chains are easily decorated on the Mg/Al-LDH via interfacial assembly. The nanoscale formulations and interfacial targeting aptamers of nanovaccine facilitate their endocytosis by DC cells; the Mg/Al-LDH structure is acid-responsive and ensures the Al ions-inducing antigen cross-presentation for durable generation of antibody (Th2 immune response); and the agonist CpG in DNA chains can bind to the Toll-like receptor 9 (TLR 9) to activate cellular immunity (Th1 immune response). The bi-adjuvant nanovaccine fully combines the advantages of nanosheet alum-like adjuvant and precisely customization of DNA, and thus balances the Th1/Th2 immune response for compensating the deficiency of traditional alum adjuvant, providing a design guidance for creating next-generation of safe and efficient adjuvants in immunology.

Research question: Does the observed correlation between dyslipidaemia and endometriosis indicate a bidirectional causal association? Design: Bidirectional Mendelian randomization was used to investigate the causal association between lipid traits and endometriosis. Drug-target Mendelian randomization was used to explore potential drug-target genes for managing endometriosis. In cases where lipid-mediated effects via speci fi c drug targets were signi fi cant, aggregate analyses, such as summary-data-based Mendelian randomization and colocalization methods, were introduced to validate the outcomes. Mediation analyses supplemented these evaluations. Results: The bidirectional Mendelian randomization results suggested that genetically predicted triglyceride (OR 1.15, 95% CI 1.08 - 1.23), high-density lipoprotein cholesterol (OR 0.87, 95% CI 0.81 - 0.94), low-density lipoprotein cholesterol (OR 1.20, 95% CI 1.06 - 1.34) and apolipoprotein A (OR 0.90, 95% CI 0.83 - 0.96) concentrations were causally associated with endometriosis. Reverse Mendelian randomization results revealed that genetically proxied endometriosis was causally associated with triglyceride concentration (OR 1.02, 95% CI 1.01 - 1.02). In drug-target Mendelian randomization, genetic mimicry in proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) (OR 1.40, 95% CI 1.13 - 1.72), apolipoprotein B ( APOB ) (OR 1.49, 95% CI 1.21 - 1.86) and angiopoietinrelated protein 3 ( ANGPTL3 ) (OR 1.57, 95% CI 1.14 - 2.16) was signi fi cantly associated with the risk of endometriosis stages 1 - 2. Conclusion: There is a potential bidirectional causal association between endometriosis and dyslipidaemia. Genetic mimicry of PCSK9 , APOB and ANGPTL3 is associated with the risk of early-stage endometriosis. The development of lipid-lowering drugs to treat endometriosis is of potential clinical interest.

In recent decades, protein-based therapy has garnered valid attention for treating infectious diseases, genetic disorders, cancer, and other clinical requirements. However, preserving protein-based drugs against degradation and denaturation during processing, storage, and delivery poses a formidable challenge. Herein, we designed a novel fluoroamphiphiles polymer to deliver protein. Two different formulations of nanoparticles, cross-linked (CNP) and micelle (MNP) polymer, were prepared rationally by disulfide cross-linked and thin-film hydration techniques, respectively. The size, zeta potential, and morphology of both formulations were characterized and the delivery efficacy of both in vitro and in vivo was also assessed. The in vitro findings demonstrated that both formulations effectively facilitated protein delivery into various cell lines. Moreover, in vivo experiments revealed that intramuscular administration of the two formulations loaded with a SARS-CoV-2 recombinant receptor-binding domain (RBD) vaccine induced robust antibody responses in mice without adding another adjuvant. These results highlight the potential use of our carrier system as a safe and effective platform for the in vivo delivery of subunit vaccines.

