ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectiveTo compare the differences between wild Alpiniae Oxyphyllae Fructus（WAOF） and cultivated Alpiniae Oxyphyllae Fructus（CAOF） through a traditional quality evaluation system for medicinal materials. MethodsA total of 10 batches of WAOF and 12 batches of CAOF samples were collected from various regions of Hainan province. Relevant analytical methods from the 2020 edition of the Pharmacopoeia of the People's Republic of China were employed to observe the characteristics of WAOF and CAOF， followed by microscopic identification， thin-layer chromatography（TLC） identification， moisture content（toluene method）， total ash， acid-insoluble ash， water-soluble and alcohol-soluble extracts（hot dipping method）， water-soluble protein， total polysaccharides and total flavonoids（ultraviolet spectrophotometry）， and volatile oil content（method A under general rule 2204）. The contents of five active components（protocatechuic acid， chrysin， kaempferol， tectochrysin and nootkatone） were quantified using ultra-performance liquid chromatography（UPLC）， and the antioxidant activity was evaluated. Building upon traditional quality evaluation of AOF， quantitative measurements were conducted on its appearance traits including diameter， length， plumpness（diameter/length ratio）， and color. Canonical correlation analysis was performed using SPSS 26.0 to explore relationships between appearance traits and intrinsic quality. ResultsNo significant differences were observed between WAOF and CAOF in microscopic observation， TLC identification， moisture content， protocatechuic acid content， kaempferol content， odor， or antioxidant activity measured by 2，2ʹ-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid）（ABTS） method. WAOF exhibited significantly higher levels in water-soluble extracts， alcohol-soluble extracts， total polysaccharide content， water-soluble protein content， 100-grain weight， length， and total color difference（ΔE^*ab） compared to CAOF（P<0.01）. In contrast， CAOF showed significantly higher levels of total ash， acid-insoluble ash， content of total flavonoids， volatile oil content， chrysin content， tectochrysin content， nootkatone content， diameter， plumpness， lightness（L^*）， red-green chromaticity（a^*）， yellow-blue chromaticity（b^*）， and antioxidant activity measured by 1，1-diphenyl-2-picrylhydrazyl（DPPH） method compared to WAOF（P<0.01）. Correlation analysis between 7 phenotypic traits and 8 quality traits revealed that among the phenotypic traits， plumpness， L^*， a^*， and b^* exerted significant influence on intrinsic quality. Among the quality traits， total flavonoids， volatile oils， nootkatone， chrysin， and tectochrysin contributed substantially to intrinsic quality. ConclusionPlumpness， L^*， a^*， and b^* of AOF significantly influence its intrinsic quality， and higher values of these parameters indicate relatively superior intrinsic quality. The comprehensive quality evaluation reveals that CAOF samples collected in this study are superior to their wild counterparts.

