Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

Ankylosing spondylitis (AS) combined with lower cervical fracture is often categorized into unstable fracture, with a high incidence of neurological injury and a high rate of disability and morbidity. As factors such as shoulder occlusion may affect the accuracy of X-ray imaging diagnosis, it is often easily misdiagnosed at the primary diagnosis. Non-operative treatment has complications such as bone nonunion and the possibility of secondary neurological damage, while the timing, access and choice of surgical treatment are still controversial. Currently, there are no clinical practice guidelines for the treatment of AS combined with lower cervical fracture with or without dislocation. To this end, the Spinal Trauma Group of Orthopedics Branch of Chinese Medical Doctor Association organized experts to formulate Clinical guidelines for the treatment of ankylosing spondylitis combined with lower cervical fracture in adults ( version 2024) in accordance with the principles of evidence-based medicine, scientificity and practicality, in which 11 recommendations were put forward in terms of the diagnosis, imaging evaluation, typing and treatment, etc, to provide guidance for the diagnosis and treatment of AS combined with lower cervical fracture.

The purpose of this study was to understand the prevalence of Neoehrlichia mikurensis in rodents in Yunnan commensal rodent plague foci.Lianghe Country,Mangshi City,and Mile City in Yunnan Province were chosen as sampling sites,where rodents were captured with dead-traps.The N.mikurensis groEL gene in rodent spleen samples was detected with nested PCR,and the positive products were sequenced with Sanger bidirectional assays.The infection rate of N.mikurensis a-mong plague foci,habitats,species,and sexes was compared with Chi-square tests or Fisher's exact probability method.Of 656 rodent spleen samples,12 N.mikurensis positive samples were detected in R.tanezumi,R.sladeni,N.confucianus,and B.bowersi.The positivity rate was 1.83％.No significant difference in the N.mikurensis positivity rate was observed a-mong plague foci,habitats,species,and sexes(P＞0.05).Genetic evolution analysis of the groEL gene indicated that the se-quence similarity of nucleic acid sequences in 12 positive samples was 99.5％-100％,and the nucleic acid sequences of N.mikurensis were in the same branch,belonging to cluster Ⅳ.Thus,four species of rodents were found to have low frequency infection with N.mikurensis in Yunnan commensal rodent plague foci.

