Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

OBJECTIVE: Hepatocellular carcinoma (HCC) is sensitive to ferroptosis, a new form of programmed cell death that occurs in most tumor types. However, the mechanism through which ferroptosis modulates HCC remains unclear. This study aimed to investigate the oncogenic role and prognostic value of FANCD2 and provide novel insights into the prognostic assessment and prediction of immunotherapy. METHODS: Using clinicopathological parameters and bioinformatic techniques, we comprehensively examined the expression of FANCD2 macroscopically and microcosmically. We conducted univariate and multivariate Cox regression analyses to identify the prognostic value of FANCD2 in HCC and elucidated the detailed molecular mechanisms underlying the involvement of FANCD2 in oncogenesis by promoting iron-related death. RESULTS: FANCD2 was significantly upregulated in digestive system cancers with abundant immune infiltration. As an independent risk factor for HCC, a high FANCD2 expression level was associated with poor clinical outcomes and response to immune checkpoint blockade. Gene set enrichment analysis revealed that FANCD2 was mainly involved in the cell cycle and CYP450 metabolism. CONCLUSION To the best of our knowledge, this is the first study to comprehensively elucidate the oncogenic role of FANCD2. FANCD2 has a tumor-promoting aspect in the digestive system and acts as an independent risk factor in HCC; hence, it has recognized value for predicting tumor aggressiveness and prognosis and may be a potential biomarker for poor responsiveness to immunotherapy.
Humans ; Carcinoma, Hepatocellular/diagnosis* ; Liver Neoplasms/diagnosis* ; Immunotherapy ; Fanconi Anemia Complementation Group D2 Protein/metabolism* ; Prognosis ; Male ; Female ; Middle Aged ; Biomarkers, Tumor/metabolism*

The imprint desorption electrospray photoionization mass spectrometry was employed to locally image the spatial distribution of chemical components in dried tobacco leaves after initial curing. The relative content distribution of different chemical components was obtained in tobacco leaves. The application of imprinting method could transfer tobacco internal compounds to the surface of porous polytetrafluoroethylene plate,which realized the detection and visual analysis of tobacco internal substances. Besides,the imprint desorption electrospray ionization/post-photoionization (Imprint DESI/PI) mass spectrometry imaging technique had the advantages of non-polarity discrimination,soft ionization and high ionization efficiency for plant samples,and could simultaneously detect and image rich compounds in tobacco samples. A total of 40 kinds of chemical components including alkaloids,amino acids,sugars,acids,ketones and phenols were identified based on high resolution mass spectrometry. The results showed that the representative chemical components of tobacco,such as alkaloids,amino acids and sugars,were mainly distributed near the leaf tip from the vertical analysis and at the left and right leaf edges from the horizontal analysis. Amadori compound (1-Deoxy-1-L-proline-d-fructose) was detected,and the content of Amadori was found to be consistent with that of free amino acid (proline). In addition,the technique was further used to study the climate spot disease area of tobacco,and it was found that the compounds had specific distribution in the climate spot area,which further proved the superiority of this method in studying the growth stress of tobacco leaves.

