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

This study aims to reveal the effect and mechanism of Gentianella turkestanorum total extract(GTI) in ameliorating non-alcoholic steatohepatitis(NASH). UPLC-Q-TOF-MS was employed to identify the chemical components in GTI. SwissTarget-Prediction, GeneCards, OMIM, and TTD were utilized to screen the targets of GTI components and NASH. The common targets shared by GTI components and NASH were filtered through the STRING database and Cytoscape 3.9.0 to identify core targets, followed by GO and KEGG enrichment analysis. AutoDock was used for molecular docking of key components with core targets. A mouse model of NASH was established with a methionine-choline-deficient high-fat diet. A 4-week drug intervention was conducted, during which mouse weight was monitored, and the liver-to-brain ratio was measured at the end. Hematoxylin-eosin staining, Sirius red staining, and oil red O staining were employed to observe the pathological changes in the liver tissue. The levels of various biomarkers, including aspartate aminotransferase(AST), alanine aminotransferase(ALT), hydroxyproline(HYP), total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), malondialdehyde(MDA), superoxide dismutase(SOD), and glutathione(GSH), in the serum and liver tissue were determined. RT-qPCR was conducted to measure the mRNA levels of interleukin 1β(IL-1β), interleukin 6(IL-6), tumor necrosis factor α(TNF-α), collagen type I α1 chain(COL1A1), and α-smooth muscle actin(α-SMA). Western blotting was conducted to determine the protein levels of IL-1β, IL-6, TNF-α, and potential drug targets identified through network pharmacology. UPLC-Q-TOF/MS identified 581 chemical components of GTI, and 534 targets of GTI and 1 157 targets of NASH were screened out. The topological analysis of the common targets shared by GTI and NASH identified core targets such as IL-1β, IL-6, protein kinase B(AKT), TNF, and peroxisome proliferator activated receptor gamma(PPARG). GO and KEGG analyses indicated that the ameliorating effect of GTI on NASH was related to inflammatory responses and the phosphoinositide 3-kinase(PI3K)/AKT pathway. The staining results demonstrated that GTI ameliorated hepatocyte vacuolation, swelling, ballooning, and lipid accumulation in NASH mice. Compared with the model group, high doses of GTI reduced the AST, ALT, HYP, TC, and TG levels(P<0.01) while increasing the HDL-C, SOD, and GSH levels(P<0.01). RT-qPCR results showed that GTI down-regulated the mRNA levels of IL-1β, IL-6, TNF-α, COL1A1, and α-SMA(P<0.01). Western blot results indicated that GTI down-regulated the protein levels of IL-1β, IL-6, TNF-α, phosphorylated PI3K(p-PI3K), phosphorylated AKT(p-AKT), phosphorylated inhibitor of nuclear factor kappa B alpha(p-IκBα), and nuclear factor kappa B(NF-κB)(P<0.01). In summary, GTI ameliorates inflammation, dyslipidemia, and oxidative stress associated with NASH by regulating the PI3K/AKT/NF-κB signaling pathway.
Animals ; Non-alcoholic Fatty Liver Disease/genetics* ; Mice ; Network Pharmacology ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Chromatography, High Pressure Liquid ; Liver/metabolism* ; Mice, Inbred C57BL ; Humans ; Mass Spectrometry ; Tumor Necrosis Factor-alpha/metabolism* ; Disease Models, Animal ; Molecular Docking Simulation

OBJECTIVES: To explore the factors related to prognosis and treatment failure in children with acute lymphoblastic leukemia (ALL). METHODS: A retrospective study was conducted to collect and analyze clinical data of ALL children admitted to the Department of Pediatric Hematology at Tongji Hospital, Huazhong University of Science and Technology, from January 2012 to December 2019, with follow-up until June 2024. RESULTS: A total of 341 children with ALL were included. Among the 69 children with treatment failure, 55 (80%) experienced relapse, while 14 (20%) had non-relapse-related deaths, and no secondary tumors were observed. Initial WBC count ≥50×10⁹/L, positive minimal residual disease, and severe adverse events were identified as independent risk factors for treatment failure (P<0.05). Among the 55 relapsed patients, early relapses were predominant (36%), and the primary site of relapse was the bone marrow (56%). Immunophenotyping (P=0.009), initial WBC count (P=0.011), and fusion genes (P=0.040) were associated with the timing of relapse. High-risk status, T-cell ALL, relapse, and severe adverse events were independent risk factors affecting long-term survival (P<0.05). CONCLUSIONS The prognosis of children with ALL is related to risk stratification, immunophenotyping, relapse status, and occurrence of severe adverse events. Among these factors, relapse is the primary cause of treatment failure. Actively preventing relapse may reduce the treatment failure rate and improve long-term survival.
Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy* ; Male ; Female ; Child ; Child, Preschool ; Retrospective Studies ; Prognosis ; Treatment Failure ; Adolescent ; Infant ; Risk Factors

