In this study, Saccharomyces cerevisiae R0 was used as the chassis cell to synthesize oleanolic acid from scratch through the heterologous expression of β-amyrin synthase(β-AS) from Glycyrrhiza uralensis, cytochrome P450 enzyme CYP716A154 from Catharanthus roseus, and cytochrome P450 reductase AtCPR from Arabidopsis thaliana. The engineered strain R1 achieved shake flask titres of 5.19 mg·L~(-1). By overexpressing enzymes in the pentose phosphate pathway(PPP)(ZWF1, GND1, TKL1, and TAL), the NADH kinase gene in the mitochondrial matrix(POS5), truncated 3-hydroxy-3-methylglutaryl-CoA reductase(tPgHMGR1) from Panax ginseng, and farnesyl diphosphate synthase gene(SmFPS) from Salvia miltiorrhiza, the precursor supply and intracellular reduced nicotinamide adenine dinucleotide phosphate(NADPH) supply were enhanced, resulting in an 11.4-fold increase in squalene yield and a 3.6-fold increase in oleanolic acid yield. Subsequently, increasing the copy number of the heterologous genes tPgHMGR1, β-AS, CYP716A154, and AtCPR promoted the metabolic flow towards the final product, oleanolic acid, and increased the yield by three times. Shake flask fermentation data showed that, by increasing the copy number, precursor supply, and intracellular NADPH supply, the final engineered strain R3 could achieve an oleanolic acid yield of 53.96 mg·L~(-1), which was 10 times higher than that of the control strain R1. This study not only laid the foundation for the green biosynthesis of oleanolic acid but also provided a reference for metabolic engineering research on other pentacyclic triterpenoids in S. cerevisiae.
Oleanolic Acid/biosynthesis* ; Saccharomyces cerevisiae/metabolism* ; Industrial Microbiology ; Microorganisms, Genetically-Modified/metabolism* ; Plants/enzymology* ; Fermentation ; Metabolic Engineering

Many triterpenoid compounds have been successfully heterologously synthesized in Saccharomyces cerevisiae. To increase the yield of triterpenoids, various metabolic engineering strategies have been developed. One commonly applied strategy is to enhance the supply of precursors, which has been widely used by researchers. Squalene, as a precursor to triterpenoid biosynthesis, plays a crucial role in the synthesis of these compounds. This study primarily investigates the effect of different squalene levels in chassis strains on the synthesis of triterpenoids(oleanolic acid and ursolic acid), and the underlying mechanisms are further explored using real-time quantitative PCR(qPCR) analysis. The results demonstrate that the chassis strain CB-9-5, which produces high levels of squalene, inhibits the synthesis of oleanolic acid and ursolic acid. In contrast, chassis strains with moderate to low squalene production, such as Y8-1 and CNPK, are more conducive to the synthesis of oleanolic acid and ursolic acid. The qPCR analysis reveals that the expression levels of ERG1, βAS, and CrCYP716A154 in the oleanolic acid-producing strain CB-OA are significantly lower than those in the control strains C-OA and Y-OA, suggesting that high squalene production in the chassis strains suppresses the transcription of certain genes, leading to a reduced yield of triterpenoids. Our findings indicate that when constructing S. cerevisiae strains for triterpenoid production, chassis strains with high squalene content may suppress the expression of certain genes, ultimately lowering their production, whereas chassis strains with moderate squalene levels are more favorable for triterpenoid biosynthesis.
Squalene/analysis* ; Saccharomyces cerevisiae/genetics* ; Triterpenes/metabolism* ; Metabolic Engineering ; Oleanolic Acid/biosynthesis* ; Ursolic Acid