Objective To compare the differences in lipid metabolism between platinum-resistant and platinum-sensitive ovarian cancer patients at stage Ⅲ-Ⅳ,to analyze the differential intestinal flora using 16S rRNA sequen-cing,and to explore the associations among intestinal flora,lipid metabolism characteristics and platinum resistance in ovarian cancer.Methods Patients diagnosed with ovarian cancer at stage Ⅲ-Ⅳ through surgical pathology were selected,including a platinum-resistant group(11 cases)and a platinum-sensitive group(11 cases).The differences in lipid metabolism between the two groups were compared.The differences in gut microbiota between the two groups were investigated using fecal 16S rRNA sequencing.The association among gut microbiota,lipid metabolism characteristics,and platinum resistance in ovarian cancer was analyzed.Results Significant differ-ences were observed in lipid metabolism-related indicators[total cholesterol(TC),high-density lipoprotein choles-terol(HDL-C),non-high-density lipoprotein cholesterol(n-HDL),low-density lipoprotein cholesterol(LDL-C),apolipoprotein(B)]between the two groups,with higher levels in the platinum-resistant group.The Shannon in-dex(P=0.008 3)and Simpson index(P=0.008 2)both showed higher diversity of gut microbiota in platinum-resistant ovarian cancer patients compared to the platinum-sensitive group.However,based on OTUs species clus-tering and relative abundance statistics,certain bacterial abundances differed significantly between the groups.Spe-cies such as Parabacteroides,Akkermansia,Blautia,Lachnoclostridium,Fusicatenibacter,and Megamonas had sig-nificantly higher abundances in the platinum-sensitive ovarian cancer group,and Akkermansia(a lipid metabolism-related bacterial group)was the most prevalent.Conclusion The platinum-resistant group of ovarian cancer ex-hibits significantly higher levels of lipid metabolism and gut microbiota diversity compared to the platinum-sensitive group.This suggests that the increase in lipid metabolism levels and fecal microbiota diversity may be associated with the development of platinum resistance.However,certain microbial taxa are reduced in abundance in the plat-inum-resistant group,such as the distinct Akkermansia genus(a lipid metabolism-related microbial community),which may serve as one of the factors inducing platinum-resistance in ovarian cancer.

BACKGROUND:Minimally invasive surgery is developing rapidly.Robot-assisted minimally invasive transforaminal lumbar interbody fusion and robot-assisted unilateral biportal endoscopic transforaminal/posterior lumbar interbody fusion are important posterior minimally invasive surgical approaches to treat lumbar degenerative diseases.However,it is worth discussing which operation method is more advantageous. OBJECTIVE:To compare the clinical efficacy and imaging examination between different operation groups,and discuss the clinical application value of robot-assisted minimally invasive lumbar posterior fusion technology to treat lumbar degenerative diseases. METHODS:Clinical data of 83 patients with lumbar degenerative diseases from January 2018 to June 2022 at the Department of Orthopedics,Sichuan Academy of Medical Sciences&Sichuan Provincial People's Hospital were retrospectively analyzed.Of them,27 patients received robot-assisted minimally invasive transforaminal lumbar interbody fusion treatment(group A);30 patients received robot-assisted unilateral biportal endoscopic transforaminal/posterior lumbar interbody fusion treatment(group B),and 26 traditional minimally invasive transforaminal lumbar interbody fusion patients were selected as the control group(group C).There were no significant differences in gender,age,body mass index,surgical segment,preoperative visual analog scale score and Oswestry Disability Index among the three groups(P＞0.05).The operation time,intraoperative blood loss,complications,fluoroscopic dose,fluoroscopic time,and fluoroscopic frequency were compared among the three groups.Gertzbein-Robbins'classification was used to evaluate the accuracy of percutaneous pedicle screw.Visual analog scale and Oswestry Disability Index scores were evaluated after surgery.The excellent and good rate of the three surgical options was evaluated using Macnab's criteria. RESULTS AND CONCLUSION:(1)The operation time of group A was significantly shorter than that of groups B and C(P＜0.05),but there was no significant difference between group B and group C(P＞0.05).The intraoperative blood loss in group B was significantly less than that in group A,and that in group A was significantly less than that in group C(P＜0.05).(2)The fluoroscopic dose,fluoroscopic time,and fluoroscopic frequency of group C were significantly higher than those of groups A and B(P＜0.05).(3)Visual analog scale score and Oswestry Disability Index in the three groups significantly improved after operation when compared with that before operation(P＜0.05),but there was no significant difference among the three groups 1 day and 6 months after surgery(P＞0.05).(4)Postoperative imaging showed that the accuracy of percutaneous pedicle screw placement in groups A and B was better than that in group C(P＜0.05).(5)There was no significant difference in the excellent and good rate of MacNab criteria among the three groups(P＞0.05).(6)There was no significant difference in complications among the three groups(P＞0.05).(7)The results indicated that robot-assisted minimally invasive transforaminal lumbar interbody fusion and robot-assisted unilateral biportal endoscopic transforaminal/posterior lumbar interbody fusion are effective surgery methods for lumbar degenerative diseases.Compared with traditional minimally invasive transforaminal lumbar interbody fusion,robot-assisted minimally invasive transforaminal lumbar interbody fusion surgery has higher efficiency,less intraoperative radiation and higher internal fixation accuracy,which has a good clinical application value.

