Background: Tuberculosis (TB) remains a major global health challenge, highlighting the urgent need for more effective vaccines. This study aimed to develop an artificial intelligence-guided epitope prediction and prioritization pipeline to identify immunodominant peptides from Mycobacterium tuberculosis (Mtb) and to evaluate the immunogenicity and protective efficacy of the resulting vaccine candidates. Methods: An AI-guided framework was used to predict and prioritize immunodominant Mtb epitopes, leading to the generation of 72 recombinant immunogens. Among these, RI-13, RI-20, and RI-31 were selected as the leading candidates. Their protective efficacy was assessed in a zebrafish TB infection model and in BALB/c mice following DNA vaccination. Humoral and cellular immune responses were further evaluated in C57BL/6 mice. Results: RI-13, RI-20, and RI-31 markedly reduced infection-associated pathology and lowered bacterial burden by up to 1.5 log10 in the zebrafish TB infection model, outperforming benchmark antigen combinations, including the Ag85A plus ESAT6/CFP10 cocktail used in the phase III vaccine candidate GamTBvac. In BALB/c mice, DNA vaccination with each construct reduced pulmonary mycobacterial burden by approximately 0.3 log10 and alleviated lung tissue damage. In addition, all three candidates elicited robust humoral and cellular immune responses, with RI-13 showing the strongest overall immunogenicity and inducing a balanced Th1, Th2, and Th17 response profile in C57BL/6 mice. Conclusions: These findings identify RI-13, derived from Rv1174c, as a promising next-generation TB vaccine candidate. More broadly, this study supports the utility of an AI-guided framework for the rational design and preclinical prioritization of novel TB immunogens.

The 2022 global mpox outbreak and subsequent viral evolution have exposed critical limitations in current therapeutic strategies, which predominantly rely on repurposed smallpox drugs targeting a narrow range of viral proteins. This review provides a timely and comprehensive evaluation of the mpox antiviral landscape, spanning from established clinical agents to cutting-edge preclinical candidates. We critically analyse the 'clinical translation gap' observed in recent clinical trials (e.g., STOMP and PALM007), attributing the limited efficacy in mild cases to host immunopathological responses versus primary viral replication. Highlighting a strategic paradigm shift, we also discuss the 2025 breakthrough in covalent inhibitors targeting the conserved CorePro. Furthermore, we explore the integration of AI-assisted drug design and host-targeted therapies (HTTs) to elevate resistance barriers. By synthesising these emerging targets and multidimensional strategies, this review serves as a strategic roadmap for developing next-generation anti-mpox therapeutics with optimised efficacy and broader resistance profiles.

Target RNAs have emerged as key regulators of miRNA stability, influencing development and physiology in both plants and animals. We previously identified the F-box protein HAWAIIAN SKIRT (HWS) as a key player in target mimicry directed miRNA degradation in plants (pTDMD) using a STTM160-triggered miR160 degradation reporter system. However, the precise mechanism by which HWS functions remains unclear. Here, we conducted an extensive forward genetic screen and identified P14 additional hws alleles that near-completely restored STTM160-induced developmental defects, underscoring the central role of HWS in pTDMD. Intriguingly, we discovered two adjacent amino acid substitutions (R421K and G422D) in the PAZ domain of AGO1 that strongly suppressed the STTM160 phenotype. Although the R/G substitutions only impacted a small set of endogenous miRNA levels, they were sufficient to rescue the developmental defects caused by HWS overexpression (HWS-OE). Similar to HWS dysfunction, the AGO1 R421K substitution significantly retained target mimicry RNAs in AGO1 immunoprecipitates, suggesting that the R421 residue of AGO1 may coordinate with HWS for the clearance of AGO1-miRNA-target mimicry complexes. Our findings thus provide solid genetic evidence for the coordination of HWS and AGO1 in pTDMD.

Background/Objectives: Over the past two centuries, tuberculosis (TB) has been responsible for approximately one billion deaths and continues to represent a significant global health challenge. Despite extensive research efforts, fully effective strategies for the prevention or eradication of TB remain elusive, highlighting the urgent demand for novel vaccines with enhanced safety profiles and efficacy. Lipoproteins, integral surface proteins of mycobacteria, are frequently associated with virulence and display notable immunogenicity, rendering them promising candidates for vaccine development. This study investigates the potential of the mycobacterial lipoprotein, LprO, as a vaccine antigen against TB. Methods: A pcDNA-lprO DNA vaccine was constructed, and its immunogenicity was evaluated using a murine model. Its protective efficacy was further assessed using a Mycobacterium marinum (M. marinum)-infected zebrafish model. Additionally, a recombinant BCG vaccine strain, BCG Japan::pNBV1-lprO, was generated. Its immunogenicity was tested in mice, and its safety was evaluated in SCID mice. Both vaccine candidates were further assessed in regard to their protective efficacy in a murine Mycobacterium tuberculosis (M. tb) infection model. Results: The pcDNA-lprO vaccine increased the M. tb-specific IFN-γ-secreting lymphocytes in murine spleens and prolonged the survival of zebrafish infected with M. marinum. The recombinant BCG Japan::pNBV1-lprO vaccine elicited M. tb-specific Th1-type immune responses in mice compared to the standard BCG Japan strain. Both vaccines effectively reduced the bacterial burden of M. tb in murine lungs, offering superior protection relative to the control groups. Conclusions: These findings establish LprO as a compelling candidate for TB vaccine development, with both LprO-based DNA and recombinant BCG vaccines demonstrating robust protective effects against TB.

