Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

Bacterial biofilms gave rise to persistent infections and multi-organ failure, thereby posing a serious threat to human health. Biofilms were formed by cross-linking of hydrophobic extracellular polymeric substances (EPS), such as proteins, polysaccharides, and eDNA, which were synthesized by bacteria themselves after adhesion and colonization on biological surfaces. They had the characteristics of dense structure, high adhesiveness and low drug permeability, and had been found in many human organs or tissues, such as the brain, heart, liver, spleen, lungs, kidneys, gastrointestinal tract, and skeleton. By releasing pro-inflammatory bacterial metabolites including endotoxins, exotoxins and interleukin, biofilms stimulated the body’s immune system to secrete inflammatory factors. These factors triggered local inflammation and chronic infections. Those were the key reason for the failure of traditional clinical drug therapy for infectious diseases.In order to cope with the increasingly severe drug-resistant infections, it was urgent to develop new therapeutic strategies for bacterial-biofilm eradication and anti-bacterial infections. Based on the nanoscale structure and biocompatible activity, nanobiomaterials had the advantages of specific targeting, intelligent delivery, high drug loading and low toxicity, which could realize efficient intervention and precise treatment of drug-resistant bacterial biofilms. This paper highlighted multiple strategies of biofilms eradication based on nanobiomaterials. For example, nanobiomaterials combined with EPS degrading enzymes could be used for targeted hydrolysis of bacterial biofilms, and effectively increased the drug enrichment within biofilms. By loading quorum sensing inhibitors, nanotechnology was also an effective strategy for eradicating bacterial biofilms and recovering the infectious symptoms. Nanobiomaterials could intervene the bacterial metabolism and break the bacterial survival homeostasis by blocking the uptake of nutrients. Moreover, energy-driven micro-nano robotics had shown excellent performance in active delivery and biofilm eradication. Micro-nano robots could penetrate physiological barriers by exogenous or endogenous driving modes such as by biological or chemical methods, ultrasound, and magnetic field, and deliver drugs to the infection sites accurately. Achieving this using conventional drugs was difficult. Overall, the paper described the biological properties and drug-resistant molecular mechanisms of bacterial biofilms, and highlighted therapeutic strategies from different perspectives by nanobiomaterials, such as dispersing bacterial mature biofilms, blocking quorum sensing, inhibiting bacterial metabolism, and energy driving penetration. In addition, we presented the key challenges still faced by nanobiomaterials in combating bacterial biofilm infections. Firstly, the dense structure of EPS caused biofilms spatial heterogeneity and metabolic heterogeneity, which created exacting requirements for the design, construction and preparation process of nanobiomaterials. Secondly, biofilm disruption carried the risk of spread and infection the pathogenic bacteria, which might lead to other infections. Finally, we emphasized the role of nanobiomaterials in the development trends and translational prospects in biofilm treatment.

Objective To establish an ultra-high performance liquid phase tandem triple quadrupole mass spectrometry method for the determination of L-synephrine in rat plasma and to study its pharmacokinetics.Methods The plasma samples were precipitated by acetonitrile vortex,centrifuged,dried by nitrogen,dissolved by mobile phase,and then analyzed.Chromatographic column:Shiseido CAPCELL PAK CR(1∶4)column(2.0 mm × 150.0 mm,5 μm),temperature:25 ℃.Mobile phase:Acetonitrile:0.1％formic acid solution(containing 10 mmoL·L-1 ammonium formate)(73∶27)with flow rate:0.3 mL·min-1 and injection volume was 2 μL.The electrospray ion source ionization and positive ion multiple reaction monitoring mode were used for mass spectrometry detection,and biological sample methodology verification was carried out according to requirements.After intragastric administration with 5 mg·kg-1 dose of L-synephrine,blood samples were collected at different time points to investigate the pharmacokinetic characteristics of L-synephrine in rats.Results L-synephrine showed a good linear relationship in the range of 2-800 ng·mL-1,the lowest limit of quantification of L-synephrine was 2 ng·mL-1.Precision,extraction recovery,matrix effect,accuracy,dilution effect and residual effect all meet the requirements all met the requirements.The main pharmacokinetic parameters:Cmax were(464.83±76.68)ng·L-1,tmax were(0.67±0.26)h,t1/2z were(1.89±0.48)h,AUC0-t were(602.26±42.25)ng·mL-1·h-1,AUC0-∞ were(612.28±41.18)ng·mL-1·h-1.Conclusion The established ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for the determination of L-synephrine in rat plasma is simple and rapid,and can be used for the determination of L-synephrine in plasma.

