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 To explore the characteristics and prognosis of follicular lymphoma(FL)with bulky disease under rituximab-based first-line treatment.Methods A retrospective analysis was conducted on 525 FL patients diagnosed between September 2009 and September 2021 who received rituximab as a first-line treat-ment[342 patients received rituximab combined with chemotherapy(R-chemo),183 patients received rituximab plus lenalidomide(R2)].The clinicopathologic characteristics,gene mutations,and prognosis of bulky FL patients were analyzed.Results Compared to non-bulky FL patients,bulky FL patients had a significantly higher proportion of lymph node≥5 sites,≥2 extranodal involvement,bone marrow involvement,elevated LDH,and a higher proportion in the high-risk group of FLIPI1 and FLIPI2.Gene sequencing revealed a significantly higher mutation rate of ZNF608 in bulky FL patients compared to non-bulky FL patients.In patients receiving R-chemo as the first-line treatment,there was no significant difference in progression-free survival(PFS)and overall survival(OS)between bulky and non-bulky FL patients.However,in patients treated with R2,the PFS and OS of bulky FL patients was significantly shorter.Conclusions Bulky FL patients compared to non-bulky FL patients have a significantly higher proportion of high-risk baseline characteristics.For bulky FL at diagnosis,chemo-free regimens require further exploration on the basis of R2.

Colorectal cancer(CRC)is a malignant tumor formed in the colon or rectum,usually caused by uncontrolled growth and division of normal cells in the body.Cell apoptosis,pyroptosis,and necroptosis are key pathways of cell death in colorectal cancer.The comprehensive treatment strategy includes the synergistic effect of cell death inducers with chemotherapy,immunotherapy,and targeted therapy.Traditional Chinese medicine plays an important role as an adjuvant therapy in regulating cell death.The combination of traditional Chinese and western medicine has shown significant effects in precancerous lesions,improving efficacy,reducing adverse reactions,and reducing drug resistance.Although the research on the mechanism of cell death is not yet sufficient,emphasizing the unique characteristics of traditional Chinese medicine,clarifying the anti-tumor mechanism of traditional Chinese medicine,and achieving modern scientific internationalization of integrated traditional Chinese and western medicine treatment for colorectal cancer have become future research directions.This article will comprehensively review the molecular mechanisms of cell apoptosis,pyroptosis,and necroptosis from the perspective of combining traditional Chinese and Western medicine,as well as their regulatory role in the treatment of colorectal cancer.

Objective To explore the characteristics and prognosis of follicular lymphoma(FL)with bulky disease under rituximab-based first-line treatment.Methods A retrospective analysis was conducted on 525 FL patients diagnosed between September 2009 and September 2021 who received rituximab as a first-line treat-ment[342 patients received rituximab combined with chemotherapy(R-chemo),183 patients received rituximab plus lenalidomide(R2)].The clinicopathologic characteristics,gene mutations,and prognosis of bulky FL patients were analyzed.Results Compared to non-bulky FL patients,bulky FL patients had a significantly higher proportion of lymph node≥5 sites,≥2 extranodal involvement,bone marrow involvement,elevated LDH,and a higher proportion in the high-risk group of FLIPI1 and FLIPI2.Gene sequencing revealed a significantly higher mutation rate of ZNF608 in bulky FL patients compared to non-bulky FL patients.In patients receiving R-chemo as the first-line treatment,there was no significant difference in progression-free survival(PFS)and overall survival(OS)between bulky and non-bulky FL patients.However,in patients treated with R2,the PFS and OS of bulky FL patients was significantly shorter.Conclusions Bulky FL patients compared to non-bulky FL patients have a significantly higher proportion of high-risk baseline characteristics.For bulky FL at diagnosis,chemo-free regimens require further exploration on the basis of R2.

Colorectal cancer(CRC)is a malignant tumor formed in the colon or rectum,usually caused by uncontrolled growth and division of normal cells in the body.Cell apoptosis,pyroptosis,and necroptosis are key pathways of cell death in colorectal cancer.The comprehensive treatment strategy includes the synergistic effect of cell death inducers with chemotherapy,immunotherapy,and targeted therapy.Traditional Chinese medicine plays an important role as an adjuvant therapy in regulating cell death.The combination of traditional Chinese and western medicine has shown significant effects in precancerous lesions,improving efficacy,reducing adverse reactions,and reducing drug resistance.Although the research on the mechanism of cell death is not yet sufficient,emphasizing the unique characteristics of traditional Chinese medicine,clarifying the anti-tumor mechanism of traditional Chinese medicine,and achieving modern scientific internationalization of integrated traditional Chinese and western medicine treatment for colorectal cancer have become future research directions.This article will comprehensively review the molecular mechanisms of cell apoptosis,pyroptosis,and necroptosis from the perspective of combining traditional Chinese and Western medicine,as well as their regulatory role in the treatment of colorectal cancer.

