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

BACKGROUND:Inflammation is one of the important factors that induce cerebral ischemia-reperfusion injury.Studies have shown that electroacupuncture can effectively reduce inflammation after ischemic stroke and improve the symptoms of neurological deficits,but the mechanism is not clear. OBJECTIVE:To observe the effect of electroacupuncture on Toll-like receptor 4/nuclear factor-κB in rats with cerebral ischemia-reperfusion injury. METHODS:Forty-eight male Sprague-Dawley rats were randomly divided into sham operation group,model group and electroacupuncture group,with 16 rats in each group.The rat model of cerebral ischemia-reperfusion injury was prepared by middle cerebral artery occlusion.At 24 hours after modeling,the rats in the electroacupuncture group were treated with electroacupuncture,once a day,20 minutes each time,for a total of 5 days.The sham operation group and the model group did not do any intervention.After 5 days of intervention,Longa method was used to evaluate the degree of neurological injury in rats.Triphenyl tetrazolium chloride staining and hematoxylin-eosin staining were used to measure the volume of cerebral infarction and the pathological changes of brain tissue in rats.Serum interleukin-6,interleukin-18 and tumor necrosis factor-α were detected by ELISA.Expressions of Toll-like receptor 4 and nuclear factor-κB in the cerebral cortex at mRNA and protein levels were detected by fluorescence quantitative PCR and western blot,respectively. RESULTS AND CONCLUSION:Compared with the sham operation group,the neurological function scores,serum interleukin-6,interleukin-18,and tumor necrosis factor-α levels,Toll-like receptor 4 and nuclear factor-κB mRNA and protein expression levels were significantly higher in the model group(P＜0.01).Compared with the model group,electroacupuncture significantly reduced the neurological function scores,serum interleukin-6,interleukin-18,and tumor necrosis factor-α levels,Toll-like receptor 4 and nuclear factor-κB mRNA and protein expression levels(P＜0.05,P＜0.01).Compared with the sham operation group,the volume of cerebral infarction in the model group increased significantly(P＜0.01).Compared with the model group,the volume of cerebral infarction in the electroacupuncture group decreased(P＜0.05).In the model group,the arrangement of neurons was disordered,some nerve cells disappeared,nuclei presented with pyknosis and incomplete structure.After electroacupuncture intervention,the degree of neuronal degeneration and neuronal loss in the cerebral cortex of rats were reduced compared with those in the model group.To conclude,electroacupuncture can significantly improve the neurobehavior of rats with cerebral ischemia-reperfusion injury,reduce brain tissue injury,and effectively reduce the level of serum inflammatory factors.The mechanism may be related to the inhibition of Toll-like receptor 4/nuclear factor-κB signaling pathway.

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 whether activation of mitochondrial acetal dehydrogenase 2(ALDH2)alleviates hypoxic pulmonary hypertension by regulating the SIRT1/PGC-1α signaling pathway.Methods Thirty 8-week-old C57 BL/6 mice were randomized into control,hypoxia,and hypoxia+Alda-1(an ALDH2 activator)group(n=10),and the mice in the latter two groups,along with 10 ALDH2 knockout(ALDH2-/-)mice,were exposed to hypoxia(10%O2,90%N2)with or without daily intraperitoneal injection of Alda-1 for 4 weeks.The changes in right ventricular function and pressure(RVSP)of the mice were evaluated by echocardiography and right ventricular catheter test,and pulmonary artery pressure was estimated based on RVSP.Pulmonary vascular remodeling,right ventricular injury,myocardial α-SMA expression,distal pulmonary arteriole muscle normalization,right ventricular cross-sectional area,myocardial cell hypertrophy,and right cardiac hypertrophy index were assessed with HE staining,immunofluorescence staining and WGA staining,and the expressions of ALDH2,SIRT1,PGC-1α,P16INK4A and P21CIP1 were detected.In pulmonary artery smooth muscle cells with hypoxic exposure,the effect of Alda-1 and EX527 on cell senescence and protein expressions was evaluated using β-galactose staining and Western blotting.Results The wild-type mice with hypoxic exposure showed significantly increased RVSP,right ventricular free wall thickness and myocardial expressions of P16INK4A and P21CIP1,which were effectively lowered by treatment with Alda-1 but further increased in ALDH2-/-mice.In cultured pulmonary artery smooth muscle cells,hypoxic exposure significantly increased senescent cell percentage and cellular expressions of P16INK4A and P21CIP1,which were all lowered by treatment with Alda-1,but its effect was obviously attenuated by EX527 treatment.Conclusion ALDH2 alleviates hypoxia-induced senescence of pulmonary artery smooth muscle cells by upregulating the SIRT1/PGC-1α signaling pathway to alleviate pulmonary hypertension in mice.

