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 investigate lymph node metastasis (LNM) in the prostatic anterior fat pad (PAFP) of PCa patients after robot-assisted radical prostatectomy (RARP), and analyze the clinicopathological features and prognosis of LNM in the PAFP. METHODS: We retrospectively analyzed the clinicopathological data on 1 003 cases of PCa treated by RARP in the Department of Urology of PLA General Hospital from January 2017 to December 2022. All the patients underwent routine removal of the PAFP during RARP and pathological examination, with the results of all the specimens examined and reported by pathologists. Based on the presence and locations of LNM, we grouped the patients for statistical analysis, compared the clinicopathological features between different groups using the Student's t, Mann-Whitney U and Chi-square tests, and conducted survival analyses using the Kaplan-Meier and Log-rank methods and survival curves generated by Rstudio. RESULTS: Lymph nodes were detected in 77 (7.7%) of the 1 003 PAFP samples, and LNM in 11 (14.3%) of the 77 cases, with a positive rate of 1.1% (11/1 003). Of the 11 positive cases, 9 were found in the upgraded pathological N stage, and the other 2 complicated by pelvic LNM. The patients with postoperative pathological stage≥T3 constituted a significantly higher proportion in the PAFP LNM than in the non-PAFP LNM group (81.8% ［9/11］ vs 36.2% ［359/992］, P = 0.005), and so did the cases with Gleason score ≥8 (87.5% ［7/8］ vs 35.5% ［279/786］, P = 0.009). No statistically significant differences were observed in the clinicopathological features and biochemical recurrence-free survival between the patients with PAFP LNM only and those with pelvic LNM only. CONCLUSION The PAFP is a potential route to LNM, and patients with LNM in the PAFP are characterized by poor pathological features. There is no statistically significant difference in biochemical recurrence-free survival between the patients with PAFP LNM only and those with pelvic LNM only. Routine removal of the PAFP and independent pathological examination of the specimen during RARP is of great clinical significance.
Humans ; Male ; Prostatectomy/methods* ; Robotic Surgical Procedures ; Lymphatic Metastasis ; Retrospective Studies ; Prognosis ; Prostatic Neoplasms/pathology* ; Adipose Tissue/pathology* ; Prostate/pathology* ; Lymph Nodes/pathology* ; Middle Aged ; Aged

The iridoid glycosides from Veronica anagallis-aquatica L. alleviate heart failure by modulating the gut microbiota and influencing the production of two metabolites with potential antihypertrophic effects, HVA and 2OH-VA.Image 1.

BACKGROUND:As tissue engineering brings new hope to the worldwide problem of articular cartilage repair,the construction of light-curing 3D printed hydrogel scaffolds with biomimetic composition is of great significance for cartilage tissue engineering. OBJECTIVE:To construct a biomimetic methacryloylated hyaluronic acid/acellular Wharton's jelly composite hydrogel scaffold by digital light processing 3D printing technology,and to evaluate its biocompatibility. METHODS:Wharton's jelly was isolated and extracted from human umbilical cord,then decellulated,freeze-dried,ground into powder,and dissolved in PBS to prepare 50 g/L acellular Wharton's jelly solution.Methylallylated hyaluronic acid was prepared,lyophilized and dissolved in PBS to prepare 50 g/L methylallylated hyaluronic acid solution.Acellular Wharton's jelly solution was mixed with methacrylyacylated hyaluronic acid solution at a volume ratio of 1:1,and was used as bio-ink after adding photoinitiator.Methylacrylylated hyaluronic acid hydrogel scaffolds(labeled as HAMA hydrogel scaffolds)and methylacrylylated hyaluronic acid/acellular Wharton's jelly gel scaffolds(labeled as HAMA/WJ hydrogel scaffolds)were prepared by digital light processing 3D printing technology,and the microstructure,swelling performance,biocompatibility,and cartilage differentiation performance of the scaffolds were characterized. RESULTS AND CONCLUSION:(1)Under scanning electron microscope,the two groups of scaffolds showed a three-dimensional network structure,and the fiber connection of HAMA/WJ hydrogel scaffold was more uniform.Both groups achieved swelling equilibrium within 10 hours,and the equilibrium swelling ratio of HAMA/WJ hydrogel scaffold was lower than that of HAMA hydrogel scaffold(P＜0.05).(2)CCK-8 assay showed that HAMA/WJ hydrogel scaffold could promote the proliferation of bone marrow mesenchymal stem cells compared with HAMA hydrogel scaffold.Dead/live staining showed that bone marrow mesenchymal stem cells grew well on the two groups of scaffolds,and the cells on the HAMA/WJ hydrogel scaffolds were evenly distributed and more cells were found.Phalloidine staining showed better adhesion and spread of bone marrow mesenchymal stem cells in HAMA/WJ hydrogel scaffold than in HAMA.(3)Bone marrow mesenchymal stem cells were inoculated into the two groups for chondrogenic induction culture.The results of qRT-PCR showed that the mRNA expressions of agglutinoglycan,SOX9 and type Ⅱ collagen in the HAMA/WJ hydrogel scaffold group were higher than those in the HAMA hydrogel scaffold group(P＜0.05,P＜0.01).(4)These findings indicate that the digital light processing 3D bioprinting HAMA/WJ hydrogel scaffold can promote the proliferation,adhesion,and chondrogenic differentiation of bone marrow mesenchymal stem cells.

