italic>Cynanchum wallichii and Cynanchum otophyllum belong to the genus Cynanchum in the family Apocynaceae, and are important medicinal plants. In this study, we sequenced and assembled the chloroplast genomes of C. wallichii and C. otophyllum, and performed a phylogenetic analysis of the structural characteristics of their chloroplast genomes and their phylogenetic positions. The results showed that the chloroplast genomes of both C. wallichii and C. otophyllum had a typical tetrad structure, with 133 genes annotated, and the total GC contents of both were similar. Codon preference analysis showed that the relative synonymous codon usage in the chloroplast genomes of C. wallichii and C. otophyllum differed slightly, but the differences were not significant, and there was a strong A or U preference at the third codon position. In both chloroplast genomes, 91 and 103 simple sequence repeats were detected respectively, and the largest proportion of A/T type repeats. Nucleotide polymorphism analysis showed that the nucleotide diversity of the intergenic sequences in the chloroplast genome of genus Cynanchum were generally higher than those of the common gene sequences. A pair of primers was designed based on the high variation region of the chloroplast genome to identify C. wallichii and C. otophyllum. The phylogenetic analysis showed that the C. wallichii and Cynanchum thesioides were the closest relatives, while the C. otophyllum, Cynanchum bungei and Cynanchum wilfordii formed a stable monophyletic clade within the genus Cynanchum, and the three species were closely related. The comparative analysis of the chloroplast genomic characteristics and phylogeny of C. wallichii and C. otophyllum will provide a theoretical basis for the species identification of the two plants and for the study of genetic diversity and phylogeny of the genus Cynanchum.

Objective To explore the risk signals of adverse events(ADE)in the use of exenatide microspheres by the FDA adverse event reporting system(FAERS),and provide reference for clinical rational drug use and drug safety.Methods With exenatide microspheres as the target drug,the search keywords were Exenatide Microspheres for Injection,LY05006,AC 2 993 LAR and Bydureon.SAS software was used to extract the ADE report data from January 2,2012 to March 31,2023 in the FAERS database and the duplicates were removed.Data mining of exenatide microspheres-related ADE reports was performed by the reporting odds ratio method and the comprehensive standard method.Results A total of 27 248 exenatide microspheres-related ADE reports were retrieved,involving 27 SOCs,of which 4 719 were severe ADE reports.The reporting personnel were mainly consumers(18 435 cases,67.66%),the United States was the mainly reporting country(26 295 cases,96.50%).A total of 163 ADE risk signals were obtained by reporting odds ratio method and comprehensive standard method,including new adverse reactions such as abnormal blood cholesterol,elevated lipase and mixed hyperlipidemia.Conclusion Based on the FAERS database,the post-marketing ADE of exenatide microspheres was mined and analyzed,which could provide reference for clinical medication safety and improvement of patients'medication compliance.

Objective To investigate the role of bone marrow mesenchymal stem cells derived from diabetic mice and their paracrine roles in inducing insulin resistance(IR).Methods The mouse model of diabetes mellitus was established,bone marrow mesenchymal stem cells(BMSC)were extracted and cultured,and the culture supernatant(M-BMSC-CS)was collected.(1)Cell experiment:HepG2 hepatocytes were divided into normal low-glycemic culture group[cultured with low-glycemic DMEM(5.55 mmol·L-1)],M-BMSC-CS experimental group(M-BMSC-CS 75 μL),and high-glycemic and high-lipid control group(given 25 mmol·L-1 high-glycemic DMEM+0.25 mmol·L-1 palmitic acid);(2)Animal experiments:Mice were divided into normal mice group(0.9％NaCl by intraperitoneal injection)and M-BMSC-CS-m group(M-BMSC-CS by intraperitoneal injection of normal mice(injection dose 0.2 mL/10 g)].Glucose intake was measured by glucose oxidase method.The fluorescence intensity of Glut2 protein was detected by immunofluorescence.The expression of insulin signaling pathway protein was detected by Western blot.Test oral glucose tolerance(OGTT)and insulin tolerance(ITT).Results The glucose intakes of the normal low-glucose culture group,the M-BMSC-CS experimental group and the high-glucose and high-lipid control group were(2.96±0.05),(1.64±0.28)and(1.42±0.32)mmol·L-1,respectively;the fluorescence expressions of glucose transporter 2(Glut2)were 53.21±2.70,30.95±3.39 and 34.96±7.60,respectively;the protein expression levels of phosphorylated insulin receptor substrate 1-ser307(p-IRS-1ser307)were 0.46±0.21,1.09±0.24 and 0.91±0.16,respectively;phosphorylated protein kinase(p-AKT)protein expression levels were 0.94±0.05,0.59±0.06 and 0.53±0.05;Glut2 protein expression levels were 1.08±0.14,0.58±0.14 and 0.62±0.09,respectively.The above indexes in M-BMSC-CS experimental group were statistically significant compared with those in normal low-glycemic culture group(all P＜0.05).Fasting blood glucose levels in the normal group and M-BMSC-CS-m group were(5.23±0.57)and(9.30±1.14)mmol·L-1;p-AKT protein expression level were 1.27±0.21 and 0.51±0.19;Glut2 protein expression level were 1.17±0.17 and 0.79±0.09,respectively.The above indexes in M-BMSC-CS-m group were significantly different from those in normal mouse group(P＜0.05).Conclusion BMSC culture supernatant from diabetic mice induced insulin resistance of normal HepG2 hepatocytes in vitro and normal mice in vivo.

