OBJECTIVE To optimize the extraction process of polysaccharides from Abelmoschus manihot flowers， and to investigate the antioxidant， moisturizing and hygroscopicity activities of the polysaccharides.METHODS The optimal ratio of composite enzymes was determined by orthogonal experiments， and the extraction process of polysaccharides was optimized by single-factor experiments and Box-Behnken response surface methodology， and validated. The scavenging rates of 2，2′-azino-bis （3-ethylbenzothiazoline-6-sulfonic acid） （ABTS） free radicals， hydroxyl free radicals， and superoxide anion free radicals， as well as the total reducing ability of the polysaccharides were investigated by the ABTS method， Fenton-salicylic acid colorimetric method， pyrogallol autoxidation method， and potassium ferricyanide reduction method， respectively. The moisturizing and hygroscopicity activities of the polysaccharides were evaluated by the weighing method.RESULTS The optimal ratio of composite enzymes was 136 000 U of papain， 125 000 U of pectinase，and 5 000 U of cellulase. The optimal extraction process was as follows： enzymatic hydrolysis time of 2.8 h， enzymatic hydrolysis temperature of 57.50 ℃， pH of the sodium dihydrogen phosphate-disodium hydrogen phosphate buffer was 5.5， and solid-to-liquid ratio of 1∶40. The results of three experiments showed that the average polysaccharides extraction rate of 23.595%， with an RSD of 0.90%. The scavenging rates of ABTS free radicals， hydroxyl free radicals， superoxide anion free radicals， and total reducing ability were 81.759%， 87.110%， 54.244%， and 1.129 （the mass concentrations of polysaccharides were 10、 5、 10， and 10 mg/mL， respectively）， respectively. At relative humidity of 43% for 12 h， the moisturizing rate and hygroscopicity rate of polysaccharides were 99.387% and 100.478%， respectively； at relative humidity of 83% for 12 h， they were 99.764% and 100.455%， respectively.CONCLUSIONS The optimal extraction process of polysaccharides from A.manihot flowers based on the composite enzymes is stable and feasible； the polysaccharides obtained under this process have good antioxidant， moisturizing， and hygroscopicity activities.

ObjectiveTo investigate whether the inhibitory effect of Panax quinquefolium saponins （PQS） on non-small cell lung cancer （NSCLC） is enhanced under graphene-mediated far-infrared therapy （GFIR）， and to analyze its relationships with mitochondrial dysfunction， mitochondrial DNA （mtDNA） leakage， and the activation of cyclic GMP-AMP synthase （cGAS）/stimulator of interferon genes （STING） signaling pathways. MethodsIn the cell experiments， the cell-counting kit 8 （CCK-8） was employed to assess the inhibitory effects of PQS alone or in combination with GFIR （PQS+GFIR） on A549 cell viability. The scratch assay and the colony formation assay were adopted to evaluate cell migration and colony formation capabilities， respectively. Flow cytometry was employed to assess apoptosis， mitochondrial membrane potential， and reactive oxygen species （ROS） levels. Immunofluorescence （IF） assay was performed to assess mitochondrial localization. Real-time polymerase China reaction（PCR） was used to detect mitochondrial DNA （mtDNA） leakage. A STING knockdown cell model was established by transfecting cells with small interfering RNA （siRNA）， and the knockdown efficiency was assessed by Real-time PCR and Western blot. The cell viability and apoptosis following gene knockdown were assessed through the CCK-8 assay and flow cytometry. In the animal experiments， an allogeneic xenograft mouse model was