Pancreatic cancer is one of the most invasive malignant tumors and remains a leading cause of cancer-related deaths, among which pancreatic ductal adenocarcinoma (PDAC) serves as the most common type of pancreatic cancer. Metastasis development is one of the critical reasons for the high mortality in PDAC. Previous studies usually used single or two omics integrated methods to discover biological processes and biomarkers related to the tumorigenesis and prognosis of PDAC. However, the molecular mechanisms of PDAC metastasis remain largely unknown. Therefore, we intended to integrate multi-omics data to explore the metastasis of PDAC. To identify genes or pathways that have potential roles in PDAC metastasis at different levels, we performed whole-exome sequencing (WES), RNA sequencing (RNA-Seq) and LC-MS/MS-based proteomics analysis on primary-derived (BxPC-3, MIA PaCa-2) and metastasis-derived (CFPAC-1, HPAF-II, SW1990) PDAC cell lines. Through multi-omics integrative analysis, we found that ATAD1 (ATPase Family AAA Domain Containing 1) knockdown significantly suppressed migration and invasion capacities of PDAC cells. Finally, analysis of the TCGA pancreatic adenocarcinoma (TCGA-PAAD) cohort further revealed that high ATAD1 expression was associated with significantly shorter overall survival (log-rank p = 0.012) and progressively increased from well- to poorly differentiated tumors, implicating ATAD1 as a potential regulator of metastatic propensity and dedifferentiation in pancreatic cancer.

Antibiotic resistance has become a serious threat to global health. It frequently emerges in real-world environments such as biofilms, where antibiotic gradients impose selective pressures that drive bacterial adaptation. However, the mechanisms underlying resistance development remain poorly understood, largely due to challenges in experimentally controlling confounding parameters. Here, we report a 3D microfluidic platform that enables real-time monitoring of bacterial dynamics under well-defined antibiotic gradients, along with programmable cell retrieval to identify key factors in resistance development. We show that motile Escherichia coli migrated randomly within the gradients regardless of antibiotic class (i.e., gentamicin, ciprofloxacin, and ampicillin). Resistance emerged only when a critical mass of bacteria sustained migration through the gradient for a sufficiently duration, revealing a previously unrecognized migration-duration threshold for adaptive evolution. Whole-genome resequencing revealed gene mutations in the resistant cells, including mutations in ATP synthase-related genes (i.e., atpG, atpD, and atpF). A trade-off between resistance and cell growth was also observed. Similar phenomena were observed in other motile bacteria species, including Serratia marcescens. Overall, we demonstrate that cell motility is a critical determinant of resistance development in heterogeneous antibiotic landscapes. Targeting cell motility and growth could offer effective strategies to combat antibiotic resistance in motile bacteria.

Developing phototheranostic agents that combine high reactive oxygen species (ROS) generation, favorable fluorescence wavelength and intensity, and prolonged tumor retention remains a major challenge. Although molecular engineering enables precise tuning of molecular structures and properties, it often relies heavily on tedious organic synthesis. Herein, we introduce an intermolecular noncovalent locking strategy to construct systems with optimized photophysical properties for efficient phototheranostics. Aggregation-induced emission luminogens and aggregation-caused quenching luminogens are locked together to construct via multiple noncovalent interactions (e.g., C-H···N, C-H···S, S/O···N, etc.), suppressing π-π stacking and enhancing fluorescence, while intermolecular charge transfer induces a red-shifted emission and modulates ROS generation pathway. The strong intermolecular noncovalent locks also enforce tight molecular packing, leading to the formation of shuttle-like nanoassemblies rather than conventional nanospheres. Notably, this morphology enables effective tumor accumulation, prolonged retention, and highly efficient phototherapeutic outcome, achieving >90% tumor inhibition with a single injection. The proposed "intermolecular noncovalent locks" approach thus offers a paradigm for the development of advanced phototheranostic agents.

Ribosome display is a powerful engineering research tool for the high-throughput selection of peptides or proteins, which results in the generation of high-performance binders against nearly any antigen of interest. As a cell-free display system, ribosome display has been well developed with many outstanding achievements for over 20 years. Compared with other related display techniques, ribosome display shows unique advantages and development prospects. This tool has been successfully exploited for the selection of functional and specific binders in vitro. Here, we describe methods for the construction and characterization of ribosome-displayed natural nanoantibody libraries, by which specific nanoantibody screening can be performed.

