Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3% mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101) by Western blot.Results About 5 × 10~6 peritoneal macrophages with the purity of(99.17±0.65)%were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucerlike vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary peritoneal macrophages.ExoQuick TC.exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.

Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3% mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101) by Western blot.Results About 5 × 10~6 peritoneal macrophages with the purity of(99.17±0.65)%were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucerlike vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary peritoneal macrophages.ExoQuick TC.exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.

Objective To evaluate the clinical efficacy and safety of intensive insulin therapy in the pa-tients with acute myocardial infarction and provide guidance for improving the prognosis.Methods The articles involving the randomized controlled trials(RCT)focusing on the effects of intensive versus conventional insulin therapy on the clinical outcomes of the patients with acute myocardial infarction were retrieved from Cochrane,Embase,PubMed,CNKI,Wanfang Data,VIP,and CBM with the time interval from inception to October 2022.The data of each RCT were extracted and used for meta-analysis in RevMan5.4.Results A total of 8 arti-cles were included in this study,involving 726 patients(372 in the intensive insulin group and 354 in the nor-mal insulin group).The meta-analysis results showed that the intensive insulin group had lower incidence of major cardiovascular adverse events(RR =0.53,95%CI =0.44-0.64,P<0.001),lower all-cause mortality(RR = 0.51,95%CI =0.33-0.78,P =0.002),lower high-sensitivity C-reactive protein level on day 7(WMD =-2.00,95%CI =-2.17--1.83,P<0.001),higher left ventricular ejection fraction on day 30(WMD = 3.94,95%CI =2.45-5.43,P<0.001),and higher incidence of hypoglycemia events(RR =2.96,95%CI =1.12-7.83,P =0.030)than the normal insulin group.There was no significant difference between the two groups in terms of no-reflow event after percutaneous coronary intervention(RR =0.39,95%CI =0.14-1.13,P =0.080).Conclusion Intensive insulin therapy might be associated with more clinical benefits in the patients with acute myocardial infarction,while the conclusion remains to be confirmed by more studies.

Objective To investigate the diagnostic value of biparametric magnetic resonance imaging(bpMRI)radiomics combined with prostate-specific antigen density(PSAD)in predicting low-grade and high-grade prostate carcinoma(PCa).Methods The clinical and imaging data of patients with PCa confirmed by pathology in Taizhou Central Hospital from June 2018 to October 2022 were retrospectively analyzed.According to Gleason grade group(GGG),GGG≤2 was defined as low-grade PCa,and GGG＞2 was defined as high-grade PCa.PCa patients with different grades were randomly divided into training group and test group according to a ratio of 7∶3.Radiomics features were extracted based on T2 weighted imaging(T2WI)and apparent diffusion coefficient(ADC)sequences.Feature selection and dimensionality reduction were carried out using maximum relevance minimum redundancy,least absolute shrinkage and selection operator,and 5-fold cross validation was performed to retain the best radiomics features.Receiver operating characteristic(ROC)curve and Delong's test were used to evaluate the performance of each model.Decision curve analysis(DCA)was used to evaluate the clinical utility of the model.Results Among all the models,T2WI-ADC-PSAD combined model had the best diagnostic efficiency,the area under the curve(AUC)in training group and test group were 0.882,0.772,respectively.Delong's test showed that in training group,there was no significant difference in AUC between T2WI-ADC-PSAD model and T2WI model(P＞0.05),but there were significant differences between T2WI-ADC-PSAD model and other models(P＜0.05).In test group there were no significant differences in AUC between T2WI-ADC-PSAD model and other models(P＞0.05).The DCA showed that the T2WI-ADC-PSAD model provided a higher net benefit for clinical decision-making when the threshold probability was less than 97％.Conclusion BpMRI radiomics combined with PSAD can improve the diagnostic efficiency of low-grade and high-grade PCa,and guide the treatment decision of patients.

Tacrolimus (FK506) is a 23-membered macrolide with immunosuppressant activity that is widely used clinically for treating the rejection after organ transplantation. The research on tacrolimus production was mainly focused on biosynthesis methods, within which there are still some bottlenecks. This review summarizes the progress made in tacrolimus biosynthesis via modification of metabolic pathways and control of fermentation process, with the hope to address the technical bottlenecks for tacrolimus biosynthesis and improve tacrolimus production by fermentation engineering and metabolic engineering.
Tacrolimus ; Immunosuppressive Agents ; Fermentation ; Macrolides ; Anti-Bacterial Agents

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.
Humans ; Methionine ; Methylenetetrahydrofolate Reductase (NADPH2) ; Carbon ; Cysteine ; Escherichia coli/genetics* ; Hydroxymethyl and Formyl Transferases ; Carrier Proteins ; Escherichia coli Proteins

Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.
Adenosine Triphosphate/metabolism* ; Adenosine Monophosphate ; Polyphosphates/metabolism* ; Catalysis ; Regeneration

