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Licensed influenza vaccines primarily target the variable hemagglutinin protein and provide inadequate cross-protection against mismatched or drifted viral strains. One promising approach to enhance the breadth of protection is to target the viral neuraminidase by incorporating a stable, recombinant neuraminidase protein with an effective adjuvant. We evaluated an intramuscular recombinant neuraminidase vaccine (N1-MPP) adjuvanted with a lipidated toll-like receptor (TLR)7/8 agonist (INI-4001), a synthetic TLR4 agonist (INI-2002), or both (TRAC-478) delivered as aqueous, liposomal, or squalene oil-in-water emulsions in mice. TLR agonists offset the Th2 bias of squalene emulsions and boosted antibody responses. Combining ligands synergistically amplified CD4⁺ immunity-increasing polyfunctional cytokine-producing T cells in lungs and spleen-while promoting Th1 cytokine production and antibody class switching. Aqueous and emulsion TRAC-478 N1-MPP induced high titers of cross-reactive, functional antibodies that exhibited strong ADCC activity and conferred protection in passive-transfer experiments. TRAC-478 N1-MPP vaccination protected against both H5N1 clade 1 and clade 2.3.4.4b viruses and recent H1N1 isolates, supporting the further development of N1-MPP adjuvanted with TRAC-478 emulsion as a stand-alone vaccine or potentially as a supplement for current vaccination regimens to improve protection against both seasonal influenza virus and strains with pandemic potential.

Danshensu (DSS) is one of the water-soluble components extractable from the traditional Chinese medicine Salvia miltiorrhiza Bge., exhibiting pharmacological effects such as promoting blood circulation, dilating coronary arteries, and improving cerebral blood flow. The Danshensu derivative (OZD-1) obtained through the derivatization of DSS is a potential multi-target drug for the central nervous system, however, its mechanism of action against cerebral ischemia-reperfusion injury (CIRI) remains unclear. Systematically investigating the therapeutic potential and mechanisms of action of Danshensu derivative against cerebral ischemia-reperfusion injury. Rat brain microvascular endothelial cells (RBMVECs) were cultured in vitro to establish an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. Groups included a control group, an OGD/R model group, and OZD-1 low-dose (12.5 μmol/L), medium-dose (25 μmol/L), and high-dose (50 μmol/L) groups. Cell viability, migration capacity, and vascular lumen formation were assessed using the CCK-8 assay, cell scratch assay, and matrigel matrix gel assay, respectively. In vivo, a transient middle cerebral artery occlusion (tMCAO) model was established in rats. Animals were randomly divided into the sham, model, OZD-1 (35, 70, 140 mg/kg), Edaravone (Eda), and DSS groups. Daily oral administration was performed post-surgery for 14 consecutive days. Tissue pathology staining, behavioral tests, and regional cerebral blood flow imaging assessed brain tissue damage, cognitive function, and ischemic side cerebral blood flow recovery, respectively. Transcriptome sequencing analyzed differential gene expression and pathway enrichment patterns. Western blot detection measured expression levels of proteins related to the PI3K-AKT-CREB signaling pathway, phosphoproteins, downstream apoptosis-related proteins, and CD31, CD34, and VEGFA proteins. In vitro experiments demonstrated that OZD-1 dose-dependently enhanced the viability of RBMVECs following OGD/R injury, significantly improving cell migration and luminal formation capabilities. In vivo studies revealed that compared to the model group, rats in all OZD-1 dosage groups exhibited markedly improved cognitive function, significantly restored cerebral blood flow in the ischemic hemisphere, and substantially reduced pathological brain tissue damage. Transcriptome sequencing results indicated significant enrichment of genes associated with the PI3K-AKT signaling pathway following OZD-1 intervention. Western blot experiments confirmed that OZD-1 significantly upregulates the phosphorylation levels of proteins related to the PI3K-AKT-CREB signaling pathway in OGD/R-injured cells and brain tissue from tMCAO rats, thereby promoting VEGFA-mediated angiogenesis and inhibiting apoptosis. To further verify pathway involvement, in vitro inhibition experiments were performed in RBMVECs using the PI3K inhibitor LY294002 and CREB inhibitor 666-15. These inhibitors abolished the OZD-1-induced upregulation of p-PI3K, p-AKT, and p-CREB, and reversed its protective effects on cell viability, migration, and tube formation. These results confirm that OZD-1 protects vascular endothelial cells directly via activating the PI3K-AKT-CREB pathway. OZD-1 exhibits significant neuroprotective effects against CIRI in rats, improving cognitive function, promoting vascular regeneration in ischemic areas, repairing damaged RBMVECs, and reducing apoptosis. Its mechanism of action is associated with the activation of the PI3K-AKT-CREB-VEGFA signaling pathway.

