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

To guide clinical blood transfusion practices for pediatric patients, the National Health Commission has issued the health standard "Guideline for pediatric transfusion" (WS/T 795-2022). Blood transfusion is one of the most commonly used supportive treatments for children with hematological diseases. This guideline provides guidance and recommendations for blood transfusions in children with aplastic anemia, thalassemia, autoimmune hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, acute leukemia, myelodysplastic syndromes, immune thrombocytopenic purpura, and thrombotic thrombocytopenic purpura. This article presents the evidence and interpretation of the blood transfusion provisions for children with hematological diseases in the "Guideline for pediatric transfusion", aiming to assist in the understanding and implementing the blood transfusion section of this guideline.
Humans ; Child ; Hematologic Diseases/therapy* ; Blood Transfusion/standards* ; Practice Guidelines as Topic

To guide clinical blood transfusion practices for pediatric patients, the National Health Commission has issued the health standard "Guideline for pediatric transfusion" (WS/T 795-2022). Blood transfusion for children undergoing hematopoietic stem cell transplantation is highly complex and challenging. This guideline provides recommendations on transfusion thresholds and the selection of blood components for these children. This article presents the evidence and interpretation of the transfusion provisions for children undergoing hematopoietic stem cell transplantation, with the aim of enhancing the understanding and implementation of the "Guideline for pediatric transfusion".
Humans ; Hematopoietic Stem Cell Transplantation ; Child ; Blood Transfusion/standards* ; Practice Guidelines as Topic

To guide clinical blood transfusion practices for pediatric patients, the National Health Commission has issued the health standard "Guideline for pediatric transfusion" (WS/T 795-2022). Critically ill children often present with anemia and have a higher demand for transfusions compared to other pediatric patients. This guideline provides guidance and recommendations for blood transfusions in cases of general critical illness, septic shock, acute brain injury, extracorporeal membrane oxygenation, non-life-threatening bleeding, and hemorrhagic shock. This article interprets the background and evidence of the blood transfusion provisions for critically ill and severely bleeding children in the "Guideline for pediatric transfusion", aiming to enhance understanding and implementation of this aspect of the guidelines. Citation:Chinese Journal of Contemporary Pediatrics, 2025, 27(4): 395-403.
Humans ; Critical Illness ; Blood Transfusion/standards* ; Child ; Hemorrhage/therapy* ; Practice Guidelines as Topic

To guide clinical blood transfusion practices in pediatric patients, the National Health Commission has issued the health standard "Guideline for pediatric transfusion" (WS/T 795-2022). Children undergoing cardiac surgery are at high risk of bleeding, and the causes of perioperative anemia and coagulation disorders in neonates and children are complex and varied, often necessitating the transfusion of allogeneic blood components. This guideline provides direction and recommendations for specific measures in blood management for children undergoing cardiac surgery before, during, and after surgery. This article interprets the background and evidence for the formulation of the blood transfusion provisions for children undergoing cardiac surgery, hoping to facilitate the understanding and implementation of this guideline.
Humans ; Cardiac Surgical Procedures ; Blood Transfusion/standards* ; Child ; Practice Guidelines as Topic

OBJECTIVE: To investigate the genetic causal relationship of leukocyte telomere length (LTL) with prostatitis, orchitis and epididymitis by two-sample Mendelian randomization (MR). METHODS: Using LTL as the exposure factor and prostatitis, orchitis and epididymitis as outcome factors, we mined the Database of Genome-Wide Association Studies (GWAS). Then, we analyzed the causal relationship of LTL with prostatitis, orchitis and epididymitis by Mendelian randomization using inverse variance weighting (IVW) as the main method and weighted median and MR-Egger regression as auxiliary methods, determined the horizontal multiplicity by MR-Egger intercept test, and conducted sensitivity analysis using the leaving-one-out method. RESULTS: A total of 121 related single nucleotide polymorphisms (SNPs) were identified in this study. IVW showed LTL to be a risk factor for prostatitis (OR = 1.383, 95% CI: 1.044－1.832, P = 0.024), and for orchitis and epididymitis as well (OR = 1.770, 95% CI: 1.275－2.456, P = 0.000 6). CONCLUSION Genetic evidence from Mendelian randomized analysis indicates that shortening of LTL reduces the risk of prostatitis, orchitis and epididymitis.
Humans ; Male ; Mendelian Randomization Analysis ; Epididymitis/genetics* ; Prostatitis/genetics* ; Polymorphism, Single Nucleotide ; Leukocytes ; Orchitis/genetics* ; Genome-Wide Association Study ; Telomere ; Risk Factors