Objective To investigate the relationship between the injury and ferroptosis of bone marrow mesenchymal stem cells(BMSCs)induced by high glucose and high fat.Methods BMSCs were divided into normal group(5.50 mmol·L-1 glucose)and high glucose and high fat(HGHF)group(25.00 mmol·L-1 glucose+0.25 mmol·L-1 palmitic acid).Assessment of cellular aging via β-galactosidase staining;enzyme linked immunosorbent assay(ELISA)were used to detect tumor necrosis factor-α(TNF-α),interleukin-10(IL-10)release levels;glutathione(GSH),malondialdehyde(MDA)and ferrous ion(Fe2+)detection kits were used to detect ferroptosis related indicators;Western blotting was used to detect the expression of ferroptosis related signaling pathway protein acyl-CoA synthetase long chain family member 4(ACS14)/arachidonate 15-lipoxygenase(ALOX15)/glutathione peroxidase 4(GPX4).Results The senescence rates of normal group HGHF group were(6.80±1.60)％and(13.00±1.58)％;the levels of TNF-α were(122.54±3.94)and(169.77±2.89)pg·mL-1;the levels of IL-10 were(155.16±3.97)and(105.15±7.30)pg·mL-1;GSH levels were 4.30±0.33 and 1.55±0.14;MDA levels were 2.94±0.10 and 5.84±0.10;Fe2+levels were 6.22±0.35 and 16.13±0.36;the relative expression levels of ACSL4 protein were 0.42±0.05 and 0.84±0.10;the relative ALOX15 protein were 0.61±0.25 and 1.06±0.11;the relative expression levels of GPX4 protein were 1.13±0.17 and 0.33±0.08,respectively.The above indexes in the HGHF group were significantly different from those in the normal group(all P＜0.05).Conclusion 25 mmol·L-1 glucose combined with 0.25 mmol·L-1 palmitic acid for 24 h can be used as a suitable condition to induce BMSCs injury.ferroptosis plays an important role in BMSCs injury induced by high glucose and high fat.

In recent years, nucleic acid therapy, as a revolutionary therapeutic tool, has shown great potential in the treatment of genetic diseases, infectious diseases and cancer. Lipid nanoparticles (LNPs) are currently the most advanced mRNA delivery carriers, and their emergence is an important reason for the rapid approval and use of COVID-19 mRNA vaccines and the development of mRNA therapy. Currently, mRNA therapeutics using LNP as a carrier have been widely used in protein replacement therapy, vaccines and gene editing. Conventional LNP is composed of four components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids, which can effectively load mRNA to improve the stability of mRNA and promote the delivery of mRNA to the cytoplasm. However, in the face of the complexity and diversity of clinical diseases, the structure, properties and functions of existing LNPs are too homogeneous, and the lack of targeted delivery capability may result in the risk of off-targeting. LNPs are flexibly designed and structurally stable vectors, and the adjustment of the types or proportions of their components can give them additional functions without affecting the ability of LNPs to deliver mRNAs. For example, by replacing and optimizing the basic components of LNP, introducing a fifth component, and modifying its surface, LNP can be made to have more precise targeting ability to reduce the side effects caused by treatment, or be given additional functions to synergistically enhance the efficacy of mRNA therapy to respond to the clinical demand for nucleic acid therapy. It is also possible to further improve the efficiency of LNP delivery of mRNA through machine learning-assisted LNP iteration. This review can provide a reference method for the rational design of engineered lipid nanoparticles delivering mRNA to treat diseases.

