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

OBJECTIVE: This study aimed to explore the association between body mass index (BMI) and mortality based on the 10-year population-based multicenter prospective study. METHODS: A general population-based multicenter prospective study was conducted at four sites in rural China between 2013 and 2023. Multivariate Cox proportional hazards models and restricted cubic spline analyses were used to assess the association between BMI and mortality. Stratified analyses were performed based on the individual characteristics of the participants. RESULTS: Overall, 19,107 participants with a sum of 163,095 person-years were included and 1,910 participants died. The underweight (< 18.5 kg/m ²) presented an increase in all-cause mortality (adjusted hazards ratio [ aHR] = 2.00, 95% confidence interval [ CI]: 1.66-2.41), while overweight (≥ 24.0 to < 28.0 kg/m ²) and obesity (≥ 28.0 kg/m ²) presented a decrease with an aHR of 0.61 (95% CI: 0.52-0.73) and 0.51 (95% CI: 0.37-0.70), respectively. Overweight ( aHR = 0.76, 95% CI: 0.67-0.86) and mild obesity ( aHR = 0.72, 95% CI: 0.59-0.87) had a positive impact on mortality in people older than 60 years. All-cause mortality decreased rapidly until reaching a BMI of 25.7 kg/m ² ( aHR = 0.95, 95% CI: 0.92-0.98) and increased slightly above that value, indicating a U-shaped association. The beneficial impact of being overweight on mortality was robust in most subgroups and sensitivity analyses. CONCLUSION This study provides additional evidence that overweight and mild obesity may be inversely related to the risk of death in individuals older than 60 years. Therefore, it is essential to consider age differences when formulating health and weight management strategies.
Humans ; Body Mass Index ; China/epidemiology* ; Male ; Female ; Middle Aged ; Prospective Studies ; Rural Population/statistics & numerical data* ; Aged ; Follow-Up Studies ; Adult ; Mortality ; Cause of Death ; Obesity/mortality* ; Overweight/mortality*

Objective To evaluate the perioperative anxiety state and analyze the influencing factors of burned patients.Methods A total of 110 burned patients undergoing selective surgery under general anesthesia were included in the Fourth Medical Center of Chinese PLA General Hospital from February to August 2022.All patients were evaluated with self-rating anxiety scale(SAS),visual analogue scale-anxiety(VAS-a),visual analogue scale-pain(VAS-p),mini-mental state examination(MMSE),and Ramsay sedation score 1-day before and after operation.The patients'parameters were recorded including mean arterial pressure(MAP)and heart rate(HR)at admission(T0),before anesthesia induction(T1),2 min after intubation(T2),15 min after surgery(T3),during surgery(T4),at surgery end(T5),and immediately after leaving the operating room(T6).The occurrence and the influencing factors of perioperative anxiety in burn patients were analyzed using the univariate and multivariate logistic regression.Results The incidence of preoperative and postoperative anxiety in burn patients was 29.1%and 22.3%respectively.Univariate logistic analysis showed that gender(P=0.002),burn time(P=0.046),burn area(P=0.005),burn site(P=0.035),and degree of preoperative pain(P=0.001)were related with preoperative anxiety status in burn patients;while burn time(P=0.030),burn area(P=0.001),burn site(P=0.016),degree of preoperative pain(P=0.021),and preoperative anxiety status(P＜0.001)were related with postoperative anxiety state in burn patients.Multivariate logistic regression analysis showed that gender and degree of preoperative pain were the independent influencing factors of preoperative anxiety status in burn patients(P=0.002,0.022),and preoperative anxiety status was the independent influencing factor of postoperative anxiety status in burn patients(P＜0.001).Compared with the preoperative non-anxious patients(n=73),preoperative anxious patients(n=30)showed no significant difference in MAP at each time point(P＞0.05),but HR was accelerated(P＜0.05),and the dosage of sufentanil,remifentanil and propofol increased significantly during the operation(P＜0.05).Conclusions The anxiety state of burn patients was significantly higher before operation than that after operation,and their consumption of anesthetic drugs during operation was higher,and there was no significant correlation with the type and number of operation.Gender,degree of preoperative pain and anxiety state were the independent influencing factors of perioperative anxiety state in burn patients.Early intervention against relevant factors will help patients recover quickly.

Three 2,3-diketoquinoxaline alkaloids were isolated from Heterosmilax yunnanensis Gagnep. Their structures were determined through 1D and 2D NMR, HR-ESI-MS, UV, and IR as 1-[5′-(3″-hydroxy-3″-methyl) glutaryl] ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (1), 1-[2′-(3″-hydroxy-3″-methyl) glutaryl]ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (2), and 1-ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (3). Compounds 1 and 2 are novel compounds, and 3 was isolated from H. yunnanensis for the first time. The hepatoprotective activity of these three compounds was evaluated, with compound 3 showing promising hepatoprotective activity.