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

Objective:To examine the perioperative safety of laparoscopic cholecystectomy in human immunodeficiency virus (HIV)-positive patients undergoing antiretroviral therapy.Methods:A retrospective cohort study method was employed to collect clinical data from 388 patients with gallstones who were admitted to Department of General Surgery of the Provincial Hospital of Anhui Medical University between January 2020 and December 2023. The cohort comprised 135 males and 253 females. For the purpose of analysis, the participants were divided into HIV-positive group ( n=25) and HIV-negative group ( n=363). The distinction between the two groups was based on whether the participants were co-infected with HIV. The clinical and pathological data pertaining to the two groups of patients, along with the intraoperative and postoperative data, were collated. The propensity score matching method was employed to control for confounding bias, and the data on anaesthesia methods, intraoperative blood loss, duration of surgery, necessity for conversion to laparotomy, postoperative complications, postoperative drainage volume, postoperative hospital stay, and total treatment costs were compared between the two groups. Measurement data with normal distribution were expressed as mean±standard deviation ( ± s) and compared using the t-test. Measurement data with skewed distribution were expressed as M( Q1, Q3) and compared using the rank-sum test. The Fisher exact probability method or the chi-square test was employed for the comparison of count data. Results:The two groups of general data were found to be statistically significant in terms of gender, preoperative red blood cells, neutrophils, alanine aminotransferase, and indirect bilirubin exhibited statistically significant differences( P<0.05). Following propensity score matching, a total of 59 cases were identified. Of these, 25 cases were observed in the HIV-positive group, while 34 cases were observed in the HIV-negative group. No statistically significant differences were observed between the two groups with respect to the general data ( P>0.05). No statistically significant difference was observed between the two groups in terms of anaesthesia method, intraoperative blood loss, duration of surgery, need for conversion to laparotomy, postoperative complication rate, postoperative drainage volume and postoperative hospital stay ( P>0.05). The total length of hospital stay and total hospital expenses in the HIV-positive group were 5.0 (3.0, 7.0) days and (14 829.3±4 852.8) yuan, respectively. HIV-negative group were 3.0 (2.0, 4.8) days and (10 762.3±1 850.3) yuan, respectively. The HIV-negative group had a significantly shorter hospital stay and lower hospital expenses, these differences were statistically significant ( P<0.05). There was no occupational exposure of medical personnel in the perioperative period among those with HIV. Conclusions:Laparoscopic cholecystectomy is a safe and feasible procedure for HIV-positive patients who are receiving antiretroviral therapy. Adherence to established protocols by healthcare personnel can mitigate the risk of occupational exposure.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Objective:To investigate the causal correlation between depression and stress urinary incontinence(SUI)using Mendelian randomization(MR)analysis.Methods:We searched the FinnGen Consortium database for genome-wide association studies(GWAS)on depression and obtained 23 424 case samples and 192 220 control samples,with the GWAS data on SUI provided by the UK Biobank,including 4 340 case samples and 458 670 control samples.We investigated the correlation between depression and SUI based on the depression data collected from the Psychiatric Genomics Consortium(PGC).We employed inverse-variance weighting as the main method for the MR study,and performed sensitivity analysis to verify the accuracy and stability of the findings.Results:Analysis of the data from the UK Biobank and FinnGen Consortium showed that depression was significantly correlated with an increased risk of SUI(P=0.005),but not SUI with the risk of depression(P=0.927).And analysis of the PGC data verified the correlation of depression with the increased risk of SUI(P=0.043).Conclusion:Depression is associated with an increased risk of SUI,while SUI does not increase the risk of depression.

Objective:To investigate the effect of miRNA-3653-3p (miR-3653-3p) on the proliferation and invasion ability of endometrial cancer cells and its related mechanisms.Methods:The data of 356 endometrial cancer patients were downloaded from the OncoLnc database (http://www.oncolnc.org, updated version 2020), and the Kaplan-Meier method was used to analyze the relationship between the expression level of miR-3653-3p and the overall survival of endometrial cancer patients. The miRGator database (https://bio.tools/mirgator_v2.0, updated version 2019) was used to predict the target gene binding to miR-3653-3p. Human endometrial cancer cell lines AN3CA, HEC-1A, HEC-1B, Ishikawa and human normal endometrial epithelial cell line ESC were selected, and the relative expression level of miR-3653-3p was detected by using quantitative real-time fluorescent polymerase chain reaction (qRT-PCR). The cell line with the lowest expression of miR-3653-3p was selected as the research object, which was divided into the negative control group and miR-3653-3p group, and transfected with the control empty vector plasmid and miR-3653-3p overexpression plasmid. CCK-8 method was used to detect the proliferation ability of cells, Transwell method was used to detect the invasion ability of cells, and qRT-PCR and Western blot were used to detect the expression of miR-3653-3p target gene. The effect of miR-3653-3p on the related protein expression of Wnt- β-catenin signaling pathway was detected by using Western blot.Results:Data analysis in the OncoLnc database showed that compared with endometrial cancer patients with low miR-3653-3p expression, patients with high miR-3653-3p expression had better overall survival ( P < 0.01). Compared with human normal endometrial epithelial ESC, the expression levels of miR-3653-3p in endometrial cancer cell lines AN3CA, HEC-1A, HEC-1B, and Ishikawa were all decreased (all P < 0.05), and the relative expression level of miR-3653-3p was the lowest in HEC-1A cells, and HEC-1A cells were selected for subsequent experiments. The result of CCK-8 showed that compared with the negative control group, the ability of HEC-1A cells in the miR-3653-3p group decreased on the 2nd, 3rd, 4th, and 5th days (all P < 0.05). The result of the Transwell chamber invasion test showed that the number of HEC-1A cell invasion after culturing for 26 h in the negative control group and the miR-3653-3p group was (80±11) and (21±4), respectively, and the difference was statistically significant ( t = 5.18, P < 0.01); compared with the negative control group, the number of cell invasion in the miR-3653-3p group decreased. The miRGator database was used to predict that the target gene of miR-3653-3p might be placenta-specific protein 8 (PLAC8). The relative expression levels of PLAC8 mRNA in HEC-1A cells in the negative control group and miR-3653-3p group were (6.26±0.83) and (0.97±0.31), respectively, and the difference was statistically significant ( t = 6.00, P < 0.01); the relative expression level of PLAC8 mRNA in the miR-3653-3p group was lower than that in the negative control group. Compared with the negative control group, the PLAC8 protein of HEC-1A cells decreased, and the expression of Wnt-β-catenin signaling pathway related proteins β-catenin, transforming growth factor β (TGF-β), GSK-3β, and Rac1 decreased in the miR-3653-3p group. Conclusions:miR-3653-3p may inhibit the proliferation and invasion of endometrial cancer cells by regulating the PLAC8-Wnt-β-catenin signaling pathway.