The substantial antigenic diversity of Influenza A virus (IAV) presents significant challenges to the development of broadly protective vaccines for swine. Moreover, pigs vaccinated with whole-inactivated virus or hemagglutinin (HA) subunit vaccines may experience more severe lung consolidation than non-vaccinated pigs when exposed to antigenically mismatched IAV strains, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). We recently developed a lipid nanoparticle-encapsulated DNA (LNP-DNA) vaccine encoding the HA of IAV, which elicited robust immune responses following a single immunization and protected pigs against homologous IAV challenges. In this study, we compared the immunogenicity and protective efficacy between the HA protein-based vaccine and the HA DNA-based vaccine against an antigenically mismatched IAV strain in pigs. Neither vaccine induced cross-reactive hemagglutination inhibition (HI) antibodies nor prevented viral shedding in nasal secretions following heterologous challenge. However, while the HA protein-based vaccine exacerbated lung lesions compared to non-vaccinated controls, the HA DNA-based vaccine prevented the development of gross lung pathology. Transcriptomic analyses revealed distinct gene expression profiles between the two vaccine groups. These findings suggest that the LNP-DNA vaccine platform may offer a safer and more effective strategy for developing vaccines against IAV in swine.

How host organisms adapt their defense systems to newly invading transposable elements remains poorly understood. Here, we show how Drosophila melanogaster acquired PIWI-interacting RNA (piRNA)-mediated immunity against the endogenous retrovirus Tirant. We uncover two distinct modes of de novo piRNA biogenesis by combining genetics, small RNA profiling, and population genomics. The primary route involves antisense insertions into the flamenco cluster, a master locus for transposon control. Unexpectedly, a second, equally potent mechanism arises from antisense Tirant insertions within host gene 3' UTRs. This process requires host gene transcription but is independent of host gene identity. Our findings challenge prevailing models that tie piRNA precursor specification to genomic origin or nuclear RNA processing context. Instead, they reveal a flexible mechanism that turns a critical vulnerability of transposons into an advantage for the host. When transposition occurs into host gene exons, chimeric antisense transcripts are exported to the cytoplasm, inadvertently initiating piRNA production and enabling rapid, adaptive genome defense against new invaders.

Broad-spectrum resistance genes are highly valuable for sustainable crop protection, yet the molecular basis of their activity is often unknown. The Pm3 allelic series in wheat encodes NLR receptors that recognize avirulence (AVR) effectors of wheat powdery mildew. Here, we show that near-identical Pm3 alleles vary greatly in resistance efficacy and broadness against a global mildew isolate collection and subsequently use this model system to study the mechanisms underlying broad-spectrum resistance. We demonstrate that two alleles, Pm3d and Pm3e, provide resistance against most isolates worldwide, by each recognizing two AVR genes, thereby lowering the risk of resistance breakdown. Pm3d recognizes two highly similar RNase-like AVRs, encoded by gene paralogs. In contrast, Pm3e detects two structurally diverse AVRs: one shared with Pm3d, the other originating from a large, uncharacterized protein family with a discrete structural fold. Using chimeric Pm3 NLRs, we identify specificity-defining polymorphisms of Pm3d and Pm3e against their diverse effector targets. Lastly, we demonstrate that Pm3d and Pm3e activities can be combined in engineered Pm3 NLRs, thereby further extending their recognition spectrum. Our findings highlight the potential of Pm3 immune receptors for long-lasting wheat protection by demonstrating their versatility in recognizing structurally diverse effectors and their amenability to NLR engineering.