In recent years,with the continuous progress of DNA extraction and detection technology,cell-free DNA(cfDNA)has been widely used in the life science field,and its potential application value in forensic identification is becoming more and more obvious.This paper reviews the concept,formation mechanism,and classification of cfDNA,etc.,and describes the latest research progress of cfDNA in personal identification of crime scene touch DNA samples and non-invasive prenatal pater-nity testing(NIPPT).Meanwhile,this paper summarizes the potential application of cfDNA in injury inference,and discusses the advantages and disadvantages of common cfDNA analysis methods and techniques,and its application prospects,to provide a new idea for the wide application of cfDNA in the field of forensic science.

Objective This study aimed to evaluate whether the onset of the plateau phase of slow hepatitis B surface antigen decline in patients with chronic hepatitis B treated with intermittent interferon therapy is related to the frequency of dendritic cell subsets and expression of the costimulatory molecules CD40,CD80,CD83,and CD86. Method This was a cross-sectional study in which patients were divided into a natural history group(namely NH group),a long-term oral nucleoside analogs treatment group(namely NA group),and a plateau-arriving group(namely P group).The percentage of plasmacytoid dendritic cell and myeloid dendritic cell subsets in peripheral blood lymphocytes and monocytes and the mean fluorescence intensity of their surface costimulatory molecules were detected using a flow cytometer. Results In total,143 patients were enrolled(NH group,n = 49;NA group,n = 47;P group,n = 47).The results demonstrated that CD141/CD1c double negative myeloid dendritic cell(DNmDC)/lymphocytes and monocytes(%)in P group(0.041[0.024,0.069])was significantly lower than that in NH group(0.270[0.135,0.407])and NA group(0.273[0.150,0.443]),and CD86 mean fluorescence intensity of DNmDCs in P group(1832.0[1484.0,2793.0])was significantly lower than that in NH group(4316.0[2958.0,5169.0])and NA group(3299.0[2534.0,4371.0]),Adjusted P all<0.001. Conclusion Reduced DNmDCs and impaired maturation may be associated with the onset of the plateau phase during intermittent interferon therapy in patients with chronic hepatitis B.

Objective To explore characteristics of clinical parameters and cytokines in patients with drug-induced liver injury(DILI)caused by different drugs and their correlation with clinical indicators. Method The study was conducted on patients who were up to Review of Uncertainties in Confidence Assessment for Medical Tests(RUCAM)scoring criteria and clinically diagnosed with DILI.Based on Chinese herbal medicine,cardiovascular drugs,non-steroidal anti-inflammatory drugs(NSAIDs),anti-infective drugs,and other drugs,patients were divided into five groups.Cytokines were measured by Luminex technology.Baseline characteristics of clinical biochemical indicators and cytokines in DILI patients and their correlation were analyzed. Results 73 patients were enrolled.Age among five groups was statistically different(P=0.032).Alanine aminotransferase(ALT)(P=0.033)and aspartate aminotransferase(AST)(P=0.007)in NSAIDs group were higher than those in chinese herbal medicine group.Interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-α)in patients with Chinese herbal medicine(IL-6:P<0.001;TNF-α:P<0.001)and cardiovascular medicine(IL-6:P=0.020;TNF-α:P=0.001)were lower than those in NSAIDs group.There was a positive correlation between ALT(r=0.697,P=0.025),AST(r=0.721,P=0.019),and IL-6 in NSAIDs group. Conclusion Older age may be more prone to DILI.Patients with NSAIDs have more severe liver damage in early stages of DILI,TNF-α and IL-6 may partake the inflammatory process of DILI.