The prelimbic cortex (PL) plays a critical role in processing both the sensory and affective components of pain. However, the underlying molecular mechanisms remain poorly understood. In this study, we observed a reduction in hyperpolarization-activated cation current (I_h) in layer V pyramidal neurons of the contralateral PL in a mouse model of spared nerve injury (SNI). The expression of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channels was also decreased in the contralateral PL. Conversely, microinjection of fisetin, a partial agonist of HCN2, produced both analgesic and anxiolytic effects. Additionally, we found that cyclin-dependent kinase 5 (CDK5) was activated in the contralateral PL, where it formed a complex with HCN2 and phosphorylated its C-terminus. Knockdown of CDK5 restored HCN2 expression and alleviated both pain hypersensitivity and anxiety-like behaviors. Collectively, these results indicate that CDK5-mediated dysfunction of HCN2 in the PL underlies nerve injury-induced mechanical hypersensitivity and anxiety.
Animals ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Hyperalgesia/metabolism* ; Cyclin-Dependent Kinase 5/metabolism* ; Neuralgia/metabolism* ; Male ; Anxiety/metabolism* ; Mice ; Potassium Channels/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Pyramidal Cells/metabolism*

Pseudorabies (PR) is an infectious disease caused by the pseudorabies virus (PRV), affecting various domesticated and wild animals. Since pigs are the only natural hosts of PRV, PR poses a serious threat to the pig farming industry. Currently, PR is primarily prevented through vaccination with inactivated vaccines or genetically modified attenuated live vaccines. Developing safe and effective genetically engineered vaccines would facilitate the eradication and control of PR. In this study, the PRV vaccine strain Bartha-K61 was used as the reference strain. The gB protein was expressed via the baculovirus-insect cell expression system. Non-denaturing gel electrophoresis confirmed that the gB protein could form a trimeric structure. The purified gB protein was used to immunize mice, and the immune effect was evaluated by a challenge test. The results showed that the gB antigen induced a strong immune response in mice, with the serum-neutralizing antibody titer above 1:70. The lymphocyte stimulation index reached more than 1.29, and the level of (interferon gamma, IFN-γ) release was higher than 100 pg/mL. After immunization, mice were challenged with the virus at a dose of 10⁴ TCID₅₀/mL, 200 μL per mouse, and the clinical protection rate was 100%. Immunohistochemistry, histopathological section, and tissue viral load results showed that the pathological damage and viral load in the gB-immunized group were significantly lower than those in the PBS group. In summary, the gB protein obtained in this study induced strong humoral and cellular immune responses in mice, laying a foundation for developing a recombinant gB protein subunit vaccine.
Animals ; Mice ; Baculoviridae/metabolism* ; Viral Envelope Proteins/biosynthesis* ; Herpesvirus 1, Suid/genetics* ; Pseudorabies/immunology* ; Swine ; Pseudorabies Vaccines/genetics* ; Antibodies, Viral/blood* ; Insecta/cytology* ; Mice, Inbred BALB C ; Female ; Viral Vaccines/immunology*