Eimeria necatrix, a member of the Apicomplexa phylum, is one of the most pathogenic parasites, causing high mortality in chickens. Microneme proteins (MICs) play essential roles in host cell recognition and invasion by apicomplexan parasites and are also attractive candidates for vaccine development. However, comprehensive studies on E. necatrix MICs remain limited. Eimeria necatrix MIC3 gene (EnMIC3) was amplified and expressed in Escherichia coli. The recombinant protein (rEnMIC3) was characterized via SDS-PAGE and Western blot. The antigenicity of rEnMIC3 and its localization in sporozoites (SZ) and second-generation merozoites (MZ-2) of E. necatrix were determined by Western blot and indirect immunofluorescence analyses (IFAs). The dynamic expression of EnMIC3 across different developmental stages and its impact on sporozoite invasion of host cells were analyzed. The immune protection provided by rEnMIC3 was evaluated in chickens using weight gain, lesion scores, oocyst production, anticoccidial index (ACI), and antibody levels. The open reading frame of EnMIC3 was 798 bp, encoding a 265-amino acid protein with a predicted molecular weight of 28.50 kDa. EnMIC3 contained a signal peptide and a single epidermal growth factor (EGF)-like domain. The rEnMIC3 with an approximate molecular weight of 36 kDa could be specifically recognized by convalescent sera from chickens infected with E. necatrix. The molecular mass of the native protein was approximately 35 kDa, and it localizes to the apical region in SZ but exhibits a cytoplasmic distribution in MZ-2. EnMIC3 mRNA was expressed at significantly higher levels in SZ than in MZ-2, whereas protein expression displayed an inverse pattern. Anti-rEnMIC3 polyclonal antibodies inhibited sporozoite invasion of DF-1 cells in a dose-dependent manner. Vaccination with rEnMIC3 conferred effective protection against E. necatrix challenge, with the high-dose group (200 µg) achieving the highest ACI value (171.32) and markedly elevated serum antibody levels. These findings not only offer a foundation for understanding the role of EnMIC3 protein in the host invasion of E. necatrix but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis.

The synergistic use of silver nanoparticles (AgNPs) and photosensitizer's offers promise biomedical improvements. This study assesses and creates the potential for photosensitizers (Chlorine e6 (Ce6), Methylene Blue (MB)) and Silver Nanoparticles to work together to enhance biological activity. AgNPs were created by the laser ablation method and characterized using methods including scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD).The antibacterial and anticancer properties of these nanoparticles, both individually and in combination with photosensitizers, were further examined. AgNPs were combined with Methylene Blue and Chlorine e6 to enhance their antibacterial activity against Gram-negative bacteria, such as Salmonella enteritidis, Pseudomonas aeruginosa, and Acinetobacter baumannii, resulting in inhibition zones of up to as large as 0.66 ± 057 mm. The anticancer properties of the combination therapy were also examined against MCF-7 breast cancer cells, where Chlorine e6 alone had an IC50 of approximately 231.2%. Another photosensitizer, Methylene blue, showed a dose-dependent reduction in cell viability, with an IC50 of around 6.52 ± 3.26%. When AgNPs and Methylene Blue combined, the IC50 decreased to 11.42 ± 5.71, indicating a synergistic increase in cytotoxicity. Similarly, Chlorine e6 and AgNPs together significantly decreased the IC50 to 80µM to 100 µM. These findings show that the combined use of Methylene Blue or Chlorine e6 with AgNPs greatly improves anticancer and antibacterial efficacy compared to their individual applications. This research highlights how AgNPs and photosensitizers have the ability to change treatment approaches by providing improved specificity and efficacy in biomedical applications.