established with Lewis lung cancer cells. Mice were randomized into a control group， a model group， a cisplatin group， a low-dose PQS （PQS-Low， 90 mg·kg^-1） group， a high-dose PQS （PQS-High， 180 mg·kg^-1） group， a low-dose PQS combined with GFIR （PQS-Low+GFIR， 90 mg·kg^-1） group， and a high-dose PQS combined with GFIR （PQS-High+GFIR， 180 mg·kg^-1） group. Treatment lasted for 14 days， and the fluorescence intensity was monitored at the tumor site by small-animal in vivo imaging during the treatment period. Flow cytometry was utilized to determine the proportions of T-lymphocyte subsets， B-lymphocyte levels， and peripheral blood NK cell levels in the spleens of mice. Immunohistochemical （IHC） staining was performed to assess the infiltration of CD3⁺， CD4⁺， and CD8⁺ T-lymphocytes in the tumor tissue. Enzyme-linked immunosorbent assay （ELISA） was conducted to measure the levels of interferon-α （IFN-α）， interferon-β （IFN-β）， tumor necrosis factor-α （TNF-α）， and interleukin-6 （IL-6） in mouse tumors. The mRNA levels of cGAS， STING， TANK-binding kinase 1 （TBK1）， interferon regulatory factor 3 （IRF3）， interferon beta 1 （IFNB1）， and nuclear factor κB p65 （NF-κB p65） were determined by Real-time PCR. The protein levels of cGAS， STING， p-STING， TBK1， p-TBK1， IRF3， p-IRF3， NF-κB p65， and p-NF-κB p65 were quantified via Western blot. ResultsThe cell experiment results showed that compared with the control group， PQS inhibited the viability and migration of A549 cells， promoted apoptosis， affected mitochondrial localization， reduced mitochondrial membrane potential， increased the ROS level， and promoted mtDNA leakage. Following STING knockdown， the reductions in cell viability and the apoptosis-inducing effects caused by PQS were partially reversed （P<0.05， P<0.01）. Compared with the PQS group， the PQS+GFIR group showed inhibited A549 cell migration and colony formation， exacerbated apoptosis， downregulated mitochondrial membrane potential， elevated ROS levels， and promoted mtDNA leakage. After STING knockdown， the reduced A549 cell viability and abnormal apoptosis phenotype were further reversed （P<0.05， P<0.01）. The animal experiment results showed that compared with the control group， the model group mice exhibited reduced proportions of T-lymphocyte subsets， CD19⁺ B-lymphocyte levels， and peripheral blood NK1.1⁺ cell levels in the spleen （P<0.01）. Compared with the model group， the PQS group showed increased immune cell parameters in mice （P<0.05， P<0.01）. Compared with the PQS group， the PQS+GFIR group exhibited enhanced levels of the aforementioned indicators （P<0.01）. Compared with the model group， the PQS group showed significantly reduced fluorescence intensity in transplanted tumors， significantly upregulated expression of CD3 and CD4 in the tumor tissue， elevated levels of IFN-α and IL-6 in tumors， and upregulated mRNA levels of cGAS， STING， TBK1， IRF3， IFNB1， and NF-κB p65 as well as upregulated protein level of cGAS and phosphorylation level of STING in tumors （P<0.05， P<0.01）. Compared with the PQS group， the PQS+GFIR group demonstrated further reduced tumor fluorescence intensity， elevated levels of CD3， CD4， and CD8 as well as cytokine levels in tumor tissue， and upregulated mRNA levels of genes related to the cGAS/STING pathway as well as upregulated protein level of cGAS and phosphorylation levels of STING， TBK1， IRF3， and NF-κB p65 （P<0.05， P<0.01） ConclusionGFIR enhances the inhibitory effect of PQS on NSCLC， potentially through mitochondrial dysfunction， mtDNA leakage， and activation of the cGAS/STING signaling pathway.