Krüppel-like transcription factor 2 (KLF2) plays a key regulatory role in endothelial inflammation, thrombosis, angiogenesis and macrophage inflammation and polarization, and up-regulation of KLF2 expression has the potential to prevent and treatment atherosclerosis. In this study, trichostatin C (TSC) was obtained from the secondary metabolites of rice fermentation of Streptomyces sp. CPCC 203909 as a KLF2 up-regulator by using a high throughput screening model based on a KLF2 promoter luciferase reporter assay. TSC significantly inhibited the adhesion of tumor necrosis factor-α (TNFα) induced monocytes (THP-1) to human umbilical vein endothelial cells (HUVECs). Western blot results showed that TSC decreased TNFα induced the protein expression increase of vascular cell adhesion molecule-1 (VCAM-1), and thereby inhibited endothelial inflammation. The results of histone deacetylase (HDAC) overexpression and molecular docking experiments showed that TSC upregulated the expression of KLF2 by inhibiting subtypes of HDAC 4/5/7. In conclusion, this study suggests that TSC up-regulates the expression of KLF2 through inhibiting HDAC 4/5/7 and thus inhibits TNFα induced endothelial inflammation, and it has the potential to prevent and treat atherosclerosis.

Excessive application of chemical fertilizer has caused many problems in Angelica dahurica var. formosana planting, such as yield decline and quality degradation. In order to promote the green cultivation mode of A. dahurica var. formosana and explore rhizosphere fungus resources, the rhizosphere fungi with nitrogen fixation, phosphorus solubilization, potassium solubilization, iron-producing carrier, and IAA-producing properties were isolated and screened in the rhizosphere of A. dahurica var. formosana from the genuine and non-genuine areas, respectively. The strains were identified comprehensively in light of the morphological characteristics and ITS rDNA sequences, and the growth-promoting effect of the screened strains was verified by pot experiment. The results showed that 37 strains of growth-promoting fungi were isolated and screened from the rhizosphere of A. dahurica var. formosana, mostly belonging to Fusarium. The cultured rhizosphere growth-promoting fungi of A. dahurica var. formosana were more abundant and diverse in the genuine producing areas than in the non-genuine producing areas. Among all strains, Aspergillus niger ZJ-17 had the strongest growth promotion potential. Under the condition of no fertilization outdoors, ZJ-17 inoculation significantly promoted the growth, yield, and accumulation of effective components of A. dahurica var. formosana planted in the soil of genuine and non-genuine producing areas, with yield increases of 73.59% and 37.84%, respectively. To a certain extent, it alleviated the restriction without additional fertilization on the growth of A. dahurica var. formosana. Therefore, A. niger ZJ-17 has great application prospects in increasing yield and quality of A. dahurica var. formosana and reducing fertilizer application and can be actually applied in promoting the growth of A. dahurica var. formosana and producing biofertilizer.
Fertilizers ; Rhizosphere ; Angelica/chemistry* ; Fungi/genetics* ; Phosphorus