This study explored the potential therapeutic targets and mechanisms of Danggui Shaoyao San(DSS) in the prevention and treatment of Alzheimer's disease(AD) through transcriptomics and metabolomics, combined with animal experiments. Fifty male C57BL/6J mice, aged seven weeks, were randomly divided into the following five groups: control, model, positive drug, low-dose DSS, and high-dose DSS groups. After the intervention, the Morris water maze was used to assess learning and memory abilities of mice, and Nissl staining and hematoxylin-eosin(HE) staining were performed to observe pathological changes in the hippocampal tissue. Transcriptomics and metabolomics were employed to sequence brain tissue and identify differential metabolites, analyzing key genes and metabolites related to disease progression. Reverse transcription-quantitative polymerase chain reaction(RT-qPCR) was employed to validate the expression of key genes. The Morris water maze results indicated that DSS significantly improved learning and cognitive function in scopolamine(SCOP)-induced model mice, with the high-dose DSS group showing the best results. Pathological staining showed that DSS effectively reduced hippocampal neuronal damage, increased Nissl body numbers, and reduced nuclear pyknosis and neuronal loss. Transcriptomics identified seven key genes, including neurexin 1(Nrxn1) and sodium voltage-gated channel α subunit 1(Scn1a), and metabolomics revealed 113 differential metabolites, all of which were closely associated with synaptic function, oxidative stress, and metabolic regulation. RT-qPCR experiments confirmed that the expression of these seven key genes was consistent with the transcriptomics results. This study suggests that DSS significantly improves learning and memory in SCOP model mice and alleviates hippocampal neuronal pathological damage. The mechanisms likely involve the modulation of synaptic function, reduction of oxidative stress, and metabolic balance, with these seven key genes serving as important targets for DSS in the treatment of AD.
Animals ; Alzheimer Disease/genetics* ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Mice, Inbred C57BL ; Metabolomics ; Transcriptome/drug effects* ; Maze Learning/drug effects* ; Hippocampus/metabolism* ; Humans ; Disease Models, Animal ; Memory/drug effects*

The zebrafish model has attracted much attention due to its strong reproductive ability, short research cycle, and ease of maintenance. It has always been an important vertebrate model system, often used to carry out human disease research. Its motor behavior features have the advantages of being simpler, more intuitive, and quantifiable. In recent years, it has received widespread attention in the study of traditional Chinese medicine(TCM)for the treatment of sleep disorders, neurodegenerative diseases, fatigue, epilepsy, and other diseases. This paper reviews the characteristics of zebrafish motor behavior and its applications in the pharmacodynamic verification and mechanism research of TCM extracts, active ingredients, and TCM compounds, as well as in active ingredient screening and safety evaluation. The paper also analyzes its advantages and disadvantages, with the aim of improving the breadth and depth of zebrafish and its motor behavior applications in the field of TCM research.
Zebrafish/physiology* ; Medicine, Chinese Traditional ; Drugs, Chinese Herbal/therapeutic use* ; Disease Models, Animal ; Drug Evaluation, Preclinical/methods* ; Animals ; Sleep Wake Disorders/physiopathology* ; Epilepsy/physiopathology* ; Neurodegenerative Diseases/physiopathology* ; Fatigue/physiopathology* ; Behavior, Animal/physiology* ; Motor Activity/physiology*

Objective To understand the prevalence and risk factors of hyperuricemia in electronics factory workers in Wuhan, and to provide evidence for the health protection of electronics factory workers. Methods A total of 1 415 employees in an electronics factory in Wuhan were selected as the research subjects, and the physical examination and determination of various biochemical indicators, as well as questionnaire survey were carried out. Results The detection rate of hyperuricemia among workers in the electronics factory in Wuhan was 32.43%, with 36.33% for men and 14.11% for women, and the difference was statistically significant ( χ²＝46.077，P＜0.001). The detection rate of hyperuricemia was the highest (33.77%) among those with university or college education, followed by graduate students and above (31.50%). Compared with subjects with good lifestyle habits, people with drinking habits had higher hyperuricemia detection rate (49.38%), and the difference was statistically significant (P =0.001). The detection rates of hyperuricemia in those with central obesity and elevated alanine aminotransferase were 48.23% and 61.29%, respectively, which were significantly higher than those in the subjects without the above diseases (26.91% and 27.21%, respectively), and the differences were statistically significant (P <0.001). Obese people had the highest detection rate of hyperuricemia (66.95%), followed by overweight people (43.75%), and the difference was statistically significant (P <0.001). Multivariate logistic analysis showed that alcohol drinking (OR=1.836, 95% CI=1.139-2.961, P =0.013) and body mass index ≥ 24 kg/m² (OR=2.175, 95% CI=1.686 -2.806, P <0.001) were risk factors for hyperuricemia in electronic factory workers. Elevated alanine aminotransferase (ALT) was significantly correlated with hyperuricemia (OR=2.964, 95%CI=2.146-4.095 , P <0.001). Female gender was a protective factor for hyperuricemia in workers in the electronics factory (OR=0.441, 95%CI=0.297-0.653 , P <0.001). Conclusion The detection rate of hyperuricemia among workers in an electronics factory in Wuhan is high, and the detection rate of hyperuricemia in men is higher than that in women. Alcohol consumption, overweight and obesity will increase the risk of hyperuricemia. Elevated ALT is associated with hyperuricemia. Maintaining an ideal body mass index and establishing a good lifestyle play an important role in preventing hyperuricemia.