ObjectiveTo investigate whether the inhibitory effect of Panax quinquefolium saponins （PQS） on non-small cell lung cancer （NSCLC） is enhanced under graphene-mediated far-infrared therapy （GFIR）， and to analyze its relationships with mitochondrial dysfunction， mitochondrial DNA （mtDNA） leakage， and the activation of cyclic GMP-AMP synthase （cGAS）/stimulator of interferon genes （STING） signaling pathways. MethodsIn the cell experiments， the cell-counting kit 8 （CCK-8） was employed to assess the inhibitory effects of PQS alone or in combination with GFIR （PQS+GFIR） on A549 cell viability. The scratch assay and the colony formation assay were adopted to evaluate cell migration and colony formation capabilities， respectively. Flow cytometry was employed to assess apoptosis， mitochondrial membrane potential， and reactive oxygen species （ROS） levels. Immunofluorescence （IF） assay was performed to assess mitochondrial localization. Real-time polymerase China reaction（PCR） was used to detect mitochondrial DNA （mtDNA） leakage. A STING knockdown cell model was established by transfecting cells with small interfering RNA （siRNA）， and the knockdown efficiency was assessed by Real-time PCR and Western blot. The cell viability and apoptosis following gene knockdown were assessed through the CCK-8 assay and flow cytometry. In the animal experiments， an allogeneic xenograft mouse model was established with Lewis lung cancer cells. Mice were randomized into a control group， a model group， a cisplatin group， a low-dose PQS （PQS-Low， 90 mg·kg^-1） group， a high-dose PQS （PQS-High， 180 mg·kg^-1） group， a low-dose PQS combined with GFIR （PQS-Low+GFIR， 90 mg·kg^-1） group， and a high-dose PQS combined with GFIR （PQS-High+GFIR， 180 mg·kg^-1） group. Treatment lasted for 14 days， and the fluorescence intensity was monitored at the tumor site by small-animal in vivo imaging during the treatment period. Flow cytometry was utilized to determine the proportions of T-lymphocyte subsets， B-lymphocyte levels， and peripheral blood NK cell levels in the spleens of mice. Immunohistochemical （IHC） staining was performed to assess the infiltration of CD3⁺， CD4⁺， and CD8⁺ T-lymphocytes in the tumor tissue. Enzyme-linked immunosorbent assay （ELISA） was conducted to measure the levels of interferon-α （IFN-α）， interferon-β （IFN-β）， tumor necrosis factor-α （TNF-α）， and interleukin-6 （IL-6） in mouse tumors. The mRNA levels of cGAS， STING， TANK-binding kinase 1 （TBK1）， interferon regulatory factor 3 （IRF3）， interferon beta 1 （IFNB1）， and nuclear factor κB p65 （NF-κB p65） were determined by Real-time PCR. The protein levels of cGAS， STING， p-STING， TBK1， p-TBK1， IRF3， p-IRF3， NF-κB p65， and p-NF-κB p65 were quantified via Western blot. ResultsThe cell experiment results showed that compared with the control group， PQS inhibited the viability and migration of A549 cells， promoted apoptosis， affected mitochondrial localization， reduced mitochondrial membrane potential， increased the ROS level， and promoted mtDNA leakage. Following STING knockdown， the reductions in cell viability and the apoptosis-inducing effects caused by PQS were partially reversed （P<0.05， P<0.01）. Compared with the PQS group， the PQS+GFIR group showed inhibited A549 cell migration and colony formation， exacerbated apoptosis， downregulated mitochondrial membrane potential， elevated ROS levels， and promoted mtDNA leakage. After STING knockdown， the reduced A549 cell viability and abnormal apoptosis phenotype were further reversed （P<0.05， P<0.01）. The animal experiment results showed that compared with the control group， the model group mice exhibited reduced proportions of T-lymphocyte subsets， CD19⁺ B-lymphocyte levels， and peripheral blood NK1.1⁺ cell levels in the spleen （P<0.01）. Compared with the model group， the PQS group showed increased immune cell parameters in mice （P<0.05， P<0.01）. Compared with the PQS group， the PQS+GFIR group exhibited enhanced levels of the aforementioned indicators （P<0.01）. Compared with the model group， the PQS group showed significantly reduced fluorescence intensity in transplanted tumors， significantly upregulated expression of CD3 and CD4 in the tumor tissue， elevated levels of IFN-α and IL-6 in tumors， and upregulated mRNA levels of cGAS， STING， TBK1， IRF3， IFNB1， and NF-κB p65 as well as upregulated protein level of cGAS and phosphorylation level of STING in tumors （P<0.05， P<0.01）. Compared with the PQS group， the PQS+GFIR group demonstrated further reduced tumor fluorescence intensity， elevated levels of CD3， CD4， and CD8 as well as cytokine levels in tumor tissue， and upregulated mRNA levels of genes related to the cGAS/STING pathway as well as upregulated protein level of cGAS and phosphorylation levels of STING， TBK1， IRF3， and NF-κB p65 （P<0.05， P<0.01） ConclusionGFIR enhances the inhibitory effect of PQS on NSCLC， potentially through mitochondrial dysfunction， mtDNA leakage， and activation of the cGAS/STING signaling pathway.