Supplementary Figure from HMGN5 Escorts Oncogenic STAT3 Signaling by Regulating the Chromatin Landscape in Breast Cancer Tumorigenesis

Background Low fertility and adverse pregnancy outcomes are commonly observed in women with chronic kidney disease (CKD). However, a causal relationship between low fertility and adverse pregnancy outcomes with CKD remains unclear. Besides, whether mild kidney dysfunction can affect fertility and pregnancy still needs exploration. Hence, this study aimed to investigate the causal effect of kidney damage on fertility and pregnancy using Mendelian randomization (MR). Methods We first used two-sample MR to examine the effects of kidney damage on fertility and pregnancy. Next, we introduced the Bayesian model averaging MR analysis to detect major causal relationships and render the results robust. The genetic instruments and outcome data were derived from various large genome-wide association studies. Results Adverse pregnancy outcomes: Our analyses supported a suggestive causal effect of CKD and estimated glomerular filtration rate (eGFR) rapid on stillbirth, with CKD having an odds ratio (OR) of 1.020 [95% confidence interval (CI) 1.002 to 1.038] and eGFR rapid having an OR of 1.026 (95% CI 1.004–1.048). We also discovered a suggestive causal effect of eGFR on spontaneous abortion, with an OR of 2.63 (95% CI 1.269 to 5.450). Moreover, increased urinary albumin-to-creatinine ratio (UACR) was regarded as a potential risk factor for pre-eclampsia (OR = 1.936; 95% CI 1.065 to 3.517) and gestational hypertension (OR = 1.700; 95% CI 1.002 to 2.886). Fertility assessment: The results indicated that eGFR and UACR had a suggestive causal relationship with the anti-Müllerian hormone level (eGFR beta: 1.004; UACR beta: 0.405). Conclusions Our study used MR to demonstrate a suggestive causal relationship between kidney damage and fertility and pregnancy. We reported that mild kidney dysfunction might be a risk factor for reduced fertility and adverse pregnancy outcomes. Dynamic renal detection may help preserve fertility and reduce the risk of pregnancy loss.

Supplementary Data from Increased Ectonucleotidase Expression and Activity in Regulatory T Cells of Patients with Head and Neck Cancer

Epigenetic regulations play crucial roles in the pathogenesis of metabolic-associated fatty liver disease; therefore, elucidating the biological functions of differential miRNAs helps us to understand the pathogenesis. Herein, we discovered miR-337-3p was decreased in patients with NAFLD from Gene Expression Omnibus dataset, which was replicated in various cell and mouse models with lipid disorders. Subsequently, overexpression of miR-337-3p in vivo could ameliorate hepatic lipid accumulation, reduce fasting blood glucose, and improve insulin resistance. Meanwhile, we determined miR-337-3p might influence multiple genes involved in glycolipid metabolism through mass spectrometry detection, bioinformatics analysis, and experimental verification. Finally, we selected HMGCR as a representative example to investigate the molecular mechanism of miR-337-3p regulating these genes, where the seed region of miR-3373p bound to 30UTR of HMGCR to inhibit HMGCR translation. In conclusion, we discovered a new function of miR-337-3p in glycolipid metabolism and that might be a new therapeutic target of MAFLD.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic steatosis, inflammation, and progressive fibrosis. Our previous study demonstrated that microRNA-552-3p (miR-552-3p) was down-regulated in the livers of patients with NASH and alleviated hepatic glycolipid metabolic disorders. However, whether miR-552-3p affects NASH progression remains unclear. In this current study, we found that hepatic miR-552-3p expression was negatively correlated with the degree of liver fibrosis and inflammation of NASH patients. Interestingly, the level of miR-552-3p was decreased during hepatic stellate cell (HSC) activation in vitro. Overexpression of miR-552-3p could not only inhibit the expression of fibrotic and inflammatory genes, but also restrain the activation of TGF-β1/Smad3 signaling pathway by down-regulating the expression of TGFBR2 and SMAD3 in HSCs, finally suppressing HSC activation. More importantly, overexpression of miR-552-3p ameliorated liver fibrosis and inflammation in two murine models: high fat/high fructose/high cholesterol diet-induced NASH model and carbon tetrachloride (CCl4)-treated liver fibrosis model. In conclusion, miR-552-3p plays a crucial role in the pathogenesis of NASH by limiting multiple fibrotic and inflammatory pathways in HSCs, which may shed light on its therapeutic potential in NASH.