Objective To explore early postoperative gait characteristics and clinical outcomes after total knee arthroplasty(TKA).Methods From February 2023 to July 2023,26 patients with unilateral knee osteoarthritis(KOA)were treated with TKA,including 4 males and 22 females,aged from 57 to 85 years old with an average of(67.58±6.49)years old;body mass in-dex(BMI)ranged from 18.83 to 38.28 kg·m-2 with an average of(26.43±4.15)kg·m-2;14 patients on the left side,12 pa-tients on the right side;according to Kellgren-Lawrence(K-L)classification,6 patients with grade Ⅲ and 20 patients with grade Ⅳ;the courses of disease ranged from 1 to 14 years with an average of(5.54±3.29)years.Images and videos of standing up and walking,walking side shot,squatting and supine kneeling were taken with smart phones before operation and 6 weeks after operation.The human posture estimation framework OpenPose were used to analyze stride frequency,step length,step length,step speed,active knee knee bending angle,stride length,double support phase time,as well as maximum hip flexion angle and maximum knee bending angle on squatting position.Western Ontario and McMaster Universities(WOMAC)arthritis index and Knee Society Score(KSS)were used to evaluate clinical efficacy of knee joint.Results All patients were followed up for 5 to 7 weeks with an average of(6.00±0.57)weeks.The total score of WOMAC decreased from(64.85±11.54)before op-eration to(45.81±7.91)at 6 weeks after operation(P＜0.001).The total KSS was increased from(101.19±9.58)before opera-tion to(125.50±10.32)at 6 weeks after operation(P＜0.001).The gait speed,stride frequency and stride length of the affected side before operation were(0.32±0.10)m·s-1,(96.35±24.18)steps·min-1,(0.72±0.14)m,respectively;and increased to(0.48±0.11)m·s 1,(104.20±22.53)steps·min-1,(0.79±0.10)m at 6 weeks after operation(P＜0.05).The lower limb support time and active knee bending angle decreased from(0.31±0.38)sand(125.21±11.64)° before operation to(0.11±0.04)s and(120.01±13.35)° at 6 weeks after operation(P＜0.05).Eleven patients could able to complete squat before operation,13 patients could able to complete at 6 weeks after operation,and 9 patients could able to complete both before operation and 6 weeks after operation.In 9 patients,the maximum bending angle of crouching position was increased from 76.29° to 124.11° before operation to 91.35° to 134.12° at 6 weeks after operation,and the maximum bending angle of hip was increased from 103.70° to 147.25° before operation to 118.61° to 149.48° at 6 weeks after operation.Conclusion Gait analysis technology based on artificial intelligence image recognition is a safe and effective method to quantitatively identify the changes of pa-tients'gait.Knee pain of KOA was relieved and the function was improved,the supporting ability of the affected limb was im-proved after TKA,and the patient's stride frequency,stride length and stride speed were improved,and the overall movement rhythm of both lower limbs are more coordinated.