Deep vein thrombosis (DVT) is a prevalent vascular disorder associated with high morbidity and mortality. Evidence suggests that ferroptosis plays a critical role in thrombus formation. Epigallocatechin-3-gallate (EGCG), the primary bioactive component of green tea, possesses antioxidant and anti-inflammatory properties, but its role and molecular mechanism in regulating ferroptosis in DVT remain unclear. A mouse model of DVT was established to evaluate the therapeutic effects of EGCG. Histological, biochemical, and molecular assays were used to assess thrombus formation, oxidative stress, and ferroptosis markers. Endothelial progenitor cells (EPCs) were used to explore mechanistic pathways in vitro. Functional assays were conducted to investigate the regulatory relationships between EGCG, NRF2, and NEDD4. Additionally, clinical relevance was assessed using peripheral blood and IVC tissues from DVT patients. EGCG treatment significantly reduced thrombus number and size, improved hypercoagulability, and lowered serum D-dimer levels in DVT mice. EGCG inhibited ferroptosis by reducing reactive oxygen species (ROS) production, iron accumulation, MDA levels, and restoring SLC7A11 and GPX4 expression. Mechanistically, EGCG transcriptionally activated NEDD4 expression in EPCs. NEDD4 overexpression mimicked anti-ferroptotic effects of EGCG, while its knockdown reversed them. Additionally, NRF2 was identified as the transcriptional activator of NEDD4, binding to its promoter and enhancing its expression. In DVT patient samples, NEDD4 expression was significantly downregulated and negatively correlated with ROS and iron levels. This study uncovers a novel EGCG/NRF2/NEDD4 axis that mitigates DVT progression by suppressing ferroptosis and oxidative stress. These findings highlight NEDD4 as a potential therapeutic target and suggest EGCG as a promising agent for DVT intervention.

BackgroundCD73 is a key enzyme in the adenosine-mediated immunosuppressive pathway and represents an attractive target for cancer immunotherapy. Here, we aimed to develop novel anti-CD73 antibodies with dual mechanisms of action by simultaneously inhibiting enzymatic activity and promoting receptor internalization to more effectively disrupt the adenosine barrier within the tumor microenvironment.MethodsTwo monoclonal antibodies, CR201 and CR202, were generated and characterized for cross-species reactivity, inhibition of both membrane-bound and soluble CD73 enzymatic activity, and domain-specific epitope recognition. Functional assays were performed to evaluate reversal of AMP-mediated T-cell suppression, restoration of IFN-γ secretion, and induction of CD73 internalization. In vivo efficacy was assessed using an A375 melanoma xenograft model.ResultsCR201 and CR202 specifically bound human and cynomolgus CD73 and recognized distinct structural domains, with CR202 targeting the N-terminal domain and CR201 binding the C-terminal domain. Functionally, CR202 demonstrated greater potency against membrane-bound CD73, whereas CR201 achieved complete inhibition of soluble CD73 activity. Both antibodies effectively restored T-cell proliferation and IFN-γ production and promoted internalization of surface CD73. In vivo, treatment with either antibody significantly suppressed tumor growth without observable toxicity.ConclusionCR201 and CR202 are domain-specific, dual-mechanism anti-CD73 antibodies that integrate potent enzymatic blockade with receptor internalization, thereby enhancing antitumor immune responses. These findings highlight the therapeutic potential of targeting distinct functional domains of CD73 to overcome adenosine-mediated immunosuppression in cancer.

Radiation enteritis is a common complication in patients undergoing abdominal radiotherapy. Current management strategies face significant limitations: clinical agents like amifostine are hindered by systemic side effects and demanding administration; direct supplementation with radioprotective metabolites such as propionate suffers from low bioavailability and transient action; and conventional probiotics lack targeted therapeutic output. To address these challenges, we engineered Escherichia coli Nissle 1917 to function as a living therapeutic that continuously produces and delivers propionate directly in the gut. This propionate-engineered probiotic achieved a production yield of 181.33 ± 4.27 mg/L in vitro. In a mouse model of abdominal irradiation, this engineered bacterium alleviated radiation-induced intestinal damage by continuously releasing propionate and enhancing intestinal epithelial barrier function. Multi-omics analysis revealed that the engineered bacterium could restore intestinal microbiota homeostasis, enhancing the abundance of advantageous bacteria with radioprotective properties (e.g., Dubosiella, Akkermansia). Moreover, it modulated intestinal microbiota metabolism, influencing the metabolism of ascorbic acid, aldoses, and other metabolites. Additionally, it protected the intestinal mucosal barrier from radiation-induced damage, which was associated with the modulation of the SOCS1/JAK2/STAT3 signaling pathway. This study introduces a novel biological therapy to mitigate the side effects of radiotherapy and could open new avenues for preventing and treating radiation-induced intestinal injury.