ObjectiveTo investigate the novel post-translational modifications (PTMs) of SnRK2.6, a central component in the abscisic acid (ABA) signaling pathway, such as SUMOylation, and to establish a foundation for revealing the physiological functions and molecular mechanisms of SnRK2.6 regulated by these new modifications. MethodsThe interaction between SnRK2.6 and the SUMO E3 ligase SIZ1, as well as members of the SUMO protease family, was examined using yeast two-hybrid and in vitro pull-down assays. An in vitro SUMOylation system in Escherichia coli was utilized to determine whether SnRK2.6 undergoes SUMOylation. Mass spectrometry, combined with site-directed mutagenesis of candidate lysine residues, was employed to identify potential SUMOylation sites on SnRK2.6. In vitro de-SUMOylation assays were performed to assess whether SUMO proteases interacting with SnRK2.6 could catalyze the removal of SUMO moieties from modified SnRK2.6. The protein stability of SnRK2.6 was assessed in a cell-free degradation assay using bacterial-purified SnRK2.6 incubated with total protein extracts from Col and siz1 mutant seedlings. To dissect the genetic relationship between SnRK2.6 and SIZ1, stomatal aperture assays were performed under ABA treatment using snrk2.6, siz1, and snrk2.6 siz1 double mutant plants. ResultsSnRK2.6 physically interacts with SIZ1 and the SUMO protease ESD4, with the binding domains localized to the C-terminal region of SIZ1 and the N-terminal region of ESD4, respectively. SnRK2.6 was found to be SUMOylated, exhibiting two distinct high-molecular-mass bands ranging from 70 to 100 ku, indicative of modified forms. Bioinformatics analysis predicted four putative SUMOylation sites on lysine residues K57, K63, K142, and K190. Mass spectrometry identified three SUMOylation sites on K63, K142, and K174. However, individual or combinatorial point mutations on these sites had minimal impact on the pattern or intensity of SUMOylation signals, suggesting that these residues may not be responsible for the SUMOylation on SnRK2.6. Instead, such mutations only weaken the protein stability or accelerate the protein mobility of SnRK2.6. Therefore, the exact SUMOylation sites on SnRK2.6 remain unidentified. In de-SUMOylation experiments, incubation of GST-ESD4 with SUMOylated SnRK2.6 for 1-2 h led to the near-complete disappearance of both SUMOylated bands. In contrast, neither the GST control nor the catalytically inactive mutant GST-ESD4^C448S exhibited any de-SUMOylation activity. In protein turnover experiments, SnRK2.6 exhibited markedly enhanced half-life in siz1 compared with Col, indicating that SIZ1-dependent SUMOylation promotes SnRK2.6 turnover. Phenotypically, snrk2.6 mutants were completely insensitive to ABA-induced stomatal closure; siz1 mutants displayed pronounced hypersensitivity; and the snrk2.6 siz1 double mutant phenocopied snrk2.6—showing no significant response to ABA beyond that of the snrk2.6 mutant. These data indicate that SIZ1 acts as a negative regulator of ABA-triggered stomatal closure and SnRK2.6 functions as a positive regulator, and the inhibitory activity of SIZ1 is strictly dependent on SnRK2.6, placing SnRK2.6 genetically upstream of SIZ1 in the ABA signaling pathway. ConclusionSnRK2.6 undergoes SUMOylation, although the specific SUMOylation sites have not been defined. SnRK2.6 is dynamically regulated by reversible SUMOylation—catalyzed by SIZ1 and reversed by ESD4—which controls its protein stability. SUMOylation acts as a destabilizing signal for SnRK2.6, and SIZ1 exerts its negative effect on ABA-triggered stomatal closure probably through promoting SnRK2.6 degradation via SUMOylation. These findings uncover SUMOylation as a critical regulatory layer fine-tuning SnRK2.6 abundance in ABA signaling.

ObjectiveTo investigate the distribution characteristics and correlation of paired oral and esophageal microbiota in patients with esophageal squamous cell carcinoma （ESCC）. MethodsA total of 46 pathologically confirmed ESCC patients were enrolled， and oral swabs and cancerous tissue samples were collected. The microbial composition was analyzed using 16S rRNA gene sequencing， and bioinformatics methods were employed to compare the microbiota diversity， compositional differences， and correlations between the two sites. ResultsThe Alpha diversity of the oral microbiota in ESCC patients was significantly higher than that of their esophageal microbiota， though the overall community structure was similar between the two sites. LEfSe analysis revealed that Spirochaetes， Prevotella， Catonella， Enterococcus， Fusobacterium periodonticum， Veillonella parvula， Capnocytophaga sputigena， and Neisseria subflava were significantly enriched in ESCC tumor tissues. In contrast， Actinobacteria， Proteobacteria， Streptococcus， Neisseria， Haemophilus， Streptococcus mitis， Haemophilus parainfluenzae， Porphyromonas gingivalis， Haemophilus haemolyticus， and Prevotella tannerae were significantly enriched in the oral cavity. Correlation analysis demonstrated significant associations between the two sites at taxonomic levels such as Spirochaetes， Treponema， Granulicatella elegans， Neisseria macacae， Treponema amylovorum， Fusobacterium periodonticum， and Neisseria meningitidis （P<0.05）. ConclusionThe oral and esophageal microbiota in ESCC patients exhibit both distinct differences and similarities. Oral microbiota may influence the esophageal microenvironment through migration or local immune modulation.