ObjectiveTo explore the mechanism of Buzhong Yiqitang in ameliorating inflammatory injury in autoimmune thyroiditis (AIT) mice based on the Toll-like receptor 4（TLR4）/nuclear transcription factor-kappa B（NF-κB）/absent in melanoma 2（AIM2）inflammasome signaling pathway. MethodThe 120 genetically susceptible 8-week-old NOD.H-2^h4 mice were selected and randomly divided into control group， model group， low， medium and high dose groups of Buzhong Yiqitang （4.78， 9.56， 19.12 g·kg^-1）， and western medicine group （selenium yeast tablets， 3.033×10^-5 g·kg^-1）. The AIT model mice in each group drank ad libitum 0.05% sodium iodide aqueous solution for 8 weeks to establish the AIT model， and the control group drank ad libitum distilled water. Eight weeks later， the mice in each dosing group were divided into groups and gavage. The swelling of thyroid tissue was observed with the naked eye， and the weight of spleen was weighed. The content of serum inflammatory factors interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α） was measured by enzyme-linked immunosorbent assay （ELISA）， and Real-time PCR was used to detect the expression of HMGB1， TLR4， AIM2， NF-κB p65，apoptosis-associated speck-like protein（ASC），cysteinyl aspartate-specific protease-1（Caspase-1）， IL-1β mRNA. Western blot was used to detect the expression of high motility group protein 1 （HMGB1）， TLR4， AIM2， NF-κB p65， phosphorylation（p）-NF-κB p65， ASC， Caspase-1， and IL-1β proteins in thyroid tissue， and immunofluorescence staining was used to observe the protein expression of HMGB1， AIM2， and NF-κB p65 in thyroid tissue of mice. ResultCompared with the control group， the thyroid tissue of mice in the model group was significantly swollen， the spleen quality was significantly increased， and the expression of HMGB1， TLR4， NF-κB p65， AIM2， ASC， Caspase-1， IL-1β in thyroid tissue was significantly increased （P<0.01）. Compared with the model group， the swelling of thyroid tissue in mice in each dose group of Buzhong Yiqitang was improved， the quality of spleen was significantly reduced， and the expression of HMGB1， TLR4， AIM2， NF-κB p65， p-NF-κB p65， ASC， Caspase-1， IL-1β in thyroid tissue was significantly reduced （P<0.05， P<0.01）. ConclusionBuzhong Yiqitang can effectively improve the inflammatory injury of AIT， and regulating the abnormal activation of the TLR4/NF-κB/AIM2 inflammasome signal pathway may be one of its intervention mechanisms.

Objective To observe the effect of canagliflozin combined with enalapril on diabetic nephropathy(DN).Methods DN patients were randomly divided into control group and treatment group.All patients in 2 groups received basic treatment of recombinant human insulin injection,and the control group was orally administered enalapril tablet 10 mg(qd).The treatment group was given orally canagliflozin tablet 100 mg(qd)on the basis of the control group.Both groups were treated for 8 weeks.Renal function,blood glucose index,serum vascular endothelial growth factor(VEGF),transforming growth factor-β(TGF-β),homocysteine(HCY)levels,clinical efficacy and incidence of adverse drug reactions were compared between 2 groups.Results There were 71 cases were included in the control group and 73 cases in the treatment group.After treatment,β2 microglobulin(β2-MG)in treatment group and control group were(0.21±0.03)and(0.28±0.04)mg·L-1;blood urea nitrogen(BUN)were(4.23±0.42)and(5.58±0.65)mmol·L-1;serum creatinine(SCr)were(89.32±8.29)and(101.25±10.18)pmol·L-1;24 h microalbumin(mAlb)were(49.38±5.06)and(58.21±6.43)mg;glycosylated hemoglobin(HbA1c)were(6.10±0.11)％and(6.45±0.16)％;2 h postprandial blood glucose levels were(6.05±0.78)and(7.68±1.82)mmol·L-1;fasting blood glucose(FBG)were(5.02±0.32)and(5.67±0.65)mmol·L-1;VEGF levels were(350.18±20.04)and(389.04±24.16)pg·mL-1;TGF-β were(148.32±16.57)and(168.24±20.02)pg·mL-1;HCY were(13.12±2.38)and(19.35±3.21)pmol·L-1,the differences were statistically significant(all P＜0.05).After treatment,the total effective rate of treatment group and control group were 83.56％(61 cases/73 cases)and 67.61％(48 cases/71 cases),the difference was statistically significant(P＜0.05).The total incidence of adverse drug reactions in treatment group and control group were 6.85％and 4.23％,with no significant difference(P＞0.05).Conclusion Canagliflozin combined with enalapril is effective in the treatment of diabetic nephropathy,which can improve renal function,regulate blood glucose metabolism,and down-regulate serum VEGF,TGF-β and HCY levels,and is safe and reliable.