Alzheimer’s disease (AD) is a devasting neurodegenerative disorder characterized by β-amyloid formation, further exacerbated by RIPK1/RIPK3 necrosome-induced programmed necrosis (necroptosis). We previously showed that the RIPK1/RIPK3 necrosome forms a functional amyloid complex using its RIP homotypic interaction motifs (RHIMs). Here, we discovered that the core RIPK1/RIPK3 necrosome shares strikingly structural similarity to the C-terminal region of β-amyloid (Aβ42), and the RHIM-derived tetrapeptides (IQIG or VQVG) directly inhibit Aβ aggregation, disassemble preformed Aβ fibrils (PFFs), and reduce RIPK1 polymerization. Also, the peptides exhibit effective membrane permeability and could reduce Aβ40 or Aβ42-induced neural death and TNFα-induced necroptosis in SH-SY5Y cells. IQIG and VQVG injected by ICV increase learning and memory abilities by reducing Aβ plaques and hyperphosphorylated tau in the cortex and hippocampus of APP/PS1 double-transgenic mice. Mechanistically, the peptides directly interact with Aβ to block Aβ aggregation and alleviate microglia-mediated neuroinflammation. Strikingly, single-cell RNA sequencing revealed that the peptides-treated AD transgenic mice restored neuronal homeostasis with increased GABAergic neurons and decreased glutamatergic neurons. Furthermore, total cell-cell interaction strength increased while the AD risk gene Apoe expression decreased in the specific oligodendrocyte subtype of peptides-treated AD mice. Thus, our findings revealed that the peptides could improve cognition and memory capabilities and serve as promising structural templates for potential drugs against AD. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 32161160323

RING1 is an E3 ligase component of the polycomb repressive complex 1 (PRC1) with known roles in chromatin regulation and cellular processes such as apoptosis and autophagy. However, its involvement in inflammation and pyroptosis remains elusive. Here, we demonstrate that human RING1, not RING2, promotes K48-linked ubiquitination of Gasdermin D (GSDMD) and acts as a negative regulator of pyroptosis and bacterial infection. Indeed, we showed that loss of Ring1 increased S. typhimurium infectious load and mortality in vivo. Though RING1 deletion initially reduced M. tuberculosis (Mtb) infectious load in vivo, increased lung inflammation and impaired immune defense responses were later observed. Moreover, Ring1 knockout exacerbated acute sepsis induced by lipopolysaccharide (LPS) in vivo. Mechanistically, RING1 directly interacts with GSDMD and ubiquitinates the K51 and K168 sites of GSDMD for K48-linked proteasomal degradation, thereby inhibiting pyroptosis. Inhibition of RING1 E3 ligase activity by direct mutation or with the use of small molecule inhibitors increased GSDMD level and cell death during pyroptosis. Our findings reveal that RING1 dictates GSDMD-mediated inflammatory response and host susceptibility to pathogen infection, highlighting RING1 as a potential therapeutic target for combating infectious diseases.

Parkinson disease (PD) features progressive loss of nigrostriatal dopamine neurons, but how mitochondrial damage engages programmed cell death pathways remains unresolved. Here, we identify gasdermin E (GSDME), the caspase-3-activated executor of pyroptosis, as a critical mediator of neurodegeneration in toxin-based PD models. In primary neurons and SH-SY5Y cells, the mitochondrial complex I inhibitor MPTP/MPP⁺ triggered caspase-3 activation, GSDME cleavage, and lytic membrane rupture. Genetic silencing of Gsdme or its transcriptional regulator SP1 reduced neuronal death. In vivo, Gsdme deficiency preserved substantia nigra pars compacta dopaminergic neurons, improved motor performance, and mitigated anxiety- and depression-like behaviors after MPTP administration. Loss of Gsdme also dampened microglial and astrocytic activation and lowered proinflammatory cytokines in striatum and substantia nigra. Mechanistically, cleaved GSDME localized to mitochondria, disrupted membrane potential, increased reactive oxygen species, and precipitated organelle injury, thereby coupling mitochondrial dysfunction to pyroptotic cell death. These findings identify GSDME-mediated pyroptosis as a mechanistic link between mitochondrial toxicity and neuroinflammation in PD and nominate GSDME as a therapeutic entry point to slow disease progression. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 82572011