Precancerous lesions of gastric cancer is a key stage in the development of gastric cancer.The reprogramming of glucose metabolism is a prominent feature of precancerous lesions of gastric cancer.Hypoxic microenvironment and hypoxia-inducible factors are important factors influencing the occurrence of glucose metabolic reprogramming.This article summarized the relationship between hypoxic microenvironment and the reprogramming of glucose metabolism in precancerous lesions of gastric cancer,and concluded the relevant research on TCM compounds and effective components to improve hypoxic microenvironment and further regulate glycolysis for the treatment of this disease.It was concluded that the mechanism may be the inhibition of angiogenesis,regulation of signaling pathways and key proteins of glycolysis,expression of multiple enzymes,reduction of lactate secretion,inhibition of cell malignant proliferation and invasion.It explored the mechanism of Chinese materia medica in improving hypoxic microenvironment and regulating glycolysis,so as to provide reference for the prevention and treatment of precancerous lesions of gastric cancer.

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 investigate the effect and mechanism of Houpu Sanwu decoction on subcutaneous tumors in C57BL/6J mice.Methods Mouse colorectal cancer MC-38 cells were injected subcutaneously into C57BL/6J mice to establish a subcutaneous tumor model of colorectal cancer.The model mice were then divided randomly into model control,Houpu Sanwu decoction(HPSWD)low dose,HPSWD medium dose,HPSWD high dose,5-fluorouracil(5-FU),and combination groups(5-FU+HPSWD medium dose)(n=6 mice per group).The mice were treated with normal saline and Houpu Sanwu decoction by gavage,or normal saline and 5-FU by intraperitoneal injection once a day for 24 consecutive days.During the intervention period,the mental state,diet,and other general conditions of the mice,as well as changes in tumor volume,were monitored regularly.At the end of the experiment,the subcutaneous tumors in each group were removed and weighed,and pathological features were examined by hematoxylin and eosin staining.Transmission electron microscopy,Western blot,and nanoparticle tracking analysis were used to identify exosomes derived from colorectal cancer tissues.Tumor mRNA and protein expression levels of Rab27a and the exosome markers CD63,TSG101,and ALIX were detected by reverse transcription-quantitative polymerase chain reaction and Western blot,respectively.Results Houpu Sanwu decoction inhibited tumor growth in mice.Tumor growth was inhibited to varying degrees in all the HPSWD dose groups and in the combination group.The HPSWD medium does group showed the greatest anticancer effect,and the combination group showed a synergistic effect with 5-FU.Houpu Sanwu decoction induced tumor necrosis in a dose-dependent manner,with a similar anticancer effect to 5-FU.Houpu Sanwu decoction also inhibited the protein and mRNA expression levels of Rab27a and the exosome-specific markers CD63,ALIX,and TSG101 in mice.Conclusions Houpu Sanwu decoction inhibits tumor growth and the secretion of exosomes,possibly by regulating Rab27a.

Objective:To understand the etiology,clinical characteristics and prognosis of secondary hemophagocytic syndrome(HLH),so as to improve the understanding of HLH and reduce the rates of misdiagnosis and missed diagnosis of HLH.Methods:A retrospective study was conducted to analyze the cause,clinical characteristics,laboratory findings,therapy and outcomes of 75 adult patients with secondary HLH admitted to our hospital from January 2015 to December 2021.Follow-up continued until the last discharge time.Results:Among 75 patients,infection-related HLH was the most common(45.33％),followed by lymphoma-related HLH(17.33％).Fever was the most common clinical manifestation(97.67％).Laboratory indicators such as NK cell activity(98.31％low or absent),sCD25(93.22％increased),and serum ferritin(94.44％elevated)had higher sensitivity in diagnosis.By comparing the clinical manifestations and laboratory indicators of HLH patients with different causes,sex,lymph node enlargement and bone marrow morphology were more valuable for the diagnosis of primary disease(all P＜0.05).By comparing the treatment and clinical outcomes of HLH patients with different causes,the highest clinical remission rate(83.3％)was achieved in patients with autoimmune disease-related HLH treated with hormone+cyclosporine(P＜0.05).The overall 12-month survival rate of all patients was 26.7％,in which the infection-related HLH was the lowest(14.7％)while autoimmune disease-related HLH was the highest(63.6％).Conclusion:The causes and clinical characteristics of adult secondary HLH are varied,with poor prognosis and heterogeneity in disease severity.It is important to identify HLH cause early for diagnosis and needed to further understand HLH.

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.