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.

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.

Objective To explore the predictive value of fat attenuation index(FAI)and fibrous plaque index(FPI)for the prognosis of patients with acute coronary syndrome(ACS).Methods A retrospective cohort study was conducted on 334 ACS patients undergoing percutaneous coronary intervention(PCI)in the First Affiliated Hospital of Army Military Medical University and Yongchuan Hospital of Chongqing Medical University from March 2021 to July 2023.All patients received coronary computed tomography angiography(CCTA)to measure FAI and FPI.According to the occurrence of major adverse cardiovascular events(MACE)with 1 year of follow-up,they were divided into MACE group(n=108)and non-MACE group(n=226).The baseline data,CCTA data and results of laboratory tests were collected and compared between the 2 groups.Multivariate logistic regression analysis was used to analyze the relationship of FAI and FPI with the prognosis of ACS patients,and ROC curve was drawn to evaluate its predictive efficiency.Results Among the 334 ACS patients,108(32.34%)experienced MACE.When compared with the non-MACE group,the MACE group exhibited significantly larger proportions of diabetes(72.22%vs 31.86%)and left main coronary artery disease(18.52%vs 7.08%),but lower success rate of operation(79.63%vs 93.81%,P<0.05).Radiologic results showed that the proportion of severe stenosis(20.37%vs 10.62%),FAI(-80.12±6.41 HU vs-72.34±7.09 HU)and FPI(0.58±0.41 vs 0.26±0.12)were obviously increased in the MACE group than the non-MACE group(P<0.05).Laboratory tests indicated that there were statistical differences between the 2 groups in high-density lipoprotein-cholesterol(HDL-C,1.20±0.15 vs 1.09±0.16 mmol/L),miR-126(0.91±0.12 vs 0.96±0.15)and SST2(38.45±5.67 vs 34.30±4.89 ng/mL,P<0.05).Multivariate Logistic regression analysis revealed that FAI(OR=1.200,95%CI:1.136～1.268),FPI(OR=63.157,95%CI:14.126～282.374),moderate stenosis(OR=1.332,95%CI:1.024～1.859),severe stenosis(OR=1.480,95%CI:1.074～2.039),miR-126(OR=0.007,95%CI:0.001～0.077),and sST2(OR=1.192,95%CI:1.113～1.277)were independent predictors of MACE(P<0.05).ROC curve analysis displayed that stenosis degree(AUC=0.622,95%CI:0.561～0.683,P=0.001),FAI(AUC=0.790,95%CI:0.741～0.839,P=0.001)and FPI(AUC=0.700,95%CI:0.638～0.761,P=0.001),miR-126(AUC=0.646,95%CI:0.584～0.707,P=0.001),sST2(AUC=0.700,95%CI:0.638～0.761,P=0.001)had certain predictive values for ACS prognosis.Conclusion Coronary FAI and FPI can be used as independent prognostic indicators of ACS patients,and their numerical changes are closely related to plaque stability and inflammatory state.

Organophosphorus nerve agents are the most threatening chemical warfare agents and terrorist agents.The number of nerve agents and their related chemicals involved in the verification of Chemical Weapon Convention(CWC)exceeds ten million,with the majority being isomers.Accurate structural identification of these chemicals has always been one of the challenges in CWC related verification analysis.In this work,a total of 17 kinds of alkyl phosphonate isomers and structural analogs from 5 groups were designed and synthesized,and then analyzed by gas chromatography-mass spectrometry(GC-MS)and gas chromatography-infrared spectroscopy(GC-FTIR).The spectra of isomers or structural analogs obtained from two techniques as well as the structural information provided therein were compared and analyzed.The results showed that for isomers or structural analogs with similar MS spectra,FTIR spectra could provided more structural fingerprint information of compounds and had advantages in confirming structures.Combined with the excellent separation ability of GC,GC-FTIR can be used to assist GC-MS in the structural confirmation of alkyl phosphates,achieving rapid and accurate identification of isomers or structural analogues.

C-Reactive protein(CRP)is an important acute-phase response protein,which is widely used in the assessment of inflammation and cardiovascular disease risk,and acts as a pathogenic factor directly involved in the disease process of certain conditions.Therefore,developing immunosuppressants targeting CRP or investigating its pathogenic mechanisms is of significant importance.Most B-cell epitopes are conformational epitopes,and studying conformational epitopes is typically challenging.To date,no methods have been reported for mapping the conformational epitopes of CRP in solution.In this study,a rapid strategy was developed for studying conformational epitopes by combining hydrogen/deuterium exchange mass spectrometry(HDX-MS)with multiparametric prediction of B-cell epitopes and protein secondary structure analysis.This approach was successfully applied to the binding sites and allosteric targets of the 115 kDa full pentameric CRP and the clinically used monoclonal antibody(mAb)5A8.The results showed that the amino acid residues 84-103,138-146,and 165-173 together form the potential conformational epitopes for mAb 5A8 on CRP,while the amino acid residues 21-32 and 175-178 were identified as potential allosteric targets.The discovery of the mAb 5A8 binding sites and allosteric targets was crucial for improving clinical diagnostic capabilities.Experimental results demonstrated that this workflow allowed rapid conformational epitope mapping of CRP under near-physiological conditions,with advantages such as high speed,high sensitivity,and high throughput.