Cardiac cephalalgia is a rare disease caused by underlying coronary artery disease that presents as headache with or without chest symptoms.Headache symptoms are often caused by referred pain,increased intracranial pressure and release of large amounts of pain-causing neurochemicals due to myocardial ischemia,and cortical hypoperfusion.Due to the low overall prevalence of the disease,the lack of chest pain typical of coronary heart disease,and the fact that it may be difficult to distinguish from headaches caused by neurological diseases,accurate diagnosis and treatment of the disease are often delayed.We report a case of acute coronary syndrome with headache only.Revascularization was achieved through percutaneous coronary intervention therapy,followed by standard secondary prevention pharmacotherapy.Follow-up six months postoperatively showed a significant improvement in exercise tolerance,with no further headache episodes.The purpose is to improve the understanding of patients with cardiac cephalalgia,with a view to early identification and timely intervention,and ultimately improve the symptoms and prognosis.

Objective To observe the efficacy and safety of Morinda officinalis oligosaccharides in the continuation treatment of mild and moderate depression.Methods An open,single-arm,multi-center design was adopted in our study.Adult patients with mild and moderate depression who had received acute treatment of Morinda officinalis oligosaccharides were enrolled and continue to receive Morinda officinalis oligosaccharides capsules for 24 weeks,the dose remained unchanged during continuation treatment.The remission rate,recurrence rate,recurrence time,and the change from baseline to endpoint of Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA),Clinical Global Impression-Severity(CGI-S)and Arizona Sexual Experience Scale(ASEX)were evaluated.The incidence of treatment-related adverse events was reported.Results The scores of HAMD-17 at baseline and after treatment were 6.60±1.87 and 5.85±4.18,scores of HAMA were 6.36±3.02 and 4.93±3.09,scores of CGI-S were 1.49±0.56 and 1.29±0.81,scores of ASEX were 15.92±4.72 and 15.57±5.26,with significant difference(P＜0.05).After continuation treatment,the remission rate was 54.59％(202 cases/370 cases),and the recurrence rate was 6.49％(24 cases/370 cases),the recurrence time was(64.67±42.47)days.The incidence of treatment-related adverse events was 15.35％(64 cases/417 cases).Conclusion Morinda officinalis oligosaccharides capsules can be effectively used for the continuation treatment of mild and moderate depression,and are well tolerated and safe.

Objective To investigate whether activation of mitochondrial acetal dehydrogenase 2(ALDH2)alleviates hypoxic pulmonary hypertension by regulating the SIRT1/PGC-1α signaling pathway.Methods Thirty 8-week-old C57 BL/6 mice were randomized into control,hypoxia,and hypoxia+Alda-1(an ALDH2 activator)group(n=10),and the mice in the latter two groups,along with 10 ALDH2 knockout(ALDH2-/-)mice,were exposed to hypoxia(10%O2,90%N2)with or without daily intraperitoneal injection of Alda-1 for 4 weeks.The changes in right ventricular function and pressure(RVSP)of the mice were evaluated by echocardiography and right ventricular catheter test,and pulmonary artery pressure was estimated based on RVSP.Pulmonary vascular remodeling,right ventricular injury,myocardial α-SMA expression,distal pulmonary arteriole muscle normalization,right ventricular cross-sectional area,myocardial cell hypertrophy,and right cardiac hypertrophy index were assessed with HE staining,immunofluorescence staining and WGA staining,and the expressions of ALDH2,SIRT1,PGC-1α,P16INK4A and P21CIP1 were detected.In pulmonary artery smooth muscle cells with hypoxic exposure,the effect of Alda-1 and EX527 on cell senescence and protein expressions was evaluated using β-galactose staining and Western blotting.Results The wild-type mice with hypoxic exposure showed significantly increased RVSP,right ventricular free wall thickness and myocardial expressions of P16INK4A and P21CIP1,which were effectively lowered by treatment with Alda-1 but further increased in ALDH2-/-mice.In cultured pulmonary artery smooth muscle cells,hypoxic exposure significantly increased senescent cell percentage and cellular expressions of P16INK4A and P21CIP1,which were all lowered by treatment with Alda-1,but its effect was obviously attenuated by EX527 treatment.Conclusion ALDH2 alleviates hypoxia-induced senescence of pulmonary artery smooth muscle cells by upregulating the SIRT1/PGC-1α signaling pathway to alleviate pulmonary hypertension in mice.