Objective: To explore the impacts of coal mine dust on lung function in rats, so as to provide the basis for the early prevention and treatment of coal worker's pneumoconiosis. Methods: Seventy-two SPF-grade 8-week-old male Sprague-Dawley rats were randomly divided into the coal dust group, the coal-silica dust group, the silica dust group and the control group. The rats in the first three groups of rats were administered 1 mL corresponding dust suspension into the lungs using non-exposure tracheal instillation, while the rats in the control group were administered 1 mL normal saline. Respiratory rate (f), forced vital capacity (FVC), peak expiratory flow (PEF) and dynamic pulmonary compliance (Cdyn) were measured at 1, 3 and 6 months after dust exposure. Lung tissues were collected to measure reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels using corresponding ELISA kits and ATP assay kits, respectively. The relative mRNA expressions of peroxisome proliferators-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) were detected using real-time fluorescent quantitative polymerase chain reaction assay. The relative protein expressions of PGC-1α and TFAM were detected using Western blotting. Results: There was no interaction between dust type and exposure duration on f (P>0.05), but there were interactions on FVC, PEF and Cdyn (all P<0.05). Compared with the control group at 6 months after dust exposure, the f of the rats in the silica dust group were increased, while the FVC and PEF of the rats in the coal-silica dust and silica dust groups were decreased, and Cdyn of the rats in the coal dust, coal-silica dust and silica dust groups were decreased (all P<0.05). There were interactions between dust type and exposure duration on ROS and ATP levels, the relative mRNA and protein expressions of PGC-1α and TFAM (all P<0.05). Compared with the control group at 3 and 6 months after dust exposure, the ROS levels in the rats in the coal dust, coal-silica dust and silica dust groups were increased, while the ATP levels, the relative mRNA and protein expressions of PGC-1α and TFAM were decreased (all P<0.05). Conclusion The lung function impairment in rats caused by different types of coal mine dust is related to PGC-1α-mediated mitochondrial biogenesis dysfunction, which leads to increased ROS levels, decreased ATP and TFAM levels.

[This corrects the article DOI: 10.1016/j.apsb.2022.05.019.].

The aim of this study was to investigate the mechanism of quercetin in inhibiting the fer-roptosis induced by zearalenone(ZEN)via mediating the nuclear factor erythroid-2-related factor 2(Nrf2)/glutathione peroxidase 4(GPX4)signaling pathway.IPEC-J2 cells were cultured in vitro and treated with ZEN(25 mg/L)and different concentrations of quercetin(10,20,40 μmol/L).Bi-ochemical methods were used to detect the levels of lactate dehydrogenase(LDH)in the cell cul-ture medium supernatant,total antioxidant capacity(T-AOC),superoxide dismutase(SOD),glu-tathione(GSH),and malondialdehyde(MDA).Fluorescence probes were used to detect the levels of Fe2+,reactive oxygen species(ROS),and lipid peroxidation.Western blot was performed to de-tect the expression levels of Nrf2,long chain acyl CoA synthetase 4(ACSL4),and GPX4.The IPEC-J2 cells were divided into the control group,ZEN group,quercetin group,and ML385(Nrf2 inhibitor)+quercetin group for further analysis.Except for the control group,the other groups were treated with ZEN in the prescence of quercetin and ML385.The changes of Nrf2/GPX4 path-way-related proteins and ferroptosis-related indexes(LDH,Fe2+,MDA,and GSH)were detected.Compared with the control group,IPEC-J2 cells in the ZEN group exhibited a decrease in cell via-bility,T-AOC,and GSH,SOD levels,Nrf2,and GPX4 protein expressions(P＜0.05),while LDH release rate,Fe2+and ROS,lipid peroxidation,MDA levels,and ACSL4 protein expression de-creased in the ZEN group(P＜0.05).Compared with ZEN group,the cell viability,the levels of T-AOC,SOD and GSH,the protein expression of Nrf2 and GPX4 in quercetin groups were increased(P＜0.05),while the LDH release rate,the levels of Fe2+,ROS,lipid peroxidation,and MDA as well as the protein expression of ACSL4 were decreased(P＜0.05).Compared with the quercetin group,the protein expression of Nrf2 and GPX4 and the level of GSH in ML385+quercetin group reduced(P＜0.05),the LDH release rate and the levels of Fe2+and MDA increased(P＜0.05).In summary,quercetin could inhibit ZEN-induced ferroptosis of IPEC-J2 cells,and its mechanism may be related to the induction of Nrf2/GPX4 signaling pathway.