BACKGROUND: Osteoarthritis (OA), a degenerative joint disorder, is a major reason of disability in adults. Accumulating evidences have proved that bone marrow mesenchymal stem cells (BMSCs)-carried exosomes play a significant therapeutic effect on OA. However, the precise regulatory network remains unknown. METHODS: OA and normal cartilage samples were acquired from patients, and chondrocytes were exposed to IL-1b to conduct a cellular OA model. Exosomes prepared from BMSCs were identified using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Cell viability was determined with CCK-8 assay. Inflammatory injury was assessed by LDH and inflammatory factors (TNF-a and IL-6) using corresponding ELISA kits, respectively. Ferroptosis was evaluated by GSH, MDA and iron levels using corresponding kits, and ROS level with DCFH-DA. The expressions of genes/proteins were determined with RT-qPCR/western bolt. RNA immunoprecipitation and luciferase activity assay were conducted for testing the interactions of small nucleolar RNA host gene 7 (SNHG7)/ferroptosis suppressor protein 1 (FSP1) and miR-485-5p. RESULTS: The expressions of SNHG7 and FSP1 were both reduced in IL-1b-induced chondrocytes and OA cartilage tissues, and there was a positive correlation between them in clinical level. Moreover, SNHG7 was enriched in BMSCsderived exosomes (BMSCs-Exos) and could be internalized by chondrocytes. Functional analysis illustrated that BMSCsExos administration repressed inflammatory injury, oxidative stress and ferroptosis in IL-1b-induced chondrocytes, while these changes were reinforced when SNHG7 was overexpressed in BMSCs-Exos. Notably, FSP1 silencing in chondrocytes abolished the beneficial effects mediated by exosomal SNHG7. CONCLUSIONS Exosomal SNHG7 released from BMSCs inhibited inflammation and ferroptosis in IL-1b-induced chondrocytes through miR-485-5p/FSP1 axis. This work suggested that BMSCs-derived exosomal SNHG7 would be a prospective target for OA treatment.

BACKGROUND: Osteoarthritis (OA), a degenerative joint disorder, is a major reason of disability in adults. Accumulating evidences have proved that bone marrow mesenchymal stem cells (BMSCs)-carried exosomes play a significant therapeutic effect on OA. However, the precise regulatory network remains unknown. METHODS: OA and normal cartilage samples were acquired from patients, and chondrocytes were exposed to IL-1b to conduct a cellular OA model. Exosomes prepared from BMSCs were identified using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Cell viability was determined with CCK-8 assay. Inflammatory injury was assessed by LDH and inflammatory factors (TNF-a and IL-6) using corresponding ELISA kits, respectively. Ferroptosis was evaluated by GSH, MDA and iron levels using corresponding kits, and ROS level with DCFH-DA. The expressions of genes/proteins were determined with RT-qPCR/western bolt. RNA immunoprecipitation and luciferase activity assay were conducted for testing the interactions of small nucleolar RNA host gene 7 (SNHG7)/ferroptosis suppressor protein 1 (FSP1) and miR-485-5p. RESULTS: The expressions of SNHG7 and FSP1 were both reduced in IL-1b-induced chondrocytes and OA cartilage tissues, and there was a positive correlation between them in clinical level. Moreover, SNHG7 was enriched in BMSCsderived exosomes (BMSCs-Exos) and could be internalized by chondrocytes. Functional analysis illustrated that BMSCsExos administration repressed inflammatory injury, oxidative stress and ferroptosis in IL-1b-induced chondrocytes, while these changes were reinforced when SNHG7 was overexpressed in BMSCs-Exos. Notably, FSP1 silencing in chondrocytes abolished the beneficial effects mediated by exosomal SNHG7. CONCLUSIONS Exosomal SNHG7 released from BMSCs inhibited inflammation and ferroptosis in IL-1b-induced chondrocytes through miR-485-5p/FSP1 axis. This work suggested that BMSCs-derived exosomal SNHG7 would be a prospective target for OA treatment.