ObjectiveColorectal cancer (CRC) is a highly heterogeneous malignancy, and traditional Chinese medicine (TCM) syndrome differentiation is widely used in its clinical management. However, the biological basis underlying different TCM syndromes remains insufficiently understood. This study aimed to investigate the proteomic differences between qi-blood deficiency syndrome (QBDS) and damp-heat syndrome (DHS) in CRC patients and to establish a “syndrome-differential protein-herbal component” network for exploring potential molecular mechanisms and therapeutic targets associated with syndrome-specific treatment strategies. MethodsTumor tissue samples were collected from 10 patients with pathologically confirmed CRC, including 5 patients diagnosed with QBDS and 5 with DHS according to standardized TCM syndrome differentiation criteria. Tandem mass tag (TMT)-based quantitative proteomics was employed to identify differentially expressed proteins (DEPs) between the two syndrome groups. Functional enrichment analyses, including Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were performed to characterize the biological functions and signaling pathways associated with the identified DEPs. Least absolute shrinkage and selection operator (LASSO) regression analysis was further applied to screen key syndrome-related proteins and construct a core protein prediction model. Subsequently, molecular docking was conducted to evaluate the binding affinities between representative herbal compounds, astragaloside IV and berberine, and the identified core proteins. Finally, cell counting kit-8 (CCK-8) assays were performed in HCT 116 and HT-29 colorectal cancer cell lines to validate the anti-proliferative activities of these compounds in vitro. ResultsA total of 44 DEPs were identified between QBDS and DHS tissues, including 21 upregulated and 23 downregulated proteins in the QBDS group relative to the DHS group. Functional enrichment analyses revealed that these proteins were primarily associated with antiviral immune responses, regulation of inflammatory signaling, NF‑κB signaling pathways, cholesterol metabolism, and other biological processes relevant to tumor progression and host immune regulation. LASSO regression analysis identified eight core proteins with potential syndrome-specific significance. Among them, ST7, GPCPD1, and PODXL were closely associated with QBDS, whereas FAT1, GRB7, KRT7, MAFF, and MMP8 were associated with DHS. Molecular docking demonstrated favorable interactions between astragaloside IV and QBDS-related proteins, with binding energies lower than -5 kcal/mol, indicating stable binding activity. Berberine exhibited even lower binding energies and stronger binding specificity toward DHS-related core proteins. In vitro experiments further confirmed that both compounds inhibited CRC cell proliferation, while berberine displayed significantly greater anti-proliferative efficacy. The lowest 48 h IC₅₀ value of berberine reached 15.21 mg/L in CRC cells, indicating a stronger growth-inhibitory effect than astragaloside IV. ConclusionCRC patients with QBDS and DHS exhibit distinct proteomic characteristics, suggesting that different TCM syndromes possess unique molecular signatures. The identified core proteins may serve as potential biomarkers for syndrome differentiation, while astragaloside IV and berberine may exert therapeutic effects through targeting syndrome-specific molecular networks. These findings provide preliminary proteomic evidence supporting syndrome-guided precision integrative medicine in CRC and offer new insights into the biological basis of TCM syndrome differentiation and individualized therapeutic strategies.

The Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis （GS/CACM 337-2023） was released by the China Association of Chinese Medicine on December 13^th， 2023. This expert consensus was developed by experts in methodology， pharmacy， and Chinese medicine in strict accordance with the development requirements of the China Association of Chinese Medicine （CACM） and based on the latest medical evidence and the clinical medication experience of well-known experts in the fields of respiratory medicine （pulmonary diseases） and pediatrics. This expert consensus defines the application of Qinbaohong Zhike oral liquid in the treatment of cough and excessive sputum caused by phlegm-heat obstructing lung， acute bronchitis， and acute attack of chronic bronchitis from the aspects of applicable populations， efficacy evaluation， usage， dosage， drug combination， and safety. It is expected to guide the rational drug use in medical and health institutions， give full play to the unique value of Qinbaohong Zhike oral liquid， and vigorously promote the inheritance and innovation of Chinese patent medicines.