A retrospective analysis was conducted on the clinical course of two children with PICALM-MLLT10-positive acute leukemia treated at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, between July 2021 and July 2023. The patients were diagnosed with acute T-lymphoblastic leukemia with central nervous system involvement and high-risk acute myeloid leukemia, respectively. Both achieved bone marrow complete remission after conventional chemotherapy combined with venetoclax. Following conversion to molecular negativity, they underwent sequential allogeneic hematopoietic stem cell transplantation. At the latest follow-up, both patients were alive and in good clinical condition. These observations suggest that proceeding to hematopoietic stem cell transplantation after venetoclax-based chemotherapy may improve the long-term survival of children with PICALM-MLLT10-positive leukemia.
Humans ; Hematopoietic Stem Cell Transplantation ; Male ; Female ; Child, Preschool ; Transplantation, Homologous ; Child ; Leukemia, Myeloid, Acute/genetics* ; Oncogene Proteins, Fusion/genetics*

OBJECTIVE: To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. METHODS: In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. RESULTS: Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. CONCLUSION AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.
Podocytes/metabolism* ; Animals ; Diabetic Nephropathies/metabolism* ; Saponins/therapeutic use* ; Triterpenes/therapeutic use* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Male ; Rats, Sprague-Dawley ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Endoribonucleases/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; Rats ; Diabetes Mellitus, Experimental/complications* ; Endoplasmic Reticulum/metabolism* ; Multienzyme Complexes

Combined radiation and burn injury (CRBI) is a severe syndrome, which is induced by the simultaneous or successive radiation and burn; but no appropriate clinical therapies are available. Loong oil (LO) is a traditional Chinese medicine oil composed of the oil extracts of cuttlebone, safflower, walnut oil, and rapeseed oil, which has been demonstrated to own anti-radiation and tissue healing functions. In this study, glyceryl monostearate (GMO) was used for the preparation of lyotropic liquid crystals that loaded LO to obtain Loong oil-lyotropic liquid crystals (LOL) for the treatment of skin CRBI. The hexagonal phase structure of LOL was proved by small X-ray scattering (SAXS) analysis with an approximate 7:9:11 ratio of peaks and under the polarizing microscope with the birefringence phenomenon. LOL had a high LO loading capacity (4%) and good bio-adhesive force, favoring skin administration. Animal experiments were approved by the Ethics Committee of the Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-834). Compared to triethanolamine creams, LOL had higher skin permeability with total skin penetration within 1 h, benefiting the treatment of deep CRBI. A deep II degree burn CRBI mouse model was established after whole-body irradiation with 5 Gy ⁶⁰Co γ-ray and the next 6-second 100 ℃ burn injury. LOL promoted LO skin penetration, improved epithermal cell proliferation and the formation of hair follicles, accelerated wound healing, and inhibited scarring, which benefitted the therapy of CRBI. In this study, LO application fields are expanded and LO is a promising medication for the treatment of CRBI.