Lipotoxicity is a pivotal factor that initiates and exacerbates liver injury and is involved in the development of metabolic-associated fatty liver disease (MAFLD). However, there are few reported lipotoxicity inhibitors. Here, we identified a natural anti-lipotoxicity candidate, HN-001, from the marine fungus Aspergillus sp. C1. HN-001 dose- and time- dependently reversed palmitic acid (PA)-induced hepatocyte death. This protection was associated with IRE-1α-mediated XBP-1 splicing inhibition, which resulted in suppression of XBP-1s nuclear translocation and transcriptional regulation. Knockdown of XBP-1s attenuated lipotoxicity, but no additional ameliorative effect of HN-001 on lipotoxicity was observed in XBP-1s knockdown hepatocytes. Notably, the ER stress and lipotoxicity amelioration was associated with PLA2. Both HN-001 and the PLA2 inhibitor MAFP inhibited PLA2 activity, reduced lysophosphatidylcholine (LPC) level, subsequently ameliorated lipotoxicity. In contrast, overexpression of PLA2 caused exacerbation of lipotoxicity and weakened the anti-lipotoxic effects of HN-001. Additionally, HN-001 treatment suppressed the downstream pro-apoptotic JNK pathway. In vivo, chronic administration of HN-001 (i.p.) in mice alleviated all manifestations of MAFLD, including hepatic steatosis, liver injury, inflammation, and fibrogenesis. These effects were correlated with PLA2/IRE-1α/XBP-1s axis and JNK signaling suppression. These data indicate that HN-001 has therapeutic potential for MAFLD because it suppresses lipotoxicity, and provide a natural structural basis for developing anti-MAFLD candidates.

Objective To investigate the environmental contamination related to first patient with carbapenem-re-sistant Acinetobacter baumannii(CRAB)infection and the infection status of relevant patients in a newly established intensive care unit(ICU)of a hospital in Tibetan area,and analyze the transmission risk.Methods From the ad-mission in ICU of a patients who was first detected CRAB on November 15,2021 to the 60th day of hospitalization,all patients who stayed in ICU for＞48 hours were performed active screening on CRAB.On the 30th day and 60th day of the admission to the ICU of the first CRAB-infected patient,environment specimens were taken respectively 2 hours after high-frequency diagnostic and therapeutic activities but before disinfection,and after disinfection but before medical activities.CRAB was cultured with chromogenic culture medium.Results Among the 13 patients who were actively screened,1 case was CRAB positive,he was transferred from the ICU of a tertiary hospital to the ICU of this hospital on November 19th.On the 40th day of admission to the ICU,he had fever,increased frequency for sputum suction,and CRAB was detected.The drug sensitivity spectrum was similar to that of the first case,and he also stayed in the adjacent bed of the first case.64 environmental specimens were taken,and 9 were positive for CRAB,with a positive rate of 14.06％,8 sampling points such as the washbasin,door handle and bed rail were positive for CRAB after high-frequency diagnostic and therapeutic activities.After routine disinfection,CRAB was detected from the sink of the washbasin.Conclusion For the prevention and control of CRAB in the basic-level ICU in ethnic areas,it is feasible to conduct risk assessment on admitted patients and adopt bundled prevention and con-trol measures for high-risk patients upon admission.Attention should be paid to the contaminated areas(such as washbasin,door handle,and bed rail)as well as the effectiveness of disinfection of sink of washbasin.