Objective To observe the clinical efficacy and safety of alteplase combined with rivaroxaban in the treatment of acute deep vein thrombosis(DVT)in elderly obese patients.Methods Elderly obese patients with DVT were divided into control group and treatment group according to random number table method.The control group was treated with 10 mg·d-1 rivaroxaban tablets orally.The treatment group was treated with 0.9 mg·kg-1·d-1 alteplase on the basis of the control group.Both groups were treated for 1 week.Clinical efficacy,coagulation function indexes,lower limb venous blood flow mechanics indexes,inflammatory factors and vascular endothelial function related indexes,relative expression level of miR-374b-5p,occurrence of adverse drug reactions and recurrence within 3 months were compared between the two groups.Results A total of 80 patients were enrolled in the treatment group and the control group,respectively.After treatment,the total effective rate of treatment group and control group were 92.50％(74 cases/80 cases)and 81.25％(65 cases/80 cases),respectively,and the difference was statistically significant(P＜0.05).After treatment,the fibrinogen levels of treatment group and control group were(3.58±0.83)and(3.91±1.04)g·L-1,respectively;D-dimer levels were(2.18±0.74)and(2.49±0.93)μg·mL-1,respectively;platelet activating factor were(115.42±10.43)and(119.74±11.37)μg·L-1,respectively;prothrombin time were(12.95±1.16)and(13.41±1.27)s,respectively;the maximum blood flow velocity were(29.19±6.74)and(26.93±4.58)cm·s-1,respectively;the blood flow were(92.17±9.61)and(88.63±8.75)mL·min-1,respectively;endothelial vasoconstricting peptide-1 were(51.97±4.86)and(54.02±4.92)pg·mL-1;the relative expression levels of miR-374b-5p were 2.18±0.73 and 2.49±0.85,respectively.The above indexes of the treatment group were statistically significant compared with the control group(all P＜0.05).Bleeding symptoms were the main adverse drug reactions in both groups.The total incidence of adverse drug reactions in treatment group and control group was 10.00％and 13.75％,respectively,with no statistical significance(P＞0.05).The 3-month recurrence rates of treatment group and control group was 6.25％and 16.25％,respectively,and the difference was statistically significant(P＜0.05).Conclusion Alteplase combined with rivaroxaban has good clinical efficacy in the treatment of elderly obese patients with DVT,which can improve the blood flow of patients,reduce inflammatory response,and has high safety.

AIM To establish a two reference substances for determination of multiple components(TRSDMC)method for the simultaneous content determination of neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,luteolin-7-O-glucuronide,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,deoxyelephantopin,isodeoxyelephantopin,isoscabertopin and scabertopin in Elephantopus scabre L..METHODS The analysis was performed on a 35℃thermostatic Waters Symmetry C18,Phenomenex C18,Agilent ZORBAX Eclipse Plus C18 columns(4.6 mm×250 mm,5.0 μm),with the mobile phase comprising of acetonitrile and 0.1%phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 220,326 nm.Chlorogenic acid was used as an internal standard to calculate the relative correction factors of neochlorogenic acid,cryptochlorogenic acid,luteolin-7-O-glucuronide,isochlorogenic acid B,isochlorogenic acid A and isochlorogenic acid C,while isodeoxyelephantopin was used as an internal standard to calculate the relative correction factors of deoxyelephantopin,scabertopin and isoscabertopin,after which the content determination was made.Subsequently,cluster analysis,principal component analysis and orthogonal partial least squares-discriminant analysis were conducted.RESULTS Eleven constituents showed good linear relationships within their own ranges(r≥0.999 0),whose average recoveries were 95.3%-103.4%with the RSDs of 0.32%-3.45%.The result obtained by TRSDMC approximated those obtained by external standard method.Isochlorogenic acid A,isochlorogenic acid C,isochlorogenic acid B,chlorogenic acid,luteolin-7-O-glucuronide and cryptochlorogenic acid were taken as quality differential constituents.CONCLUSION This reliable and stable method can be used for the quality control of E.scabre.