Objective This study aimed to comprehensively analyze and compare the clinicopathological features and prognosis of Chinese patients with human epidermal growth factor receptor 2(HER2)-low early breast cancer(BC)and HER2-IHC0 BC. Methods Patients diagnosed with HER2-negative BC(N=999)at our institution between January 2011 and December 2015 formed our study population.Clinicopathological characteristics,association between estrogen receptor(ER)expression and HER2-low,and evolution of HER2 immunohistochemical(IHC)score were assessed.Kaplan-Meier method and log-rank test were used to compare the long-term survival outcomes(5-year follow-up)between the HER2-IHC0 and HER2-low groups. Results HER2-low BC group tended to demonstrate high expression of ER and more progesterone receptor(PgR)positivity than HER2-IHC0 BC group(P<0.001).The rate of HER2-low status increased with increasing ER expression levels(Mantel-Haenszel χ2 test,P<0.001,Pearson's R=0.159,P<0.001).Survival analysis revealed a significantly longer overall survival(OS)in HER2-low BC group than in HER2-IHC0 group(P=0.007)in the whole cohort and the hormone receptor(HR)-negative group.There were no significant differences between the two groups in terms of disease-free survival(DFS).The discordance rate of HER2 IHC scores between primary and metastatic sites was 36.84%. Conclusion HER2-low BC may not be regarded as a unique BC group in this population-based study due to similar clinicopathological features and prognostic roles.

In the study, to explore the anti-tumor effects and mechanisms of chlorpromazine (CPZ) and perphenazine (PPZ) combined with temozolomide (TMZ) on human glioma cell lines, we performed MTT assays to determine the growth inhibitory rate of CPZ, PPZ and TMZ in mono and combined treatments. The anti-tumor effects of CPZ and PPZ alone or in combination with TMZ were determined by colony formation, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) detection (JC-1). The expression level of p53 was detected by immunofluorescence assay. Furthermore, autophagy under different administrations was detected by flow cytometry and confocal imaging to explore the anti-tumor mechanism of CPZ and TMZ. Protein phosphatase 2A (PP2A), cancerous inhibitor of protein phosphatase 2A (CIP2A) and proto-oncogene protein (c-Myc) were detected by immunofluorescence assay, tumor stem cell markers (CD44, CD133) and aldehyde dehydrogenase (ALDH) were detected by flow cytometry to explore the anti-tumor mechanism of PPZ and TMZ. The results showed that after 72 h treatments of combinations, the values of half maximal inhibitory concentration (IC₅₀) of TMZ on U87 and U251 cells were reduced, and the ability of TMZ to induce apoptosis and cycle arrest was improved. In addition, the combination of CPZ and TMZ could induce an increased effect of autophagy via activating the relevant pathway of p53 gene in glioma cells. The combination of PPZ and TMZ increased the sensitivity of glioma cells to TMZ, and the underlying mechanism might be related to the inhibition of CIP2A/PP2A/c-Myc signaling pathway. In conclusion, CPZ and PPZ combined with TMZ, showed the significant synergistic effects in cancer treatment, which are the novel and potential therapeutic regimens providing a new treatment strategy for human glioma.