The precise control of optical properties in molecular systems remains a challenge for phototherapy. Herein, the strategic combination of aggregation-caused quenching (ACQ) and aggregation-induced emission (AIE) molecule creates ACQ@AIE bimolecular systems with tunable optical properties, which are almost unattainable by single-component materials. Through systematic investigation of three ACQ@AIE bimolecular systems, it is established that molecule structure size differentials dictate their intermolecular interactions and consequent optical behaviors. Crucially, AIE molecule with a smaller structure size promotes ACQ molecule clustering to enhance the photothermal effect, while when the size becomes larger, particularly approaching that of ACQ molecule, facilitating π-π stacking and boosting the photodynamic effect. These distinct assembly modes revealed through combined experimental and theoretical analyses, enable precise regulation of photothermal versus photodynamic effects by simply regulating the structure size and ratio of ACQ and AIE molecules. Building on these mechanistic insights, the optimal molecule combination of ACQ@AIE bimolecular system is engineered into nanoparticles that exhibit mild photothermal effect, strong photodynamic effect, and excellent tumor accumulation and retention, achieving near-complete tumor eradication with minimal treatment cycles while maintaining good biosafety. This work not only elucidates the fundamental structure size-interaction-property relationships in ACQ@AIE bimolecular systems but also provides generalizable strategies for developing intelligent photo theranostic materials through controlled intermolecular interaction.

Recent progress in synthetic biology has empowered engineered probiotics to sense tumor-specific physicochemical signals, thereby facilitating targeted in situ drug delivery. Here, an engineered probiotic consortium capable of integrating multiple tumor microenvironment (TME) signals and orchestrating multi-therapeutic payloads release through an orthogonal quorum-sensing system is designed. The probiotic consortium can respond to three characteristic TME parameters, pH, hypoxia, and high-lactate levels, in order to achieve controlled release of lactate depletion enzyme (LdhA) for metabolic environment improvement and the programmed death ligand 1 (PD-L1) nanobody for immune checkpoint inhibition. Using the humanized PD-1 mouse model bearing hPD-L1 MC38 tumor and the humanized peripheral blood mononuclear cells (PBMC) mouse model bearing HT-29 tumor, it is demonstrated that this self-regulating microbial consortium achieves sustained oscillations and significantly suppresses tumor progression. Mechanistic studies reveal that the antitumor efficacy activates CD8+, CD4+, and IFN-γ+ T cells, coupled with diminished immunosuppressive Foxp3+ regulatory T cell infiltration. This work advances the development of engineered live biotherapeutic products for cancer therapy and provides a modular platform for microbial consortium-based precision medicine.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality, and the incidence of early-onset colon cancer has been increasing globally in recent years. The development of immunotherapies for colon cancer is critical for providing new treatment strategies to combat drug resistance. Here, a bispecific nanobody against PD-L1 (BsNb-PD-L1) constructed using genetically encoded noncanonical amino acids (ncAAs) is reported. A computational protocol was developed to identify appropriate sites in the nanobody for incorporating p-acetylphenylalanine (pAcF). Variants of nanobodies PV2 and PV3 with pAcF incorporated were conjugated with linkers containing an aminooxy functionality to enable oxime ligation. The resulting PV2-S71 + PV3-N77 bispecific nanobody (BsNb-ncAA) exhibited higher thermostability and binding affinity compared to the nanobody monomers and the BsNb constructed by simply fusing two proteins. Moreover, in an in vitro phagocytosis model, the BsNb-ncAA exhibited improved capability to inhibit immune evasion and showed stronger biological activity compared to the fusion protein PV2-PV3. Furthermore, the BsNb-ncAA resulted in a marked increase in the number of CD8+ T cells within tumor tissues and demonstrated efficient inhibitory effects against colon tumor growth in vivo. Our study provides a general strategy for constructing BsNbs, which has potential applications in other cancer immunotherapy. A bispecific nanobody (BsNb-ncAA) with sites found by computation to improve binding, inhibit immune evasion, boost CD8+ T cells, and suppress colon tumor growth, offering promising colorectal cancer immunotherapy strategies.