The development of synthetic biology has greatly promoted the construction of microbial cell factories, providing an important strategy for green and efficient chemical production. However, the bottleneck of poor tolerance to harsh industrial environments has become the key factor hampering the productivity of microbial cells. Adaptive evolution is an important method to domesticate microorganisms for a certain period by applying targeted selection pressure to obtain desired phenotypic or physiological properties that are adapted to a specific environment. Recently, with the development of technologies such as microfluidics, biosensors, and omics analysis, adaptive evolution has laid the foundation for efficient productivity of microbial cell factories. Herein, we discuss the key technologies of adaptive evolution and their important applications in improvement of environmental tolerance and production efficiency of microbial cell factories. Moreover, we looked forward to the prospects of adaptive evolution to realize industrial production by microbial cell factories.
Metabolic Engineering ; Industrial Microbiology/methods* ; Synthetic Biology ; Environment ; Industry

Major natural disasters seriously threaten human life and health. After earthquakes and other catastrophes, survivors are often trapped in the confined spaces caused by the collapse of ground and buildings, with relative separation from the outside world, restricted access, complex environment, and oncoming or ongoing unsafety, leading to the rescue extremely difficult. In order to save lives and improve the outcome more efficiently in the confined spaces after natural disasters, it is very important to standardize and reasonably apply the trauma assessment and first aid workflow. This study focuses on trauma assessment and first aid. From the aspects of trauma assessment, vital signs stabilization, hemostasis and bandaging, post-trauma anti-infection, and the transportation of patients, a trauma first aid work process suitable for a small space of a major natural disaster is formed, It is helpful to realize the immediate and efficient treatment of trauma in the confined spaces after natural catastrophes, to reduce the rate of death and disability and improve the outcome of patients.
Humans ; Disasters ; First Aid ; Confined Spaces ; Earthquakes

Objective:To investigate the influencing factors for anastomotic biliary stric-ture after liver transplantation.Methods:The retrospective case-control study was conducted. The clinical data of 428 recipients who underwent allogeneic orthotopic liver transplantation in the First Hospital of Jilin University from September 2014 to August 2021 were collected. There were 324 males and 104 females, aged (52±10)years. Observation indicators: (1) surgical conditions of recipients; (2) occurrence of anastomotic biliary stricture after liver transplantation and its treat-ment; (3) analysis of influencing factors for anastomotic biliary stricture after liver transplantation. Follow-up was conducted using outpatient examination to detect occurrence of anastomotic biliary stricture and treatment up to August 30, 2021. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was analyzed using the t test. Measure-ment data with skewed distribution were represented as M( Q1, Q3) or M(range), and comparison between groups was analyzed using the Mann-Whitney U test. Count data were represented as absolute numbers, and the chi-square test was used for comparison between groups. Logistic regression model was used for multivariate analysis. Results:(1) Surgical conditions of recipients: the operation time of 428 recipients was 465(420,520)minutes, the cold ischemia time was 368(320,450)minutes, and the volume of intraoperative blood loss was 2 500(1 500,4 000)mL. Of the 428 recipients, 142 cases were performed continuous biliary posterior wall anastomosis + interrup-ted anterior wall anastomosis by polygluconate sutures, 286 cases were anastomosed with polypro-pylene sutures, including 169 cases undergoing continuous biliary posterior wall anastomosis combined with interrupted anterior wall anastomosis, 73 cases undergoing completely interrupted biliary anterior and posterior wall anastomosis, and 44 cases undergoing completely continuous biliary anterior and posterior wall anastomosis. None of the 428 recipients had indwelling T tubes. (2) Occurrence of anastomotic biliary stricture after liver transplantation and its treatment:all the 428 recipients were followed up for 3 to 72 months, with a median follow-up time of 28 months. During the follow-up, 50 patients developed anastomotic biliary stricture, of which 41 patients were treated with endoscopic retrograde cholangiopancreatography, 8 patients were treated with percutaneous transhepatic cholangial drainage, and 1 patient was treated with surgery, showing no recurrence. (3)Analysis of influencing factors for anastomotic biliary stricture after liver transplanta-tion: results of univariate analysis showed that anastomosis method and donor liver cold ischemia time were related factors for postoperative anastomotic biliary stricture of recipients undergoing allogeneic orthotopic liver transplantation ( χ2=15.74, Z=-2.04, P<0.05). Results of multivariate analysis showed that completely interrupted biliary anterior and posterior wall anastomosis and donor liver cold ischemia time were independent influencing factors for postoperative anastomotic biliary stricture of recipients undergoing allogeneic orthotopic liver transplantation ( odds ratio=0.25, 1.00, 95% confidence interval as 0.08-0.85, 1.00-1.01, P<0.05). Conclusions:Suture type is not an influencing factor for postoperative anastomotic biliary stricture of recipients undergoing allogeneic orthotopic liver transplantation. Completely interrupted biliary anterior and posterior wall anastomosis and donor liver cold ischemia time were independent influencing factors.