Palmatine, the main effective ingredient of Fibraurea recisa, is a typical berberine isoquinoline alkaloid with extensive anti-inflammatory and antibacterial activities. In this work, the studies of metabolomics and transcriptomics were utilized to detect differentially expressed genes (DEGs) that are significantly associated with the synthesis of palmatine. In addition, eight of these DEGs were verified by quantitative real-time PCR (qRT-PCR). A total of 106 alkaloids were detected in the metabolomics study, including 23 isoquinoline alkaloids. Palmatine ranked in the top ten of differential metabolites in the group of root vs leaf, and its relative content in root was about 47.5 times higher than that in leaf. In the transcriptomics study, a total of 188 genes were annotated to the pathway of isoquinoline alkaloid biosynthesis. Among them, there were 36 DEGs were significantly different. In the comparison group of root and leaf, a total of 33 DEGs were significantly different, and 30 DEGs were annotated on the biosynthetic pathway of palmatine. Finally, the results of the correlation analysis between metabolomics and transcriptomics showed that the expression patterns of four gene sequences were screened to be significantly correlated with palmatine. The results of qRT-PCR experiments showed that the expression trends of eight DEGs were consistent with the results of transcriptomic. This study not only enriched the omics data of F. recisa, but also established the foundation for the study of the synthetic biology of palmatine. It further provided a reference for the analysis of the key enzyme genes on the biosynthetic pathway of other isoquinoline alkaloids.

Lipopolysaccharides (LPS) are major outer membrane components of Gram-negative bacteria. Unlike most Gram-negative bacteria, Acinetobacter baumannii can still survive and acquire polymyxin resistance after complete loss of LPS. Previous studies of LPS-deficient Acinetobacter baumannii mostly focused on LPS-deficient strains induced by polymyxin, whose background was complex and unstable. To investigate LPS loss-mediated polymyxin resistant-Acinetobacter baumannii, this study constructed a stable and clear background LPS-deficient strain by knocking out lpxC gene in Acinetobacter baumannii ATCC 19606 with CIRSPR/Cas9, and then studied the phenotypic changes of the lpxC-deficient strain including morphology, growth rate, antibiotic susceptibility, virulence, membrane permeability, and membrane potential. Animal experiments were approved by the Animal Care and Welfare Committee Institute of Medicinal Biotechnology, CAMS and PUMC (approval number: IMB-20240119D₉). The results indicated that the lpxC gene of Acinetobacter baumannii ATCC 19606 was successfully knocked out. After losing the lpxC, the strain underwent the morphological change from rod-shaped to spherical. Furthermore, it leads to reduced growth rate, enhanced membrane permeability, decreased membrane potential, lower virulence, and increased antibiotic susceptibility to β-lactams, quinolones, aminoglycosides, macrolides, glycopeptides. The lpxC deletion results in significant changes in membrane homeostasis and adaptability of Acinetobacter baumannii. Understanding the phenotypic changes of colistin-resistant Acinetobacter baumannii mediated by LPS loss is useful for exploring the resistance mechanism of Acinetobacter baumannii and developing new therapeutic strategies.

Objective:To explore the predictive value of methyltetrahydrofolate reductase (MTHFR) gene polymorphism in collaboration with serum ANG2 expression on the efficacy of estrogen receptor (ER) positive breast cancer chemotherapy.Methods:Sixty-six patients with ER-positive breast cancer who underwent chemotherapy were selected from Changshu First People’s Hospital from Mar. 2017 to Mar. 2022. According to the efficacy of chemotherapy, patients were divided into effective group and ineffective group. Clinical data of two groups of patients were collected, and PCR was used to detect MTHFR gene polymorphisms in leukocyte DNA and ANG2 expression in serum of patients before chemotherapy. The general data, MTHFR gene polymorphism and ANG2 level of the two groups were compared, and the factors influencing the efficacy of chemotherapy for ER-positive breast cancer were analyzed by Logistic regression. The predictive value of MTHFR gene polymorphism and ANG2 level in ER-positive breast cancer chemotherapy was analyzed according to receiver operating characteristic (ROC) curve.Results:There were 11 complete responses (CR), 38 partial responses (PR), 10 stable disease (SD), and 7 progressive disease (PD) after chemotherapy in 66 ER-positive breast cancer patients. The proportion of lymphatic metastases, the proportion of CC in C677T MTHFR gene and the level of ANG2 in serum in the ineffective group were higher than those in the effective group ( P<0.05). Logistic analysis showed that lymph metastases, C677T MTHFR gene and ANG2 level were independent risk factors for chemotherapy efficacy of ER-positive breast cancer by Logistic regression analysis ( P<0.05). ROC analysis showed that the area under the curve (AUC) values of C677T MTHFR gene and ANG2 in predicting the efficacy of chemotherapy for ER-positive breast cancer were 0.779 and 0.726, respectively, and 95% CI were 0.660-0.872 and 0.596-0.824. The combined predicted AUC value was 0.920, and 95% CI was 0.826-0.972. Conclusions:MTHFR gene polymorphism and ANG2 level are of reference value for chemotherapy efficacy of ER positive breast cancer. C677T type MTHFR gene has higher value in predicting the efficacy of ER-positive breast cancer chemotherapy in collaboration with serum ANG2 level.