Glioblastoma (GBM), a highly aggressive form of glioma, poses serious harm to patients due to its extremely poor prognosis and severe resistance to chemotherapeutic agents. Although programmed necrosis (necroptosis) has been implicated in GBM progression, its precise function and biological significance in GBM remain incompletely defined. Here, we show that elevated expression of key necroptotic machinery proteins, including RIPK1 and MLKL, is positively associated with disease progression and predicts poor prognosis in glioma patients. Functionally, RIPK1 promotes glioblastoma cell proliferation, migration, and invasion. Genetic ablation of RIPK1 induces cell-cycle arrest and suppresses tumor growth in subcutaneous xenograft models, whereas pharmacological inhibition of RIPK1 with necrostatin-1 fails to restrict GBM cell expansion, suggesting that RIPK1 exerts oncogenic effects independent of its canonical necroptotic role. Notably, dual apoptosis- and necroptosis-inducing agents, ZZW115 and citronellol, synergize with temozolomide (TMZ)—the first-line chemotherapy for GBM—to enhance glioma cell death and increase tumor clearance in an orthotopic mouse glioma model. Collectively, these findings identify RIPK1 as a critical driver of glioma malignancy and underscore the therapeutic potential of activating necroptosis to augment TMZ efficacy, providing a framework for novel prognostic and treatment strategies in glioma.

The mpox virus (MPXV) mRNA cap N7 methyltransferase (RNMT) methylates guanosine at mRNA 5'-cap N7 positions to enable immune evasion. Here, we present a protocol for E1CTD-E12 complex purification and crystallization. We describe steps for rational sequence design of the complex, co-expression in E. coli, affinity chromatography purification, gel filtration, and crystallization optimization using vapor diffusion. We further outline X-ray diffraction data collection and structure determination. This reproducible framework enables structural analysis of viral mRNA-modifying enzyme complexes. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

![Graphic Abstract][1] Graphic Abstract Mycobacterium tuberculosis (Mtb) remains a major global health threat, partly due to the extensive cytotoxicity induced during infection. Although GSDMD-mediated excessive pyroptosis promotes pathogen dissemination and tissue damage in tuberculosis, the mechanism remains poorly understood. Here, we identify the effector EccB5, a component of the Mtb ESX-5 secretion system, as a key driver of pyroptosis and hyperinflammatory responses. EccB5 enhances Mtb virulence by inducing pyroptosis of macrophages, promoting bacterial dissemination and exacerbating lung pathology. Conditional knockdown EccB5 increases host cell viability. Mechanistically, EccB5 directly interacts with GSDMD, strengthens its association with caspase-1, and facilitates caspase-1-mediated cleavage of GSDMD both in vitro and in vivo . In summary, our findings uncover a precise mechanism by which Mtb modulates host responses and advances its pathogenicity. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, https://ror.org/01h0zpd94, 32161160323 [1]: pending:yes

Mpox, caused by monkeypox virus (MPXV), remains a global public-health threat, with limited vaccine options and few mechanism-based therapeutics. MPXV P1L, a Bcl-2 like immune-evasion protein, suppresses antiviral signaling and regulates apoptosis, whereas its structural basis keeps unknown. Here we report the crystal structures of P1L and its mutant D14 N at 2.4 Å and 2.9 Å resolution, respectively. P1L forms a homodimer stabilized by a salt-bridge network at the dimer interface, notably involving Asp14 and Arg7. Mutation D14 N disrupts the dimeric interface. Structural comparison with cellular antiapoptotic Bcl-2 protein reveals a conserved groove characteristic of BH3-ligand binding, consistent with evolutionary mimicry of host regulatory motifs. Functionally, P1L protects cells from TNFα-induced apoptosis and interacts with the BH3-only protein BID, linking the conserved groove to apoptosis blockade. These findings identify P1L as a viral immune-evasion factor and provide a structural framework for discovering groove-targeting inhibitors with potential anti-poxvirus activity.