Adenosine (Ado) is a key signaling molecule in the central nervous system. Under cellular stress, extracellular Ado accumulation drives neuroinflammation-induced neuronal damage and sleep dysfunction. Tianwang Buxin Decoction (TWBXD) demonstrates long-term therapeutic efficacy for insomnia. However, the related pharmacological pathways require further mechanistic studies. To elucidate the therapeutic effects of TWBXD against insomnia and its mechanisms in mitigating Ado-induced neuroinflammation. TWBXD components were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An in vivo insomnia model was established by inducing adenosine A2A receptor (A2aR) overexpression in the basal forebrain. Sleep architecture was monitored via 24-h polysomnography, followed by analyses of histopathology, Ado levels, A2aR expression, inflammatory mediators, and AMPK/SIRT1 activity. In vitro, neuroinflammation was modeled in a neuron-astrocyte co-culture using A2aR-overexpressing plasmids. TWBXD-containing serum effects on cell viability, Ado dynamics, and inflammatory responses were assessed. Ado accumulation induced neuroinflammation and disrupted sleep. TWBXD exhibited optimal efficacy at 17.6 g/kg compared with other doses. It reversed Ado-mediated suppression of the AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1) axis to improve sleep architecture, inhibiting the expression of downstream pro-inflammatory mediators such as nuclear factor-κB, interferon-γ, and interleukin-1β while promoting that of interleukin-10. In vitro experiments and molecular docking verified that TWBXD's neuroprotective effects against Ado overload are mediated by functional A2aR antagonism. Neuroinflammation in insomnia is promoted by Ado-driven suppression of the AMPK/SIRT1 signaling pathway. TWBXD restores this axis to alleviate insomnia, representing a novel therapeutic strategy for managing neuroinflammation-related sleep disorders.

Triple negative breast cancer (TNBC) is a cancer with significant unmet medical needs and comprises 10-15 % of all breast cancers and 15-20 % of advanced breast cancers due to the limited therapeutic options. Many TNBC tumors are driven by aberrant activities of epidermal growth factor receptor, mesenchymal epithelial transition factor (cMet), and vascular endothelial growth factor (VEGF). We demonstrated how TAVO412, a tri-specific antibody that recognized cMet, dual epitopes of EGFR, and VEGF could elicit antitumor activity in TNBC. TAVO412 showed inhibition of EGFR- and cMet- mediated tumor proliferation, enhanced Fc-mediated effector functions, and suppression of angiogenesis. The avidity of the dual-epitope based anti-EGFR and anti-cMet design had better signaling inhibition and cytotoxicity against EGFR-low expressing tumor cell lines than the JNJ-61186372 analogue, a marketed EGFR/cMet bispecific antibody . Furthermore, TAVO412 exhibited anti-tumor activities in multiple TNBC cell line-derived xenograft models. Overall, TAVO412 demonstrated great preclinical utility against TNBC.

Cis-regulatory elements (CREs) drive tissue- and cell-specific gene expression and are essential for safe, sustainable genetic control strategies in pest and vector insects, including the engineering of gene drives in the primary human-malaria vector Anopheles gambiae. Yet CREs remain poorly defined in mosquitoes due to limited computational tools and practical methods for identification and validation. We present a systematic in silico approach for CRE discovery, correlating targeted DNA-motif searches with gene expression, followed by frequency and distribution analysis within putative promoter regions. Applied to the A. gambiae germline, this approach identified hundreds of putative CREs significantly correlated with germline expression in one or both sexes, often linked to distinct sperm developmental stages and chromosomal locations, suggesting roles in broader regulatory mechanisms such as dosage compensation and meiotic silencing. When mapped onto pre-characterised germline promoters, CRE distribution aligned with regions associated with experimental expression patterns. Finally, we validated a top-ranked testis-enriched CRE using an in vivo dual-reporter assay, showing that mutation of conserved nucleotides drastically altered male germline expression. To the best of our knowledge this work provides the first nucleotide-resolution regulatory genome annotation of the A. gambiae germline, offering a transferable framework to aid promoter design for genetic control strategies against malaria mosquitoes and other insect pests.