Objective: To reveal the molecular basis of the cisAB (p. Met266Leu) glycosyltransferase by studying a proband with cisAB subtype and his family. Methods: A male newborn was selected as the research subject. Tube methods were used to identify ABO blood types of the proband and his family members. PCR-SSP detection, ABO gene sequencing, and cloning analysis were performed on the proband and some family members. The inheritance pattern of the subtype gene in the family was determined through pedigree analysis. Homology modeling was used to analyze the impact of amino acid variations on the structure of the transferase, and molecular docking was used to demonstrate the bifunctional activity of the transferase and the donor-receptor binding conformation. Results: Serological tests showed that the proband and his father had enhanced anti-H agglutination, and the grandmother had a forward and reverse discrepancy. Sequencing of the proband revealed heterozygous variations of c. 297A>G, c. 526C>G, c. 657C>T, c. 703G>A, c. 803G>C, and c. 930G>A compared with A1. 01 (compared with B. 01, lacking the c. 796C>A variation, namely harboring the c. 796A>C variation) and c. 261delG. Combined with cloning analysis, the proband's genotype was determined to be ABO cisAB (c. 796A>C)/ABO O. 01. 01, the father's genotype was ABO cisAB (c. 796A>C)/ABO O. 01. 02, and the grandmother's genotype was ABO cisAB (c. 796A>C)/ABO B102. Pedigree analysis indicated that the cisAB allele in this newborn was inherited from his father and grandmother rather than a natural mutation. Homology modeling showed that the side chain orientation and intermolecular forces of Leu266 in the cisAB (p. Met266Leu) transferase changed, and molecular docking demonstrated that the "binding pocket" of the active center of the variant enzyme could accommodate both UDP-GalNAc and UDP-Gal, indicating that the cisAB enzyme structure has bifunctional activity. Conclusion: The bifunctional activity of this cisAB (p. Met266Leu) enzyme is related to the nucleotide variation of c. 796A>C, and molecular docking indicates that the enzyme has dual affinity for A/B sugar donors.

Objective:To construct a debriefing and reflection training program for nursing undergraduate interns, and to provide a basis for implementing such a program and improve the practical clinical skills of the interns.Methods:Based on competency theory, the draft of the debriefing and reflection training program for undergraduate nursing interns was constructed through literature review and expert conference discussion. Delphi expert consultation was conducted from August to October 2024, and the final version of the program was established based on the experts' opinions. The weight of each indicator was determined using the analytic hierarchy process. Excel 2019 was used for data entry, and SPSS 19.0 was used for data analysis.Results:Two rounds of expert consultation were conducted, with a questionnaire recovery rate of 100.00% for both. The experts consulted in the second round had an authority coefficient of 0.861, a coefficient of variation for each index of 0.048 to 0.237, and a Kendall's concordance coefficient of 0.137 ( P<0.05). The final program included 5 primary indicators, 16 secondary indicators, and 73 tertiary indicators. Conclusions:The debriefing and reflection training program for undergraduate nursing interns proposed in this study has high scientifical validity and reliability. It can provide a reference for debriefing and reflection training of nursing undergraduate interns in China, thereby cultivating qualified clinical nursing talents.