Phthalate esters (PAEs) are prevalent environmental contaminants, with their biodegradation efficiently driven by microorganisms through ester bond hydrolysis. This study investigates the mechanism of Poc14, a novel family IV esterase, using x-ray crystallography, bioinformatics, biochemistry and site-directed mutagenesis. Phylogenetic analysis classifies Poc14 as a family IV esterase with conserved catalytic motifs crucial for its activity. Poc14 retains over 80% activity at 50°C for 4 h and tolerates up to 5% methanol or DMF, though surfactants like Tweens inhibit its function. Poc14 activity is independent of metal ions, and the addition of EDTA further enhances its activity by approximately 130%. The 1.8 Å crystal structure reveals a CAP domain and two substrate channels. Enzyme assays show Poc14 hydrolyses short-chain diethyl phthalate (DEP) (Km = 0.068 mM, Vmax = 9975 μM/min/mg) but not long-chain di(2-ethylhexyl) phthalate (DEHP) due to steric hindrance. Molecular docking assessed Poc14's potential to hydrolyse DEP and DEHP after residue mutations, resulting in the Poc14-AAG variant. Poc14-AAG could hydrolyse one bond of DEHP and diester bonds of DEP. Our study positions Poc14 as a promising enzyme for environmental remediation, with potential for optimising DEHP degradation and exploring dimerisation effects.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Z-DNA binding protein 1 (ZBP1) is a crucial player in the intracellular recognition of Z-form nucleic acids (Z-NAs) through its Zαβ domain, initiating downstream interactions with RIPK1 and RIPK3 via RHIM domains. This engagement leads to the assembly of PANoptosomes, ultimately inducing programmed cell death to curb pathogen dissemination. How Zαβ and RHIM domain cooperate to trigger Z-NAs recognition and signal transduction remains unclear. Here, we show that ZBP1 condensate formation facilitates Z-NAs binding and antiviral signal transduction. The ZBP1 Zαβ dimerizes in a concentration-dependent manner, forming characteristic condensates in solutions evidenced by DLS and SAXS methods. ZBP1 exhibits a binding preference for 10-bp length CG (10CG) DNA and Z-RNA ligand, which in turn enhanced Zαβ dimerization, expediting the formation of droplet condensates in vitro and amyloid-like puncta in cells. Subsequent investigations reveal that Zαβ could form condensates with liquid-liquid phase separation property upon HSV and IAV infections, while full-length ZBP1 forms amyloid-like puncta with or without infections. Furthermore, ZBP1 RHIM domains show typical amyloidal fibril characterizations and cross-polymerize with RIPK1 depending on the core motif of 206IQIG209, while mutated ZBP1 could impede necroptosis and antiviral immunity in HT-29 cells. Thus, ZBP1 condensate formation facilitates the recognition of viral Z-NAs and activation of downstream signal transduction via synergic action of different domains, revealing its elaborated mechanism in innate immunity.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

Amyloidosis is characterized by the abnormal accumulation of misfolded proteins, called amyloid fibrils, leading to diverse clinical manifestations. Recent studies on the amyloidogenesis of SARS-CoV-2 protein segments have raised concerns on their potential link to post-infection neurodegeneration, however, the mechanisms remain unclear. Herein, we investigated the structure, stability, and amyloidogenic propensity of a nine-residue segment (SK9) of the SARS-CoV-2 envelope protein and their impact on neuronal protein α-synuclein (αSyn) aggregation. Specifically, the amino acid sequence of the SK9 wildtype has been modified from a basic and positively charged peptide (SFYVYSRVK), to a nearly neutral and more hydrophobic peptide (SAAVASAVK, labelled as SK9 var1), and to an acidic and negatively charged peptide (SDAVANAVK, labelled as SK9 var2). Our findings reveal that the SK9 wildtype exhibited a pronounced amyloidogenic propensity due to its disordered and unstable nature, while the SK9 variants possessed more ordered and stable structures preventing the amyloid formation. Significantly, the SK9 wildtype demonstrated distinct effect on αSyn aggregation kinetics and aggregate morphology to facilitate the formation of αSyn aggregates with enhanced resistance against enzymatic degradation. This study highlights the potential of modifying short peptide sequences to fine-tune their properties, providing insights into understanding and regulating viral-induced amyloid aggregations.

Gasdermin-E (GSDME), the executioner of pyroptosis when cleaved by caspase 3, plays a crucial role in tumor defense and the response to chemotherapy drugs in cells. So far, there are poorly known mechanisms for the expression regulation of GSDME during cell death. Here, we identify the transcription factor Sp1 (Specificity protein 1) as a positive regulator of GSDME-mediated pyroptosis. Sp1 directly interacts with the GSDME promoter at −36 ~ −28 site and promotes GSDME gene transcription. Further, Sp1 knockdown or inhibition suppresses GSDME expression, thus reducing chemotherapy drugs (topotecan, etoposide, doxorubicin, sorafinib and cisplatin) induced cell pyroptosis. The regulation process synergizes with STAT3 (Signal transducer and activator of transcription 3) activity and antagonizes with DNA methylation but barely affects GSDMD-mediated pyroptosis or TNF-induced necroptosis. Our current finding reveals a new regulating mechanism of GSDME expression, which may be a viable target for the intervention of GSDME-dependent inflammatory diseases and cancer therapy.