Objective To compare the application effects of plankton multiplex polymerase chain reac-tion-capillary electrophoresis(PCR-CE),SYBR Green Ⅰ real-time quantitative PCR(qPCR)and microwave digestion-vacuum filtration-automated scanning electron microscopy(MD-VF-Auto SEM)in the diagnosis of drowning.Methods Lung,liver and kidney tissues from 212 drowned corpses and 30 non-drowned corpses were examined respectively by the three drowning-related plankton testing methods,and the detection rates of plankton in each tissue by three methods were compared.Results In drowned corpses,the total detection rates of PCR-CE,qPCR,and MD-VF-Auto SEM were 93.9%,96.2%,and 95.3%,respectively,with no statistically significant difference(P>0.05).The detection rate of lung tissue by MD-VF-Auto SEM(100%)was higher than those of PCR-CE and qPCR(P<0.05),and there was no significant difference in the detection rates of the three methods in liver or kidney tissues(P>0.05).In non-drowning corpses,a small number of diatoms(less than 10 cells/10 g)were detected by MD-VF-Auto SEM method,only in liver and kidney tissues,while the other two methods yielded negative results for all tissues.Conclusion All three methods have good efficacy in the examination of drowned corpses.The MD-VF-Auto SEM method directly observes diatom morpho-logical characteristics through scanning electron microscopy,and the qualitative and quantitative analy-ses are intuitive and accurate.It has great advantages in the examination of difficult degradation samples.The PCR-CE method and qPCR method have a low sample demand(0.5 g),are easy to operate and have short detection time(4-7 h).They are easy to be applied in the grassroots depart-ments and are suitable for the rapid determination of drowned corpses in routin cases.The combina-tion of the two DNA methods with the MD-VF-Auto SEM method can increase the detection rate of plankton,ensuring the reliability of examination results.This combined use is of significant importance in the application of drowning diagnosis.

Objective To verify the efficacy of a domestic human DNA quantification kit based on real-time fluorescence quantitative PCR in detecting the total human DNA concentration,male DNA concen-tration in mixed male/female DNA samples,the degree of DNA degradation and inhibitor tolerance.Methods Samples with different concentrations,different male/female ratios,different concentrations of inhibitors,and different degradation degrees were tested using the domestic human DNA quantification kit based on real-time fluorescence quantitative PCR.This kit was compared with a similar product on the market and was applied to the detection of DNA from real cases.Results This human DNA quan-tification kit can effectively detect human DNA as low as 0.001 65 ng/μL,and 6.25 pg/μL of male DNA in mixed samples with a male-to-female ratio of 1∶15 000.Even when the sample contains as high as 400 ng/μL of humic acid or 1 000 μmol/L of hemin alone,the DNA concentration can still be accurately detected.The degradation index can effectively characterize the degradation degree of the sample.This kit has been successfully applied in forensic practice.Conclusion This human DNA quan-tification kit is accurate and reliable in detection.It can accurately reflect the degradation of DNA and inhibitor tolerance.It has good performance in quantitative accuracy,determination of the male/female ratio in mixed samples,and inhibitor tolerance.It has application potential in forensic case examination.

Objective To explore the effect of enhancer of Zeste homolog 2(EZH2)on hepatocellular car-cinoma HepG2 cell proliferation by inhibiting Wnt/β-catenin signaling pathway.Methods The expression lev-els of mRNA and protein of EZH2 in hepatocellular carcinoma HepG2,SMMC-7721,BEL-7402 cells and nor-mal hepatocytes were detected by real time fluorescence quantitative reverse transcription polymerase chain reaction and Western blot.Transfected siRNA EZH2 and siRNA control in hepatocellular carcinoma HepG2 cells.siRNA EZH2 in HepG2 cells and siRNA control were transfected,MTT was used to measure the HepG2 cells' ability to proliferate.Western blot technique was used to identify the expression levels of proteins.By transfecting siRNA EZH2 in HepG2 cells and administering an activator of the Wnt/β-catenin signaling path-way,MTT assay was used to determine the proliferation ability of cells.Results The EZH2 mRNA and EZH2 protein levels in hepatocellular carcinoma HepG2,SMMC-7721 and BEL-7402 cells were higher than those in normal hepatocytes(P＜0.05),and the mRNA and protein levels of EZH2 in HepG2 cells were high-er than those in SMMC-7721 and BEL-7402 cells(P＜0.05).siRNA EZH2 could significantly inhibit the mR-NA and protein levels of EZH2 in hepatocellular carcinoma HepG2 cells.After EZH2 expression inhibition,the cell viability of HepG2 cells was lower at 24,48,72 h(P＜0.05),Wnt 1 and β-catenin protein levels were lower(P＜0.05).Conclusion EZH2 is overexpressed in hepatocellular carcinoma HepG2 cells,and knock-down of EZH2 significantly inhibits the proliferative ability of HepG2 cells and decreases the levels of proteins related to the Wnt/β-catenin signaling pathway.

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.