Objective:To analyze the related factors of treatment failure in children with acute lymphoblastic leukemia (ALL)in real-world.Methods:The clinical data of 1414 newly diagnosed children with ALL admitted to five hospital in Fujian province from April 2011 to December 2020 were retrospectively analyzed.Treatment failure was defined as relapse,non-relapse death,and secondary tumor.Results:Following-up for median time 49.7 (0.1-136. 9)months,there were 269 cases (19.0％)treatment failure,including 140 cases (52.0％)relapse,and 129 cases (48.0％)non-relapse death.Cox univariate and multivariate analysis showed that white WBC≥50 ×109/L at newly diagnosis,acute T-cell lymphoblastic leukemia (T-ALL),BCR-ABL1,KMT2A-rearrangement and poor early treatment response were independent risk factor for treatment failure (all HR>1.000,P<0.05).The 5-year OS of 140 relapsed ALL patients was only 23.8％,with a significantly worse prognosis for very early relapse (relapse time within 18 months of diagnosis).Among 129 patients died from non-relapse death,71 cases (26.4％)were died from treatment-related complications,56 cases (20.8％)died from treatment abandonment,and 2 cases (0.7％)died from disease progression.Among them,treatment-related death were significantly correlated with chemotherapy intensity,while treatment abandonment were mainly related to economic factors.Conclusion:The treatment failure of children with ALL in our province is still relatively high,with relapse being the main cause of treatment failure,while treatment related death and treatment abandonment caused by economic factors are the main causes of non-relapse related death.

Objective To observe the improvement of motor dysfunction and serum levels of C-reactive protein(CRP),blood glucose and blood lipid in post-stroke patients treated with scalp acupuncture at different needle retention time.Methods A total of 120 patients with motor dysfunction after stroke were randomly divided into control group,observation group 1 and observation group 2,with 40 cases in each group.The patients in the 3 groups were treated with scalp acupuncture,body acupuncture and routine rehabilitation exercise,once a day and 6 times a week,lasting for 2 weeks.The control group was given scalp acupuncture with retaining of needles for 30 minutes,the observation group 1 was given scalp acupuncture with retaining of needles for one hour,and the observation group 2 was given scalp acupuncture with retaining of needles for 2 hours.Before and after treatment,the 3 groups were observed in the changes of the scale scores of National Institutes of Health Stroke Scale(NIHSS),Fugl-Meyer Assessment(FM A),Berg Balance Scale(BBS)and modified Barthel Index(MBI),and the levels of laboratory indicators of peripheral blood CRP,fasting plasma glucose(FPG),total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C).After treatment,the clinical safety of the three groups was evaluated.Results(1)After treatment,the scale scores of NIHSS in the three groups were lower(P＜0.01)and the scale scores FMA,BBS and MBI were higher than those before treatment(P＜0.05 or P＜0.01).The comparison of post-treatment scale scores showed that the differences among the three groups were statistically significant(P＜0.01).The intergroup comparison showed that the decrease of NIHSS score and the increase of FMA,BBS and MBI scores in the observation group 2 were significantly superior to those in the control group and the observation group 1(P＜0.01);the improvement of FMA score in the observation group 1 was significantly superior to that in the control group(P＜0.01),while the improvement of NIHSS,BBS and MBI scores tended to be superior to that in the control group without statistically significant differences(P＞0.05).The results indicated that the curative effect of scalp acupuncture plus exercise regimen was positively correlated with the duration of needle retention for scalp acupuncture.(2)After treatment,the laboratory indicator levels of CRP and FPG in the peripheral blood of the three groups,the levels of TG and LDL-C in the two observation groups and the level of HDL-C in the observation group 2 were improved compared with those before treatment(P＜0.05 or P＜0.01).Statistically significant differences were presented in the post-treatment levels of CRP and TG in peripheral blood among the three groups(P＜0.05 or P＜0.01).The intergroup comparison showed that the improvement of CRP and TG levels in the observation group 2 was significantly superior to that in the control group,and the improvement of CRP level in the observation group 2 was significantly superior to that in the observation group 1,the differences being statistically significant(P＜0.05 or P＜0.01).The TC level in the three groups after treatment did not differ from that before treatment,and there was no significant difference in TC level after treatment among the three groups either(P＞0.05).(3)During the treatment,no adverse reactions such as fainting,needle breaking and hematoma occurred in the three groups,the vital signs of the patients were stable,and there were no obvious abnormal changes in pulse,blood pressure and respiratory rate.Conclusion Scalp acupuncture can effectively improve the motor function of post-stroke patients in a pasitive time-effect relationship with the needle retention,and better the curative effect can be achieved by retaining of the needle for 2 h.