Background/Aims: Despite the high efficacy of direct-acting antivirals (DAAs), approximately 1–3% of hepatitis C virus (HCV) patients fail to achieve a sustained virological response. We conducted a nationwide study to investigate risk factors associated with DAA treatment failure. Machine-learning algorithms have been applied to discriminate subjects who may fail to respond to DAA therapy. Methods: We analyzed the Taiwan HCV Registry Program database to explore predictors of DAA failure in HCV patients. Fifty-five host and virological features were assessed using multivariate logistic regression, decision tree, random forest, eXtreme Gradient Boosting (XGBoost), and artificial neural network. The primary outcome was undetectable HCV RNA at 12 weeks after the end of treatment. Results: The training (n=23,955) and validation (n=10,346) datasets had similar baseline demographics, with an overall DAA failure rate of 1.6% (n=538). Multivariate logistic regression analysis revealed that liver cirrhosis, hepatocellular carcinoma, poor DAA adherence, and higher hemoglobin A1c were significantly associated with virological failure. XGBoost outperformed the other algorithms and logistic regression models, with an area under the receiver operating characteristic curve of 1.000 in the training dataset and 0.803 in the validation dataset. The top five predictors of treatment failure were HCV RNA, body mass index, α-fetoprotein, platelets, and FIB-4 index. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the XGBoost model (cutoff value=0.5) were 99.5%, 69.7%, 99.9%, 97.4%, and 99.5%, respectively, for the entire dataset. Conclusions Machine learning algorithms effectively provide risk stratification for DAA failure and additional information on the factors associated with DAA failure.

The medicinal value of vanilla planifolia is of great interest.We analyzed the common princi-pal components of VOCs in different parts of vanilla planifolia,and the human serum albumin (HSA),β-lactoglobulin (β-La) andα-lactalbumin (α-La) were used as template proteins to establish a chain a-nalysis approach with the 'solid phase microextraction gas chromatography-mass spectrometry-multispec-troscopy-physical modelling-pharmacokinetics' (S-M-P-P ) .The mechanisms of the transport and phar-macodynamics for the common principal components of vanilla planifolia were analyzed.The results showed that the common primary VOCs in different parts of vanilla planifolia was vanillin (Van),which attenuated the endogenous fluorescence of HSA/β-La/α-La by static bursting,and formed hydrogen bonds and Van der Waals forces with HSA,and noncovalent complexes with β-La/α-La through hydro-phobic forces.Their interaction facilitates the transport of Van in vivo to intestinal and hepatic tissues and its metabolism by CYP1A2 and CYP2C9 enzymes to exert its pharmacological effects.This study provides a comprehensive and in-depth investigation of the transport mechanisms and pharmacological effects of VOCs from vanilla planifolia,which provides an important reference for understanding the medicinal po-tential of plant derived VOCs.

Objective:To investigate the effect of feeder layer cells expressing interleukin(IL)-21 on the amplification of NK cells in vitro.Methods:The K562 cell line with IL-21 expression on its membrane was constructed by electroporation,and co-cultured with NK cells after inactivation.The proliferation of NK cells was observed.The killing function of the amplified NK cells in vitro was evaluated by the lactate dehydrogenase(LDH)and interferon-γ(IFN-y)release assay.A colorectal cancer xenograft model in NOD/SCID mice was established,and a blank control group,a NK cell group and an amplified NK cell group were set up to detect the tumor killing effect of amplified NK cells in vivo.Results:K562 cells expressing IL-21 on the membrane were successfully constructed by electroporation.After co-culturing with K562 cells expressing IL-21 on the membrane for 17 days,the NK cells increased to 700 times,which showed an enhanced amplification ability compared with control group(P＜0.001).In the tumor cell killing experiment in vitro,there was no significant difference in the killing activity on tumor cells between NK cells and amplified NK cells,and there was also no significant difference in mice in vivo.Conclusion:K562 cells expressing IL-21 on the membrane can significantly increase the amplification ability of NK cells in vitro,but do not affect the killing function of NK cells in vitro and in vivo.It can be used for the subsequent large-scale production of NK cells in vitro.

Objectives:To understand the current situation of the sense of uselessness of the elderly in China and the impact of Internet use on the sense of uselessness of the elderly,exploring the moderating role of age and social participation in it,so as to provide empirical evidence for improving the mental health of the elderly and promoting active aging.Method:T-test and analysis of variance were used for univariate analysis,multiple linear regression was used for multivariate analysis,and point selection and Johnson Neyman methods were used to analyze the moderating effects of age and social participation.Results:At present,the level of the sense of uselessness of the elderly in China is relatively high.Internet use affects the sense of uselessness of the elderly(b=-0.10,P<0.001);With the increase of age and the level of social participation,the alleviating effect of Internet use on the sense of uselessness of the elderly has gradually weakened(b=0.01,P<0.05;b=0.02,P<0.01).Internet use can alleviate the sense of uselessness of the elderly and is regulated by age and social participation.It is suggested that the Internet access intervention should be carried out as soon as possible for the elderly at a lower age,and the elderly with a lower level of social participation should be focused,so as to play the positive role of the Internet in reducing the sense of uselessness of the elderly and improving their mental health.