Ectonucleotidases, including NTPDases and ecto-5'-nucleotidase (e-5'NT/CD73), regulate extracellular purinergic signaling by converting ATP to adenosine, a pathway critically involved in immune response, inflammation, and cancer progression. In this study, a novel library of 22 N-propylsulfonyl-substituted indole-based hydrazinecarbothioamides (5a-5v) was synthesized and structurally characterized. Biological evaluation against human e-5'NT and NTPDase1, -2, -3, and - 8 revealed that several compounds exhibited low micromolar inhibitory activity, with 5n (IC50 = 1.7 µM), 5o (IC50 = 1.7 µM), 5f (IC50 = 1.0 µM), and 5i (IC50 = 1.6 µM) emerging as the most promising derivatives, showing strong potency and isoform selectivity. Structure-activity relationship analysis indicated that both electronic and steric features of substituents significantly influence activity and enzyme preference. Molecular docking studies performed on e-5'NT demonstrated that active compounds adopt consistent binding modes within the catalytic pocket, stabilized by key residues such as Asp-506, Phe-500, Phe-417 and Arg-395. Binding free energy calculations (MM-GBSA) supported strong ligand-protein interactions ( ~ - 70 kcal/mol). The docking protocol was validated by redocking, yielding an RMSD value well below the accepted threshold. Molecular dynamics simulations (500 ns) confirmed stable complex formation, with low RMSD values (~ 1-3 Å), limited residue fluctuations, and persistent interactions with catalytic residues. Surface and compactness parameters (rGyr, SASA) remained stable, indicating consistent ligand accommodation. In silico ADME analysis suggested favorable drug-like properties for most compounds, particularly for the lead candidates. Overall, these findings identify 5n and 5o as the most promising lead compounds, supported by both experimental and computational results, and highlight this scaffold as a valuable platform for the development of selective ectonucleotidase inhibitors.

Disability assessment in dementia is important for care planning, but the full World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0) is time-intensive and may limit clinical use. This study developed machine learning (ML)-based short forms of the WHODAS 2.0 and examined their reliability, concurrent validity, and responsiveness. Using data from 51,245 persons with dementia (training set: n = 31,952; validation set: n = 19,293), we developed two ML-based short forms, ML-WHODAS-16 and ML-WHODAS-10, with Extreme Gradient Boosting and bootstrap-based item selection under a lock-down training/validation workflow. Their performance was compared with the full WHODAS-32 and the conventional 12-item short form adapted to 11 items after excluding work-related items (Standard-12). Anchor-based longitudinal validity was also examined using deterioration in official Disability Severity Grade. Both ML short forms showed high internal consistency (α = 0.96 for ML-WHODAS-16 and 0.93 for ML-WHODAS-10) and excellent concurrent validity with the full WHODAS-32 (r = 0.98 for both). Compared with the Standard-12, they showed lower error, negligible Bland-Altman bias, and met predefined equivalence criteria, including ±0.5 points. Anchor-based Responsiveness was broadly comparable to the Disability Severity Grade (anchor) (r = 0.66-0.67; standardized response mean = 0.37-0.40). Anchor-based minimal clinically important differences were 9.26 for ML-WHODAS-16 and 9.95 for ML-WHODAS-10. The ML-WHODAS-16 and ML-WHODAS-10 substantially reduced assessment burden while maintaining scores that closely reflected those of the full WHODAS-32, particularly for group-level assessment and longitudinal monitoring. These findings support their use as practical, low-burden alternatives in dementia disability assessment. However, external validation, validation against harder clinical outcomes, formal non-inferiority testing, and clinically anchored longitudinal thresholds remain needed before individual-level interchangeability can be inferred.

This study evaluated the polyphenol content of leaf extracts from Artemisia monosperma (AM) and investigated their antioxidant properties, cytotoxic effects, and potential to induce DNA damage in human cancer cell lines. High-performance liquid chromatography (HPLC) quantified polyphenols in methanolic (AMM), ethanolic (AME), and aqueous (AMA) extracts, identifying 13 compounds in AME and 12 in AMA. AMM exhibited the strongest antioxidant activity (IC50 = 24 µg/ml). Both AME and AMM demonstrated potent anticancer activity against HCT-116 (IC₅₀ = 0.38 µg/mL for AMM) and HUH-7 (IC₅₀ = 21.95 µg/mL for AMM) cells, while exhibiting minimal cytotoxicity toward normal skin fibroblast cells (BJ-1; IC₅₀ = 13.05 µg/mL for AMM), with AMM demonstrating particular selectivity for HCT-116 cells. AMM induced DNA fragmentation and modulated apoptosis-related gene expression (Bax, Bcl-2, p53) in HUH-7 cells and caused cell cycle arrest at G0/G1 phase in HCT-116 cells. Molecular docking further supported AMM's apoptosis activity. These results position A. monosperma as a rich source of bioactive polyphenols and antioxidants, with AMM showing promise as a therapeutic agent, especially for colorectal cancer.