ObjectiveAsthma is a common chronic inflammatory airway disease, and severe asthma poses a significant challenge in its diagnosis and management. Immune cells are involved in and altered by asthma pathogenesis, and several lipid metabolites can serve as diagnostic markers for the disease. In this study, we investigated the characterization of severe asthma at the metabolic and cellular level. MethodsDifferential metabolites in blood samples from severe asthma (41 cases) and controls (18 cases) were screened using multifactorial statistical analysis and independent samples t-tests; key pathways were identified by KEGG enrichment analysis, and biomarkers were characterized based on ROC curves; immune cell types and proportions in the blood were identified based on the results of cell-type annotations (5 severe and 3 control cases); and single-sample Gene Enrichment Analysis (ssGSEA) to investigate the characterization of differential metabolic pathways in single cells. ResultsCompared with controls, the abundance of 28 metabolites was increased and the abundance of 13 metabolites was decreased in the blood of patients with severe asthma (P<0.05); the differential metabolites were enriched in 4 pathways: sphingolipid metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, and histidine metabolism. Among them, 13 differential metabolites could be used as biomarkers for the diagnosis of severe asthma, including L-glutamic acid (AUC=0.809), nicotinamide (AUC=0.886), phytosphingosine (AUC=0.882), and sphinganine (AUC=0.893). In single-cell transcriptome analysis,5 key cell types were identified: CD4⁺ T cells, CD8⁺ T cells, NK cells, B cells, and monocytes. The number of NK cells was increased in patients with severe asthma, and severe asthma exhibited more frequent cellular communication, particularly dense communication between CD8⁺ T cells and other cell types. In healthy samples, monocytes were the primary cells engaging in dense communication. Single-sample gene enrichment analysis (ssGSEA) showed that 4 pathways enriched for differential metabolites had lower scores (P<0.01) in CD4⁺ T and CD8⁺ T cells in severe patients, and it was hypothesized that the expression of genes associated with these pathways was suppressed in these two types of cells. The suppressed genes included DGKA and NT5C3A, which are associated with immune processes. We observed that these genes play key roles in the regulation of T cell signaling, activation, differentiation, and immune responses. ConclusionL-glutamic acid, nicotinamide, phytosphingosine, and sphinganine can be used as biomarkers for the diagnosis of severe asthma; genes of the severe asthma-associated pathway were suppressed in CD4⁺ T cells and CD8⁺ T cells.

Objective To compare the fidelity of chronic obstructive pulmonary disease(COPD)models established using two methods:exposure to cigarette smoke(CS)and exposure to motor vehicle exhaust(MVE)in rats.Methods Twenty-four male SD rats were randomly divided into control,CS-exposed(CS),and MVE-exposed(MVE)groups,with 8 rats per group.Rats in CS and MVE groups were exposed to CS or MVE,respectively,to induce COPD models.After COPD model established,lung function of each group was assessed.Bronchoalveolar lavage fluid(BALF)was collected to measure inflammatory cell counts,levels of inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factor(TNF)-α,and expression levels of mucin 5AC(MUC5AC).Lung tissue sections were stained with hematoxylin and eosin(HE)to observe pulmonary tissue and airway pathological changes.Periodic acid-Schiff(PAS)staining was used to detect goblet cell hyperplasia in airways.Results Compared with control group,rats in CS and MVE groups showed significantly increased inspiratory resistance(RI),total lung capacity(TLC),and lung static compliance(Cchord)(P<0.05),while expiratory flow parameters FEV50/FVC were significantly decreased(P<0.05).Compared with MVE group,rats in CS group had significantly higher RI,TLC,and Cchord(P<0.05),and lower FEV50/FVC(P<0.05).HE staining of lung tissues showed that mean linear intercept(MLI)was significantly higher in both CS and MVE groups compared with control group(P<0.05),with CS group having higher MLI than MVE group(P<0.05).BALF analysis revealed that white blood cells,neutrophils,macrophages,lymphocytes,IL-6,and TNF-α levels were significantly higher in both CS and MVE groups compared with control group(P<0.05),and inflammatory cell counts,IL-6,and TNF-α levels were higher in CS group compared with MVE group(P<0.05).PAS staining of lung tissues indicated that goblet cells in large airways were significantly increased in both CS and MVE groups compared with control group(P<0.05),with CS group showing higher goblet cell counts than MVE group(P<0.05).Expression levels of MUC5AC in BALF were significantly higher in both CS and MVE groups compared with control group(P<0.05),with CS group having significantly higher MUC5AC levels than MVE group(P<0.05).Conclusions Exposure to CS or MVE can establish a rat model of COPD,with CS exposure better mimicking characteristics of acute exacerbation of COPD compared to MVE exposure.