The silencing of disease-causing genes with small interfering RNA (siRNA) offers a particularly effective therapeutic strategy for different disorders; however, its clinical efficacy relies on the development of nontoxic and tissue-specific delivery vehicles. Herein, we report that bioresponsive chimaeric polymersomes (BCP) with short poly(ethylenimine) as inner shell mediate highly efficacious, sustained, and liver-specific siRNA transfection in vivo. BCP exhibited remarkable encapsulation efficiencies of siRNA (95-100%) at siRNA-feeding contents of 15-25 wt %, to afford stable, small-sized (55-64 nm), and neutral-charged BCP-siRNA. siApoB-Loaded BCP (BCP-siApoB) outperformed lipofectamine counterparts and silenced 93% of ApoB mRNA in HepG2 cells at 50 nM siApoB without inducing cytotoxicity. Intriguingly, the in vivo studies using wild-type C57BL/6 mice revealed that BCP-siApoB preferentially accumulated in the liver, and a single dose of 4.5 mg/kg achieved over 90% downregulation of ApoB mRNA for at least 10 days. The systemic administration of BCP-siApoB at 4.5 mg/kg every 2 weeks or 1.5 mg/kg weekly in diet-induced obese mice could also achieve up to 80% silencing of ApoB mRNA. The liver specificity and silencing efficacy of BCP-siApoB could further be improved by decorating it with the trivalent N-acetylgalactosamine (TriGalNAc) ligand. These bioresponsive and liver-specific chimaeric polymersomes provide an enabling technology for siRNA therapy of various liver-related diseases.

Safe, effective, and economical vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and end the pandemic. We constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains immunodominant peptides screened from the receptor-binding domain (RBD) and is fully chemically synthesized. It has been formulated in an optimized nanoemulsion formulation and is stable at 40 °C for 1 month. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of protective neutralizing antibodies against multiple RBD mutations, SARS-CoV-2 original strain, and variants (B.1.1.7 and B.1.617.2). Specific peptides booster immunization against the B.1.351 variant has also been shown to be effective in improving protection against B.1.351. Meanwhile, CoVac501 elicited the increase of memory T cells, antigen-specific CD8+ T-cell responses, and Th1-biased CD4+ T-cell immune responses in NHPs. Notably, at an extremely high SARS-CoV-2 challenge dose of 1 × 107 TCID50, CoVac501 provided near-complete protection for the upper and lower respiratory tracts of cynomolgus macaques.

Lipotoxicity induced by the overload of lipid in the liver, especially excess free cholesterol (FC), has been recognized as one of driving factors in the transition from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). MicroRNA (miR)-379-5p has been reported to play regulatory roles in hepatic triglyceride homeostasis, but the relationship of miR-379-5p and hepatic cholesterol homeostasis has never been touched. In the current study, we found that hepatic miR-379-5p levels were decreased obviously in NAFLD patients and model mice compared with their controls. Moreover, miR-379-5p was discovered to be able to inhibit intracellular FC accumulation and alleviate mitochondrial damage induced by palmitic acid (PA) in vitro. Furthermore, overexpression of miR-379-5p in HFHC-fed db/db mice could reduce the level of hepatic total cholesterol (TC) and FC, and ameliorate hepatic injury reflected by the lower serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Subsequently, by combining spectrometry (MS) and luciferase assay, we identified miR-379-5p suppressed STAT1 through transcriptional and translational regulation. Finally, we confirmed that STAT1 was a transcriptional factor of HMGCS1. In conclusion, miR-379-5p inhibits STAT1 expression and regulates cholesterol metabolism through the STAT1/HMGCS1 axis, suggesting miR-379-5p might be applied to improve lipotoxicity in the future.