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing. Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing. Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

Objective To compare the effects of 20 mg qd and 10 mg bidadministration of iprrazole enteric-coated tablets on the control of gastric acid in healthy subjects.Methods A randomized,single-center,parallel controlled trial was designed to include 8 healthy subjects.Randomly divided into 2 groups,20 mg qd administration group:20 mg enteric-coated tablets of iprrazole in the morning;10 mg bid administration group:10 mg enteric-coated tablets of iprrazole in the morning and 10 mg in the evening.The pH values in the stomach of the subjects before and 24 h after administration were monitored by pH meter.The plasma concentration of iprazole after administration was determined by HPLC-MS/MS.The main pharmacokinetic parameters were calculated by Phoenix WinNonlin(V8.0)software.Results The PK parameters of iprrazole enteric-coated tablets and reference preparations in fasting group were as follows:The Cmax of 20 mg qd group and 10 mg bid group were(595.75±131.15)and(283.50±96.98)ng·mL-1;AUC0-t were(5 531.94±784.35)and(4 686.67±898.23)h·ng·mL-1;AUC0-∞ were(6 003.19±538.59)and(7 361.48±1 816.77)h·ng·mL-1,respectively.The mean time percentage of gastric pH＞3 after 20 mg qd and 10 mg bid were 82.64％and 61.92％,and the median gastric pH within 24 h were 6.25±1.49 and 3.53±2.05,respectively.The mean gastric pH values within 24 h were 5.71±1.36 and 4.23±1.45,respectively.The correlation analysis of pharmacokinetic/pharmacodynamics showed that there was no significant correlation between the peak concentration of drug in plasma and the inhibitory effect of acid.Conclusion Compared with the 20 mg qd group and the 10 mg bid group,the acid inhibition effect is better,the administration times are less,and the safety of the two administration regimes is good.

Objective To evaluate the effects of high-fat diet on the pharmacokinetics of metronidazole in Chinese healthy adult subjects.Methods This program is designed according to a single-center,randomized,open,single-dose trial.Forty-seven healthy subjects were assigned to receive single dose of metronidazole tablets 200 mg in either fasting and high-fat diet state,and blood samples were taken at different time points,respectively.The concentrations of metronidazole in plasma were determined by high performance liquid chromatography-mass spectromentry.Results The main pharmacokinetic parameters of metronidazole in fasting state and high-fat diet state were as follows:Cmax were(4 799.13±1 195.32)and(4 044.17±773.98)ng·mL-1;tmax were 1.00 and 2.25 h;t1/2 were(9.11±1.73)and(9.37±1.79)h;AUC0_t were(5.59±1.19)x 104 and(5.51±1.18)x 104 ng·mL-1·h;AUC0_∞ were(5.79±1.33)x 104 and(5.74±1.32)× 104 ng·mL-1·h.Compared to the fasting state,the tmaxof the drug taken after a high fat diet was delayed by 1.25 h(P＜0.01),Cmax,AUC0_t,AUC0-∞ were less or decreased in different degrees,but the effects were small(all P＞0.05).Conclusion High-fat diet has little effects on the pharmacokinetic parameters of metronidazole,which does not significantly change the degree of drug absorption,but can significantly delay the time to peak.

Objective To observe the efficacy and safety of Morinda officinalis oligosaccharides in the continuation treatment of mild and moderate depression.Methods An open,single-arm,multi-center design was adopted in our study.Adult patients with mild and moderate depression who had received acute treatment of Morinda officinalis oligosaccharides were enrolled and continue to receive Morinda officinalis oligosaccharides capsules for 24 weeks,the dose remained unchanged during continuation treatment.The remission rate,recurrence rate,recurrence time,and the change from baseline to endpoint of Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA),Clinical Global Impression-Severity(CGI-S)and Arizona Sexual Experience Scale(ASEX)were evaluated.The incidence of treatment-related adverse events was reported.Results The scores of HAMD-17 at baseline and after treatment were 6.60±1.87 and 5.85±4.18,scores of HAMA were 6.36±3.02 and 4.93±3.09,scores of CGI-S were 1.49±0.56 and 1.29±0.81,scores of ASEX were 15.92±4.72 and 15.57±5.26,with significant difference(P＜0.05).After continuation treatment,the remission rate was 54.59％(202 cases/370 cases),and the recurrence rate was 6.49％(24 cases/370 cases),the recurrence time was(64.67±42.47)days.The incidence of treatment-related adverse events was 15.35％(64 cases/417 cases).Conclusion Morinda officinalis oligosaccharides capsules can be effectively used for the continuation treatment of mild and moderate depression,and are well tolerated and safe.