Currently, the drug delivery system (DDS) based on nanomaterials has become a hot interdisciplinary research topic. One of the core issues is drug loading and controlled release, in which the key lever is carriers. Vaterite, as an inorganic porous nano-material, is one metastable structure of calcium carbonate, full of micro or nano porous. Recently, vaterite has attracted more and more attention, due to its significant advantages, such as rich resources, easy preparations, low cost, simple loading procedures, good biocompatibility and many other good points. Vaterite, gained from suitable preparation strategies, can not only possess the good drug carrying performance, like high loading capacity and stable loading efficiency, but also improve the drug release ability, showing the better drug delivery effects, such as targeting release, pH sensitive release, photothermal controlled release, magnetic assistant release, optothermal controlled release. At the same time, the vaterite carriers, with good safety itself, can protect proteins, enzymes, or other drugs from degradation or inactivation, help imaging or visualization with loading fluorescent drugs in vitro and in vivo, and play synergistic effects with other therapy approaches, like photodynamic therapy, sonodynamic therapy, and thermochemotherapy. Latterly, some renewed reports in drug loading and controlled release have led to their widespread applications in diverse fields, from cell level to clinical studies. This review introduces the basic characteristics of vaterite and briefly summarizes its research history, followed by synthesis strategies. We subsequently highlight recent developments in drug loading and controlled release, with an emphasis on the advantages, quantity capacity, and comparations. Furthermore, new opportunities for using vaterite in cell level and animal level are detailed. Finally, the possible problems and development trends are discussed.

Objective By combining biological detection and imaging evaluation, a clinical prediction model is constructed based on a large cohort to improve the accuracy of distinguishing between benign and malignant pulmonary nodules. Methods A retrospective analysis was conducted on the clinical data of the 32 627 patients with pulmonary nodules who underwent chest CT and testing for 7 types of lung cancer-related serum autoantibodies (7-AABs) at our hospital from January 2020 to April 2024. The univariate and multivariate logistic regression models were performed to screen independent risk factors for benign and malignant pulmonary nodules, based on which a nomogram model was established. The performance of the model was evaluated using receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). Results A total of 1 017 patients with pulmonary nodules were included in the study. The training set consisted of 712 patients, including 291 males and 421 females, with a mean age of (58±12) years. The validation set included 305 patients, comprising 129 males and 176 females, with a mean age of (58±13) years. Univariate ROC curve analysis indicated that the combination of CT and 7-AABs testing achieved the highest area under the curve (AUC) value (0.794), surpassing the diagnostic efficacy of CT alone (AUC=0.667) or 7-AABs alone (AUC=0.514). Multivariate logistic regression analysis showed that radiological nodule diameter, nodule nature, and CT combined with 7-AABs detection were independent predictors, which were used to construct a nomogram prediction model. The AUC values for this model were 0.826 and 0.862 in the training and validation sets, respectively, demonstrating excellent performance in DCA. Conclusion The combination of 7-AABs with CT significantly enhances the accuracy of distinguishing between benign and malignant pulmonary nodules. The developed predictive model provides strong support for clinical decision-making and contributes to achieving precise diagnosis and treatment of pulmonary nodules.

AIM To establish a UPLC-MS/MS method for the simultaneous content determination of liquiritin,liquiritin apioside,verbascoside,narirutin,isoacteoside,apigetrin,hesperidin,isoliquiritin,ononin,liquiritigenin,glycyrrhizic acid,isoliquiritigenin,honokiol,obovatol,pogostone and magnolol in Jiawei Huoxiang Zhengqi Soft Capsules.METHODS The analysis was performed on a 40 ℃ thermostatic ZORBAX Eclipse Plus C18 column(2.1 mm×100 mm,1.8 μm),with the mobile phase comprising of 0.1％formic acid-acetonitrile flowing at 0.4 mL/min in a gradient elution manner,and electron spray ionization source was adopted in positive and negative ion scanning with multiple reaction monitoring mode.RESULTS Sixteen constituents showed good linear relationships within their own ranges(r＞0.990 0),whose average recoveries were 83.74％-105.12％with the RSDs of 1.10％-4.8％.CONCLUSION This accurate,sensitive,stable and reproducible method can provide a reference for the overall quality control of Jiawei Huoxiang Zhengqi Soft Capsules.