BACKGROUND:The pathogenesis of sepsis-induced cardiomyopathy (SIC) remains unclear, presenting a complex challenge in both clinical practice and research. Gypenoside XLIX (Gyp-XLIX) has garnered significant attention for its anti-inflammatory, antioxidant, and various other pharmacological activities; however, its protective effect on SIC has not been thoroughly investigated. PURPOSE:This study aims to explore the effect of Gyp-XLIX on SIC and its potential therapeutic mechanisms. METHODS:A sepsis mouse model was established through cecal ligation and puncture surgery and treated with Gyp-XLIX. The modeling was evaluated through sepsis model scoring, survival analysis, weight changes, wet-to-dry weight ratio, H&E staining, biochemical assays and cardiac ultrasound. The mechanism exploration was deteced using proteomics sequencing, Western blotting, qPCR, biochemical analysis, and reactive oxygen species assays. Additionally, target proteins of Gyp-XLIX were identified through LC-MS/MS proteomics analysis, molecular docking, CESTA, DARTS, SPR with SIRT1 being a primary focus. Bioinformatics and Immunoprecipitation mass spectrometry analysis, and immunohistochemistry were employed to further elucidate the downstream target of SIRT1, specifically the YAP-NLRP3 pathway. The degradation mechanisms of YAP-NLRP3 were explored using MG132, chloroquine, cycloheximide, and co-immunoprecipitation. Corresponding rescue experiments were conducted in H9C2 cells for further validation. RESULTS:Gyp-XLIX effectively mitigates SIC, reduces cardiac inflammation, and restores cardiac structural, left ventricular functional abnormalities, and oxidative stress balance. Proteomics results showed that Gyp-XLIX binded to SIRT1 and alleviates inflammation and oxidative stress. Whereas, SIRT1 is low expressed and alleviated oxidative stress and NLRP3 inflammasome activation in SIC. Mechanically, Gyp-XLIX binded to SIRT1 improved its stability and promoted the deacetylation and phosphorylation of downstream YAP, which in turn regulated YAP degradation. This process subsequently affects the ubiquitination of NLRP3, downstream of YAP, at the K27 site, leading to its degradation and, consequently, alleviating inflammation and oxidative stress during SIC. CONCLUSION:Gyp-XLIX targets SIRT1 to block YAP-NLRP3 activation and improve SIC, suggesting that Gyp-XLIX is an effective protective agent against SIC and represents a promising therapeutic strategy.

Claudin18.2 (CLDN18.2) is a stomach-specific tight junction protein aberrantly exposed in multiple cancers, particularly gastric and pancreatic malignancies, making it an attractive therapeutic target. Here, we developed CR101-ADC, a novel antibody-drug conjugate (ADC) constructed from a fully human anti-CLDN18.2 monoclonal antibody conjugated to the microtubule inhibitor MMAE via a cleavable linker. The CR101 antibody exhibits high affinity and specificity for claudin18.2, demonstrating superior tumor cell-killing efficiency compared to IMAB362. Additionally, CR101 shows cross-species reactivity in humans, mice, and monkeys, simplifying preclinical pharmacology and efficacy studies.In vitro pharmacodynamic studies confirmed that CR101-ADC effectively induces targeted cytotoxicity, while multiple tumor models demonstrated its potent anti-tumor activity and favorable safety profile with minimal toxicity. Transcriptomic analysis revealed that CR101-ADC primarily affects cytoskeletal, inflammatory, and stress pathways and represents a promising candidate for the treatment of gastrointestinal malignancies. Collectively, these findings demonstrate that CR101-ADC combines high specificity, potent intracellular drug delivery, and favorable safety, representing a promising next-generation therapeutic candidate for CLDN18.2-positive gastrointestinal cancers.

Aim or purpose: Diabetes aggravates the risk and severity of periodontitis, making the treatment of periodontitis in diabetic patients (DP) a significant challenge. Intestinal butyrate levels are critical regulators of DP and a potential therapeutic target. Butyrate-producing engineered bacteria have been demonstrated as an effective strategy for intestinal butyrate delivery. This study aimed to evaluate the therapeutic effect of butyrate-producing engineered probiotic on diabetic periodontitis and analyze its effects on the ''gut-bone axis''. Materials and methods: Four-week-old male C57BL/6 mice with streptozotocin-induced diabetes and ligature-induced periodontitis received engineered probiotic for 6 weeks (approved by the Animal Ethics Committee). Periodontal repair was assessed via micro-CT and H&E staining. Gut microbiota (16S rRNA), serum cytokines (ELISA), Th17/Treg balance (flow cytometry), and metabolites (LC-MS) were analyzed. Results: The results confirmed that the engineered bacteria treated group exhibited significant increases in periodontal bone mass, bone density (BMD), bone volume (BV), trabecular volume fraction (BV/TV), and trabecular thickness (P < 0.05). Serum inflammatory cytokine levels decreased significantly, and the balance of Th17 cells and Treg cells shifted toward an anti-inflammatory phenotype in immune organs (P < 0.05). 16S rRNA sequencing and untargeted metabolomics revealed that engineered bacterial treatment alleviated dysbiosis induced by diabetic periodontitis, promoted the proliferation of beneficial bacteria, and elevated serum levels of metabolites such as Camellimidazole B (P < 0.05). Conclusions: This study demonstrates that engineered probiotic mitigate diabetic alveolar bone loss by restoring gut homeostasis, attenuating systemic inflammation, and promoting osteogenesis.