Lithocarpus litseifolius is rich in the chalcones phloridzin and trilobatin, the biosynthesis pathways of which have not been fully demonstrated. Chalcone synthase(CHS) is the first key rate-limiting enzyme in the biosynthesis of flavonoids in plants. To explore the functions of CHS gene family in chalcone synthesis of L. litseifolius, this study screened out two CHS genes(LlCHS1 and LlCHS2) from the transcriptome data of this plant, and then bioinformatics analysis and functional characterization were performed for the two genes. The bioinformatics analysis showed that LlCHS1 and LlCHS2 were acidic hydrophilic stable proteins with no transmembrane domain, composed of 395 and 390 amino acid residues, respectively. Both of them contained the characteristic amino acid sequence "WGVLFGFGPGL" and highly conserved active sites(Cys-164, Phe-215, His-303, and Asn-336) of the CHS family. The phylogenetic tree showed that LlCHS1 shared the same clade with similar genes in Aquilaria sinensis, and LlCHS2 was closely related to similar genes in Malus domestica. Under exogenous addition of phloretic acid, co-expression of LlCHS1 or LlCHS2 with Aa4CL from Aromatoleum aromaticum in Escherichia coli catalyzed the production of phloretin from phloretic acid. This study laid a theoretical foundation for revealing the functions of CHS in plants and provided new enzymatic modules for producing phloretin by synthetic biology.
Acyltransferases/chemistry* ; Phylogeny ; Plant Proteins/chemistry* ; Cloning, Molecular ; Amino Acid Sequence

The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca²⁺, Mn²⁺ and Co²⁺) showed inhibitory effect on the activity of UGT74L2, while Mg²⁺ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the K_m value was 29.84 μmol·L^-1, the k_cat was 0.02 s^-1, and the k_cat·K_m^-1 was 572.6 mol^-1·s^-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.

Objective: To explore the safety and efficacy of excimer laser coronary angioplasty (ELCA) for the treatment of degenerated great saphenous vein graft (SVG). Methods: This is a single-center, prospective, single-arm study. Patients, who were admitted to the Geriatric Cardiovascular Center of Beijing Anzhen Hospital from January 2022 to June 2022, were consecutively enrolled. Inclusion criteria were recurrent chest pain after coronary artery bypass surgery (CABG), and coronary angiography confirmed that the SVG stenosis was more than 70% but not completely occluded, and interventional treatment for SVG lesions was planned. Before balloon dilation and stent placement, ELCA was used to pretreat the lesions. Optical coherence tomography (OCT) examination was performed and postoperative index of microcirculation resistance (IMR) were assessed after stent implantation. The technique success rate and operation success rate were calculated. The technique success was defined as the successful passage of the ELCA system through the lesion. Operation success was defined as the successful placement of a stent at the lesion. The primary evaluation index of the study was IMR immediately after PCI. Secondary evaluation indexes included thrombolysis in myocardial infarction (TIMI) flow grade, corrected TIMI frame count (cTFC), minimal stent area and stent expansion measured by OCT after PCI, and procedural complications (Ⅳa myocardial infarction, no reflow, perforation). Results: A total of 19 patients aged (66.0±5.6) years were enrolled, including 18 males (94.7%). The age of SVG was 8 (6, 11) years. The length of the lesions was greater than 20 mm, and they were all SVG body lesions. The median stenosis degree was 95% (80%, 99%), and the length of the implanted stent was (41.7±16.3)mm. The operation time was 119 (101, 166) minutes, and the cumulative dose was 2 089 (1 378, 3 011)mGy. The diameter of the laser catheter was 1.4 mm, the maximum energy was 60 mJ, and the maximum frequency was 40 Hz. The technique success and the operation success rate were both 100% (19/19). The IMR after stent implantation was 29.22±5.95. The TIMI flow grade of patients after ELCA and stent implantation was significantly improved (all P>0.05), and the TIMI flow grade of all patients after stent implantation was Grade Ⅲ. The cTFC decreased significantly after ELCA (33.2±7.8) and after stent placement (22.8±7.1) than preoperative level (49.7±13.0) (both P<0.001). The minimum stent area was (5.53±1.36)mm², and the stent expansion rate was (90.0±4.3)%. Perforation, no reflow, type Ⅳa myocardial infarction and other complications were not observed. However, postoperative high-sensitivity troponin level was significantly increased ((67.937±33.839)ng/L vs. (5.316±3.105)ng/L, P<0.001). Conclusion: ELCA is safe and effective in the treatment of SVG lesions and could improve microcirculation and ensure full expansion of stent.
Male ; Humans ; Aged ; Prospective Studies ; Percutaneous Coronary Intervention ; Lasers, Excimer/therapeutic use* ; Saphenous Vein/transplantation* ; Constriction, Pathologic ; Atherectomy, Coronary/methods* ; Myocardial Infarction ; Coronary Angiography ; Stents ; Treatment Outcome