Objective: To investigate the aberrant alterations of microRNAs ( miRNAs) in exosomes derived from bone marrow stromal cells ( BMSCs) in the bone marrow supernatants of patients with acute myeloid leukemia (AML) and their impact on the prognosis of AML patients . Methods: Bone marrow supernatant samples were col- lected from three AML patients and three healthy donors . Exosomes were isolated using a commercial kit , identif- ying the morphology and marker expression , and subjected to miRNA sequencing to determine differentially ex- pressed miRNAs (DE-miRNAs) . The DE-miRNAs were then intersected with the exosomal miRNA expression pro- files of primary AML cells (GSE64029) to exclude AML cell - derived signals and to identify BMSC-derived DE - miRNAs . Subsequently , candidate miRNAs were identified through Cox regression and Lasso regression analyses based on data from The Cancer Genome Atlas (TCGA) . A prognostic risk model for AML was constructed , and pa- tients were stratified into high-risk and low-risk groups according to the median risk score . The prognostic value and clinical relevance of the model were further validated . Finally , the target genes of the candidate miRNAs were pre- dicted , followed by pathway enrichment analysis , construction of key regulatory networks , and correlation analysis between the expression levels of key miRNAs and their corresponding target genes . Results: Isolated exosomes ex- hibited a typical cup-shaped morphology with intact structures with particle size of 30 - 150 nm , and expressed exo- somal markers CD63 , ALIX , and TSG101 . miRNA sequencing identified 103 DE-miRNAs in AML patients com- pared with healthy donors; after intersection with the GSE64029 dataset , 83 BMSC-derived DE-miRNAs were re- tained . Among these , five candidate miRNAs ( miR-25-3p , miR-532-5p , miR-194-5p , miR-10a-5p , and miR- 20a-5p) were used to construct the prognostic model . Kaplan-Meier survival analysis demonstrated significantly lon- ger overall survival in the low-risk group compared with the high-risk group (P < 0. 05) . The areas under the ROC curve for the training/validation cohorts were 0. 80/0. 74 , 0. 80/0. 78 , and 0. 79/0. 64 at 1 , 2 , and 3 years , re- spectively . The prognostic model was significantly associated with risk stratification , patient age , and FAB classifi- cation (P < 0. 05) . KEGG pathway enrichment revealed that target genes of the candidate miRNAs were closely linked to cancer-related signaling pathways , including hepatocellular carcinoma , breast cancer , and non-small cell lung cancer. Correlation analysis indicated that the candidate miRNAs were significantly associated with key genes such as HIF1A , CREB1 , PIK3CA , IGF1R , PIK3R1 , TIAM1 , CRK , and PTEN (P < 0. 05) . Conclusion AML patients exhibit distinct miRNA expression profiles in BMSC-derived exosomes . A five-miRNA signature ( miR-25 - 3p , miR-532-5p , miR-194-5p , miR-10a-5p , and miR-20a-5p) demonstrates robust prognostic performance , sup- porting its potential clinical utility in risk stratification and outcome prediction for AML.

Objective:To evaluate the efficacy of eculizumab in treating hematopoietic stem cell transplantation-associated thrombotic microangiopathy (TA-TMA) .Methods:This retrospective study included 11 patients who developed TA-TMA after allogeneic hematopoietic stem cell transplantation and subsequently received eculizumab treatment at Peking University People′s Hospital between June 2018 and May 2024. The incidence of TA-TMA, treatment details, and clinical outcomes were analyzed.Results:Among the 11 included patients [4 males, 7 females; median age: 29 years (range: 9-56) ], underlying diseases were severe aplastic anemia (SAA) in 5 patients, acute lymphoblastic leukemia (ALL) in 3 patients, and acute myeloid leukemia (AML) in 3 patients. The median time to TA-TMA diagnosis was 48 days post-transplantation (range: 4-213 days), and all patients met the diagnostic criteria for high-risk TA-TMA. The median interval from TA-TMA diagnosis to the initiation of eculizumab treatment was 12 days (range: 1-56 days). Patients received a median of 3 doses of eculizumab (range: 1-14). Ten of the 11 patients were assessed as having no response (NR) to eculizumab at the end of treatment or at death. One patient achieved a partial response (PR) but subsequently died after TA-TMA relapsed due to infection. At the last follow-up, all patients were either lost to follow-up or had died. The median follow-up duration was 88 days (range: 33-326 days), and the median time from TA-TMA diagnosis to the last follow-up was 31 days (range: 21-113 days) .Conclusion:Eculizumab demonstrated poor efficacy in this TA-TMA cohort. This might be attributable to the critical and complex condition of the patients, delayed initiation of eculizumab treatment, and insufficient dosage.