Objective To investigate the nutritional status of children with Crohn's Disease (CD) at diagnosis and its association with clinical characteristics. Methods A retrospective analysis was performed for the clinical data and nutritional status of 118 children with CD who were admitted to Beijing Children's Hospital,Capital Medical University,from January 2016 to January 2024. A multivariate logistic regression analysis was used to investigate the risk factors for malnutrition. Results A total of 118 children with CD were included,among whom there were 68 boys (57.6％) and 50 girls (42.4％),with a mean age of (11±4) years. Clinical symptoms mainly included recurrent abdominal pain (73.7％,87/118),diarrhea (37.3％,44/118),and hematochezia (32.2％,38/118),and 63.6％ (75/118) of the children had weight loss at diagnosis. The incidence rate of malnutrition was 63.6％ (75/118),and the children with moderate or severe malnutrition accounted for 67％ (50/75). There were 50 children (42.4％) with emaciation,8 (6.8％) with growth retardation,and 9 (7.6％) with overweight or obesity. Measurement of nutritional indices showed a reduction in serum albumin in 83 children (70.3％),anemia in 74 children (62.7％),and a reduction in 25 hydroxyvitamin D in 15 children (60％,15/25). The children with malnutrition had significantly higher disease activity,proportion of children with intestinal stenosis,and erythrocyte sedimentation rate and a significant reduction in serum albumin (P<0.05). The multivariate logistic regression analysis showed that intestinal stenosis was an independent risk factor for malnutrition in children with CD (OR=4.416,P<0.05). Conclusions There is a high incidence rate of malnutrition in children with CD at diagnosis,which is associated with disease activity and disease behavior. The nutritional status of children with CD should be closely monitored.

Objective To understand the change in distribution and antimicrobial resistance of bacteria isolated from blood specimens of Hunan Province,and provide for the initial diagnosis and treatment of clinical bloodstream infection(BSI).Methods Data reported from member units of Hunan Province Antimicrobial Resistance Survei-llance System from 2012 to 2021 were collected.Bacterial antimicrobial resistance surveillance method was imple-mented according to the technical scheme of China Antimicrobial Resistance Surveillance System(CARSS).Bacteria from blood specimens and bacterial antimicrobial susceptibility testing results were analyzed by WHONET 5.6 soft-ware and SPSS 27.0 software.Results A total of 207 054 bacterial strains were isolated from blood specimens from member units in Hunan Province Antimicrobial Resistance Surveillance System from 2012 to 2021,including 107 135(51.7％)Gram-positive bacteria and 99 919(48.3％)Gram-negative bacteria.There was no change in the top 6 pathogenic bacteria from 2012 to 2021,with Escherichia coli(n=51 537,24.9％)ranking first,followed by Staphylococcus epidermidis(n=29 115,14.1％),Staphylococcus aureus(n=17 402,8.4％),Klebsiella pneu-moniae(17 325,8.4％),Pseudomonas aeruginosa(n=4 010,1.9％)and Acinetobacter baumannii(n=3 598,1.7％).The detection rate of methicillin-resistant Staphylococcus aureus(MRSA)decreased from 30.3％in 2015 to 20.7％in 2021,while the detection rate of methicillin-resistant coagulase-negative Staphylococcus(MRCNS)showed an upward trend year by year(57.9％-66.8％).No Staphylococcus was found to be resistant to vancomy-cin,linezolid,and teicoplanin.Among Gram-negative bacteria,constituent ratios of Escherichia coli and Klebsiella pneumoniae were 43.9％-53.9％and 14.2％-19.5％,respectively,both showing an upward trend(both P＜0.001).Constituent ratios of Pseudomonas aeruginosa and Acinetobacter baumannii were 3.6％-5.1％and 3.0％-4.5％,respectively,both showing a downward trend year by year(both P＜0.001).From 2012 to 2021,resistance rates of Escherichia coli to imipenem and ertapenem were 1.0％-2.0％and 0.6％-1.1％,respectively;presenting a downward trend(P＜0.001).The resistant rates of Klebsiella pneumoniae to meropenem and ertapenem were 7.4％-13.7％and 4.8％-6.4％,respectively,presenting a downward trend(both P＜0.001).The resistance rates of Pseudomonas aeruginosa and Acinetobacter baumannii to carbapenem antibiotics were 7.1％-15.6％and 34.7％-45.7％,respectively.The trend of resistance to carbapenem antibiotics was relatively stable,but has de-creased compared with 2012-2016.The resistance rates of Escherichia coli to the third-generation cephalosporins from 2012 to 2021 were 41.0％-65.4％,showing a downward trend year by year.Conclusion The constituent ra-tio of Gram-negative bacillus from blood specimens in Hunan Province has been increasing year by year,while the detection rate of carbapenem-resistant Gram-negative bacillus remained relatively stable in the past 5 years,and the detection rate of coagulase-negative Staphylococcus has shown a downward trend.