This study was aimed at characterizing the variations in hemagglutinin(HA)and neuraminidase(NA)genes of influenza virus subtype A(H1N1)pdm09 isolated during the 2022-2023 influenza monitoring year in Shandong Province,to provide a scientific basis for influenza prevention and control.A total of 14 A(H1N1)pdm09 subtype influenza strains were se-lected randomly by city by the influenza monitoring network laboratory.The vaccine strains recommended by the WHO served as references for whole gene sequencing analysis.A fluorescence method was used to conduct neuraminidase inhibition experi-ments to evaluate drug sensitivity.The A(H1N1)pdm09 influenza virus in Shandong Province,2022-2023 belonged to the 5a.2a evolutionary cluster in the 6B.1A branch.Nucleotide sequence analysis indicated that the HA and NA genes were closely re-lated to the Northern Hemisphere vaccine strain A/Victoria/2570/2019 in the years 2021-2023,and showed homology of 98.5％to 98.7％and 98.8％to 99.1％,respectively.Amino acid sequence analysis revealed 20 amino acid sequence mutations in the HA protein,but only one virus strain was found to have antigen drift,and three virus strains showed loss of HA protein glycosylation sites.No mutations were found at important sites affecting NA enzymes.The neuraminidase inhibition experiment indicated viral sensitivity to anti-influenza drugs.In conclusion,the monitored virus strains had high overall homology with vac-cine strains but showed some amino acid variation.In the future,continued monitoring of the genetic variation characteristics of influenza viruses will be necessary to understand the risk of influenza epidemics,and the effectiveness of influenza vaccines and therapeutic drugs.

This study was aimed at understanding the pollution distribution pattern of avian influenza virus in the environ-ment in poultry related places in Shandong Province,to provide a scientific basis for the prevention,control,prediction,and early warning regarding human infection with avian influenza.From 2020 to 2023,6 523 environmental samples were collected in 16 cities in Shandong Province from four types of poultry-related places.Fluorescence quantitative PCR was used for nucleic acid testing of influenza A virus.Positive samples were further identified for the H5,H7,and H9 subtypes of avian influenza virus.The epidemiological characteristics of avian influenza viruses in the poultry related environment of Shandong Province were described,and inter-rate comparisons were performed with the x2 test.During 2020-2023,6 523 environmental samples were collected,and 1 007 cases positive for avian influenza virus were detected,with a positivity rate of 15.44％.H5,H7,and H9 subtypesand mixed infections were detected.H9 was the main subtype(88.48％)in positive specimens.A significant difference in positivity rates was observed among regions(x2=431.956,P＜0.001),and the highest positivity rate was 28.93％.Significant differences in positivity rates were observed among monitoring sites(x2=304.604,P＜0.001),sample types(x2=109.678,P＜0.001),and quarters(x2=64.963,P＜0.001).The positive detection rate was highest at monitoring sites in urban and rural live poultry markets(20.12％),and the positive detection rate of samples collected by wiping meat cut-ting board surfaces was higher than that of other samples(22.56％).The peak positive detection rate occurred in spring(20.31％).Widespread contamination with avian influenza virus was observed in poultry environments in Shandong Prov-ince.The H9 subtype,the main pathogen,coexisted with H5 and H7 subtypes,thus posing a risk of human infection with avian influenza.Therefore,prevention and control of avian influenza must be strengthened in key seasons,areas,places,and links.