Bispecific antibodies (BsAbs) represent a promising strategy for cancer immunotherapy. Challenges in immunotherapy include inefficient early events in the immune response cycle, such as antigen presentation and T cell priming. Background stimulation of CD40 with agonistic antibodies is a promising strategy to enhance the therapeutic efficacy of immune checkpoint inhibitors (ICIs). Assisted by Alphafold2(AlphaFold-Multimer), we developed a humanized CD40 agonistic antibody that exhibits activation only in the presence of cross-linking. It also demonstrates that the current AlphaFold2(AlphaFold2-Multimer) can predict antibody-antigen complexes. Due to the unique epitope, it demonstrates superior activation compared to APX005M (S267E). Building upon this, we created a novel bispecific antibody (anti-PD-L1/CD40 bispecific antibody, referred to as ''BA4415'') designed to activate CD40 signaling specifically in the context of PD-L1 while simultaneously blocking PD-1/PD-L1 signaling. Results from functional evaluations using effector cells revealed the superior biological activity of BA4415 compared to the combination of each monoclonal antibody. BA4415 demonstrated the ability to enhance T-cell cytokine release in vitro assays, exhibiting superior functional attributes compared to the anti-PD-L1 antibody. Furthermore, in humanized transgenic mice challenged with huPD-L1-expressing tumor cells, BA4415 induced superior anti-tumor activity. This novel anti-PD-L1/CD40 bispecific antibody holds potential for strong anti-tumor therapeutic efficacy by selectively restricting CD40 stimulation in tumors.

Radiotherapy is a method of treating cancer through radiation aimed at killing cancer cells or inhibiting their growth. However, radiotherapy has numerous side effects because it kills tumors while causing damage to normal cells or tissues. The literature shows that radiation can cause damage to heart tissue. This study found that engineered yeast that produced butyrate can maintain small intestinal barrier function by recovering GPR109A to reduce intestinal damage caused by abdominal irradiation in mice. We unexpectedly found that engineered yeast could mitigate irradiation-induced heart damage via the gut-heart axis. Mechanistically, engineered yeast enhanced taurine and nicotinamide metabolism by increasing the relative abundance of Akkermansia and Lachnospiraceae_NK4A136; then, yeast modulated cardiac function by activating the Sgcg and Nppa genes to attenuate cardiac damage induced by abdominal irradiation. Finally, we confirmed that engineered yeast mitigated cardiac damage caused by total body irradiation, which protected other vital organs through the intestinal tract. This study has a profound impact on cancer treatment, the emergence of engineered yeast will alleviate radiotherapy side effects and benefit patients.

Synthetic microbial consortia (SyMCon), composed of different artificially engineered bacteria, offer a promising alternative to live biotherapeutic products for disease therapy. These microbial communities use a quorum sensing (QS) mechanism that allows for precise and low-interference communication. Compared to current therapy using only one engineered bacterium, they can reduce the metabolic load of one bacterium, thereby increase drug production, and respond to a wider variety of disease-related signals. This review summarizes recent developments and emphasizes the unique advantages of SyMCon, then proposes multiple perspectives of designs for therapeutic SyMCon. Although SyMCon possess advantages in colonization, responding to multiple environmental signals, and delivering high-yield drugs, future developments should focus on orthogonal QS systems, complex genetic circuits, and modular consortia. More complex consortia allow for better therapeutic functionality, and modular consortia allow for the rapid replacement of disease-specific components, which could unlock the potential of the next generation of personalized microbial therapy.

Cholesterol, a key steroid in cell membranes and organelles, is vital for processes like migration and apoptosis, regulated by biosynthesis, uptake, and esterification. Its metabolism significantly impacts stem cell fate via pathways like AKT/FOXO1 and Notch, influencing proliferation, differentiation, and migration. Thus, cholesterol emerges as a promising therapeutic target. This review aims to shed light on the impact of cholesterol metabolism on stem cells and explores cholesterol-targeting drugs’ potential in regenerative medicine. Our purpose is to elucidate cholesterol’s role in stem cell function and guide therapeutic development.