Objective To compare the outcomes of transplant kidneys and patient survival between simultaneous pancreas-kidney transplantation(SPKT)recipients and deceased donor kidney transplant(DDKT)recipients in patients with type 2 diabetes mellitus(T2DM)complicated with end-stage renal disease(ESRD),and to analyze the risk factors affecting patient survival post-SPKT.Methods Clinical and prognostic data of patients who underwent kidney transplantation from January 27,2003,to January 1,2021,were retrieved from the United Network for Organ Sharing(UNOS)database.A total of 50 230 cases were selected based on inclusion criteria,with 48 669 cases in DDKT group and 1561 cases in SPKT group.Kaplan-Meier analysis was employed to compare transplant kidney and patient survival between the two groups,and propensity score matching(PSM)was utilized to balance confounding factors between the groups.Cox regression model was used to analyze independent risk factors affecting patient survival post-SPKT.Results Compared with DDKT group,recipients in SPKT group had a younger median age(P<0.001),a higher proportion of males(P<0.001),lower BMI(P<0.001),shorter dialysis and transplant waiting times(P<0.001),a higher percentage of private medical insurance(P<0.001),a lower proportion of previous transplants(P<0.001),a younger age at diabetes diagnosis(P<0.001),and a lower incidence of peripheral vascular disease(P=0.033).Compared with DDKT group,the donors in SPKT group had a younger median age(P<0.001),a higher proportion of males(P<0.001),lower BMI(P<0.001),and a lower prevalence of hypertension and diabetes history(P<0.001).In terms of transplant-related factors,the SPKT group had a shorter donor kidney cold ischemia time(P<0.001),a higher degree of HLA mismatch(P<0.001),and a lower Kidney Donor Profile Index(KDPI)(P<0.001)when compared with DDKT group.The SPKT group had lower serum creatinine levels at discharge(P<0.001),lower rates of postoperative delayed graft function(DGF)and acute rejection(AR)(P<0.001),but longer hospital stays(P<0.001)when compared with DDKT group.Kaplan-Meier survival analysis curves,both original and after propensity score matching(PSM),consistently showed significantly higher transplant kidney and patient survival rates in SPKT group compared with DDKT group(P<0.001).Cox regression model analysis indicated that recipient age,recipient race,donor age,and donor kidney cold ischemia time were independent risk factors influencing patient survival post-SPKT.Conclusions For ESRD patients with T2DM,SPKT offers improved long-term graft and patient survival rates compared with DDKT.Recipient age,recipient ethnicity,donor age,and cold ischemia time for the donor's kidney are independent risk factors affecting post-SPKT patient survival.

Gorham-Stout disease(GSD)is a sporadic chronic disease characterized by progressive bone dissolution,absorption,and disappearance along with lymphatic vessel infiltration in bone-marrow cavities.Although the osteolytic mechanism of GSD has been widely studied,the cause of lymphatic hyperplasia in GSD is rarely investigated.In this study,by comparing the RNA expression profile of osteoclasts(OCs)with that of OC precursors(OCPs)by RNA sequencing,we identified a new factor,semaphorin 3A(Sema3A),which is an osteoprotective factor involved in the lymphatic expansion of GSD.Compared to OCPs,OCs enhanced the growth,migration,and tube formation of lymphatic endothelial cells(LECs),in which the expression of Sema3A is low compared to that in OCPs.In the presence of recombinant Sema3A,the growth,migration,and tube formation of LECs were inhibited,further confirming the inhibitory effect of Sema3A on LECs in vitro.Using an LEC-induced GSD mouse model,the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo.We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss,whereas the injection of lentivirus expressing Sema3A short hairpin RNA(shRNA)into the tibiae caused GSD-like phenotypes.Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment,compared with the control.Based on the above results,we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.