Objective:To explore the surgical strategies and clinical efficacy for aortic dissection combined with refractory superior mesenteric artery (SMA) ischemia.Methods:This is a retrospective case series study. Clinical data of 24 patients with aortic dissection and refractory SMA ischemia admitted to the Department of Vascular Surgery, Zhongshan Hospital, Fudan University from August 2010 to August 2020 were retrospectively collected. Of the 24 patients, 21 were males and 3 were females, with an age of (50.3±9.9) years (range: 44 to 72 years).Among them, 9 cases were Stanford type A aortic dissection, and 15 cases were type B. All patients underwent CT angiography upon admission, and based on imaging characteristics, they were classified into three types. Type Ⅰ: severe stenosis/occlusion of the SMA true lumen only; Type Ⅱ: stenosis of the true lumens in the descending aorta and SMA (isolated type); Type Ⅲ: stenosis of the true lumens in the thoracoabdominal aorta and SMA (continuation type). Surgical procedures, complications, mortality, and reintervention rates were recorded.Results:Among the 24 patients, 17 (70.8%) were classified as Type Ⅰ, 4 (16.7%) as Type Ⅱ, and 3 (12.5%) as Type Ⅲ. Fourteen cases of Type Ⅰ underwent thoracic endovascular aortic repair combined with SMA stent implantation. Additionally, 3 Type Ⅰ and 1 Type Ⅱ patients underwent only SMA reconstruction (with one case of chronic TAAD treated with iliac artery-SMA bypass surgery). Moreover, 3 Type Ⅱ and 3 Type Ⅲ patients underwent descending aorta combined with SMA stent implantation. There were 5 patients (20.8%) who underwent small bowel resection, either in the same sitting or in a staged procedure. During hospitalization, 4 patients died, resulting in a mortality rate of 16.7%. Among these cases, two patients succumbed to severe intestinal ischemia resulting in multiple organ dysfunction syndrome. The follow-up duration was (46±9) months (range: 13 to 72 months). During the follow-up, 2 patients died, unrelated to intestinal ischemia. The 5-year freedom from reintervention survival rate was 86.1%, and the 5-year cumulative survival rate was 82.6%.Conclusions:Patients with aortic dissection and refractory SMA ischemia have a high perioperative mortality. However, implementing appropriate surgical strategies according to different clinical scenarios can reduce mortality and alleviate intestinal ischemia.

Objective:To evaluate the clinical outcomes of thoracic endovascular aortic repair (TEVAR) in the treatment of Stanford type B aortic dissection (TBAD) in Marfan syndrome patients who had no history of aortic arch replacement.Methods:This is a retrospective case-series study. From January 2009 to December 2019,the clinical data of Marfan syndrome patients who underwent TEVAR for TBAD at the Department of Vascular Surgery were collected. A total of 23 patients were enrolled,including 15 males and 8 females. The age was (38.0±11.0) years (range:24 to 56 years). Among them,12 patients had history of ascending aortic surgery. Details of TEVAR,perioperative complications and reintervention were recorded and survival rate was analyzed by Kaplan-Meier curve.Results:Technical success was 91.3% (21/23). Two patients with technical failure were as follows:one patient had type Ⅰa endoleak at the completion angiography,which healed spontaneously during the follow-up,and the other patient suffered aortic intimal intussusception after the deployment of the first stent-graft, and the second stent-graft was deployed. However, type Ⅲ endoleak was detected,which disappeared during the follow-up. One patient died during hospitalization. The median follow-up time ( M(IQR)) was 60 (48) months (range:12 to 132 months). Reintervention was performed on 7 patients,including 3 distal stent-graft-induced new entry,2 distal aortic dilation,1 Ⅰa endoleak and 1 retrograde type A aortic dissection,respectively. Five-year cumulative survival rate was 86.7% (95% CI:86.6% to 86.8%) and the 5-year freedom from reintervention rate was 81.8% (95% CI:61.8% to 92.8%). Conclusions:TEVAR is feasible in the treatment of TBAD in Marfan syndrome patients who has no history of aortic arch replacement. It has high technical success rate and low perioperative complication.