Objective To investigate changes in the distribution and antimicrobial resistance of bacteria isolated from cerebrospinal fluid(CSF)specimens in Hunan Province,and provide reference for correct clinical diagnosis and rational antimicrobial use.Methods Data reported by member units of Hunan Province Antimicrobial Resistance Surveillance System from 2012 to 2021 were collected according to China Antimicrobial Resistance Surveillance Sys-tem(CARSS)technical scheme.Data of bacteria isolated from CSF specimens and antimicrobial susceptibility tes-ting results were analyzed with WHONET 5.6 and SPSS 20.0 software.Results A total of 11 837 bacterial strains were isolated from CSF specimens from member units of Hunan Province Antimicrobial Resistance Surveillance Sys-tem from 2012 to 2021.The top 5 strains were coagulase-negative Staphylococcus(n=6 397,54.0％),Acineto-bacter baumannii(n=764,6.5％),Staphylococcus aureus(n=606,5.1％),Enterococcus faecium(n=465,3.9％),and Escherichia coli(n=447,3.8％).The detection rates of methicillin-resistant coagulase-negative Staphyloco-ccus(MRCNS)and methicillin-resistant Staphylococcus aureus(MRSA)were 58.9％-66.3％and 34.4％-62.1％,respectively.No Staphylococcus spp.were found to be resistant to vancomycin,linezolid,and teicoplanin.The de-tection rate of Enterococcus faecium was higher than that of Enterococcus faecalis,and the resistance rates of En-terococcus f aecium to penicillin,ampicillin,high concentration streptomycin and levofloxacin were all higher than those of Enterococcus faecalis(all P=0.001).Resistance rate of Streptococcus pneumoniae to penicillin was 85.0％,at a high level.Resistance rate of Escherichia coli to ceftriaxone was＞60％,while resistance rates to enzyme inhibitors and carbapenem antibiotics were low.Resistance rate of Klebsiella pneumoniae to ceftriaxone was＞60％,to en-zyme inhibitors piperacillin/tazobactam and cefoperazone/sulbactam was＞30％,to carbapenem imipenem and me-ropenem was about 30％.Resistance rates of Acinetobacter baumannii to most tested antimicrobial agents were＞60％,to imipenem and meropenem were 59.0％-79.4％,to polymyxin B was low.Conclusion Among the bac-teria isolated from CSF specimens,coagulase-negative Staphylococcus accounts for the largest proportion,and the overall resistance of pathogenic bacteria is relatively serious.Bacterial antimicrobial resistance surveillance is very important for the effective treatment of central nerve system infection.