Objective:To analyze the trend of incidence and mortality of thyroid cancer and estimate its age-period-cohort effect in Shandong Province from 2012 to 2022.Methods:The Joinpoint regression was used to analyze the trend of incidence and mortality of thyroid cancer and calculate the average annual percentage change (AAPC) based on the data on thyroid cancer from 2012 to 2022. The age-period-cohort model was used to analyze the age-effect, time-effect and cohort-effect of thyroid cancer risk in the population aged over 20 years.Results:From 2012 to 2022, the incidence of thyroid cancer in Shandong province showed a significant upward trend, with an AAPC of 21.68% (95% CI: 19.14%-24.27%, P<0.001). The incidence of females was higher than that of males, and the incidence of urban areas was higher than that of rural areas. The trend of thyroid cancer mortality was relatively stable with an AAPC of -3.04% (95% CI:-8.81%-3.09%, P=0.323). The age effect of incidence increased with age before 60 years old and decreased with age after 60 years old. The incidence peaked in the age group of 55-59. The period effect increased with time. The cohort effect showed that the cohort born before 1957 had a downward trend over time, while the cohort born after 1957 had an upward trend. Conclusion:The incidence of thyroid cancer in Shandong shows a rising trend from 2012 to 2022. Age is an important factor affecting the risk of thyroid cancer. The mortality of thyroid cancer remains stable.

Objective:We aimed to analyse the trend of incidence and mortality of stomach cancer in Shandong province from 2012 to 2022 and predict the development trend from 2023 to 2030.Methods:Data on incidence and mortality of stomach cancer in Shandong province from 2012 to 2022 were obtained from Shandong Cancer Registry. The incidence, age-specific incidence, mortality and age-specific mortality in different years, sexes and urban and rural areas were calculated, the rates were standardized based on the age composition of the Chinese standard population in 2000. The average annual percent change (AAPC) of incidence and mortality was calculated using Joinpoint software. The Bayesian age-period-cohort model was used to predict the trend of stomach cancer incidence and mortality from 2023 to 2030.Results:From 2012 to 2022, the stomach cancer age-standardized incidence rate (ASIR) and age-standardized mortality rate (ASMR) showed a decreasing trend. The ASIR decreased from 27.47/100 000 in 2012 to 16.06/100 000 in 2022 (AAPC=-5.10%, P＜0.001), and the ASMR decreased from 17.69/100 000 to 11.09/100 000 (AAPC=-5.52%, P＜0.001). The ASIR and ASMR of male, female, urban and rural population also showed downward trends. The incidence and mortality rates of men were always higher than those of women, and the difference between urban and rural areas is gradually narrowing. In 2022, the ASIR (16.09/100 000 in urban and 16.03/100 000 in rural) and the ASMR (11.10/100 000 in urban and 11.08/100 000 in rural) of stomach cancer between urban and rural areas were nearly identical. The Bayesian age-period-cohort model predicted that the ASIR of stomach cancer in Shandong would further decrease from 2023 to 2030 (AAPC=-0.51%, P=0.001), but the change tended to be smooth. The incidences in male (AAPC=-1.46%, P=0.010) and rural areas (AAPC=-1.21%, P＜0.001) were still expected to have a little room for decline. The trend of incidences in female and urban areas were not statistically significant. The trend of mortality was consistent with the incidence. Conclusions:The stomach cancer incidence and mortality in Shandong shows a decreasing trend and it is expected to decrease further by 2030. However, the trend tends to be smooth, and the disease burden should be reduced as early as possible for high-risk population and high-risk factors of stomach cancer.