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

Transformer models have emerged as pivotal tools within the realm of drug discovery, distinguished by their unique architectural features and exceptional performance in managing intricate data landscapes. Leveraging the innate capabilities of transformer architectures to comprehend intricate hierarchical dependencies inherent in sequential data, these models showcase remarkable efficacy across various tasks, including new drug design and drug target identification. The adaptability of pre-trained transformer-based models renders them indispensable assets for driving data-centric advancements in drug discovery, chemistry, and biology, furnishing a robust framework that expedites innovation and discovery within these domains. Beyond their technical prowess, the success of transformer-based models in drug discovery, chemistry, and biology extends to their interdisciplinary potential, seamlessly combining biological, physical, chemical, and pharmacological insights to bridge gaps across diverse disciplines. This integrative approach not only enhances the depth and breadth of research endeavors but also fosters synergistic collaborations and exchange of ideas among disparate fields. In our review, we elucidate the myriad applications of transformers in drug discovery, as well as chemistry and biology, spanning from protein design and protein engineering, to molecular dynamics (MD), drug target identification, transformer-enabled drug virtual screening (VS), drug lead optimization, drug addiction, small data set challenges, chemical and biological image analysis, chemical language understanding, and single cell data. Finally, we conclude the survey by deliberating on promising trends in transformer models within the context of drug discovery and other sciences.

OBJECTIVE: To investigate the role and mechanism of the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon gene (cGAS/STING) pathway in oleic acid-induced acute lung injury (ALI) in mice. METHODS: Male wild-type C57BL/6J mice were randomly divided into five groups (each n = 10): normal control group, ALI model group, and 5, 50, 500 μg/kg inhibitor pretreatment groups. The ALI model was established by tail vein injection of oleic acid (7 mL/kg), while the normal control group received no intervention. The inhibitor pretreatment groups were intraperitoneally injected with the corresponding doses of cGAS inhibitor RU.521 respectively 1 hour before modeling. At 24 hours post-modeling, blood was collected, and mice were sacrificed. Lung tissue pathological changes were observed under light microscopy after hematoxylin-eosin (HE) staining, and pathological scores were assessed. Western blotting was used to detect the protein expressions of cGAS, STING, phosphorylated TANK-binding kinase 1 (p-TBK1), phosphorylated interferon regulatory factor 3 (p-IRF3), and phosphorylated nuclear factor-κB p65 (p-NF-κB p65) in lung tissue. Immunohistochemistry was performed to observe STING and p-NF-κB positive expressions in lung tissue. Serum interferon-β (IFN-β) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the normal control group, the ALI model group exhibited significant focal alveolar thickening, intra-alveolar hemorrhage, pulmonary capillary congestion, and neutrophil infiltration in the pulmonary interstitium and alveoli, along with markedly increased pathological scores (10.33±0.58 vs. 1.33±0.58, P < 0.05). Protein expressions of cGAS, STING, p-TBK1, p-IRF3, and p-NF-κB p65 in lung tissue significantly increased [cGAS protein (cGAS/β-actin): 1.24±0.02 vs. 0.56±0.02, STING protein (STING/β-actin): 1.27±0.01 vs. 0.55±0.01, p-TBK1 protin (p-TBK1/β-actin): 1.34±0.03 vs. 0.22±0.01, p-IRF3 protein (p-IRF3/β-actin): 1.23±0.02 vs. 0.36±0.01, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 1.30±0.02 vs. 0.53±0.02, all P < 0.05], positive expressions of STING and p-NF-κB in lung tissue were significantly elevated [STING (A value): 0.51±0.03 vs. 0.30±0.07, p-NF-κB (A value): 0.57±0.05 vs. 0.31±0.03, both P < 0.05], and serum IFN-β levels were also significantly higher (ng/L: 256.02±3.84 vs. 64.15±1.17, P < 0.05). The cGAS inhibitor pretreatment groups showed restored alveolar structural integrity, reduced inflammatory cell infiltration, and decreased hemorrhage area, along with dose-dependent lower pathological scores as well as the protein expressions of cGAS, STING, p-TBK1, p-IRF3 and p-NF-κB p65 in lung tissue, with significant differences between the 500 μg/kg inhibitor group and ALI model group [pathological score: 2.67±0.58 vs. 10.33±0.58, cGAS protein (cGAS/β-actin): 0.56±0.03 vs. 1.24±0.02, STING protein (STING/β-actin): 0.67±0.03 vs. 1.27±0.01, p-TBK1 protein (p-TBK1/β-actin): 0.28±0.01 vs. 1.34±0.03, p-IRF3 protein (p-IRF3/β-actin): 0.32±0.01 vs. 1.23±0.02, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 0.63±0.01 vs. 1.30±0.02, all P < 0.05]. Compared with the ALI model group, positive expressions of STING and p-NF-κB in lung tissue were significantly reduced in the 500 μg/kg inhibitor group [STING (A value): 0.40±0.01 vs. 0.51±0.03, p-NF-κB (A value): 0.43±0.02 vs. 0.57±0.05, both P < 0.05], and serum IFN-β levels were also markedly reduced (ng/L: 150.03±6.19 vs. 256.02±3.84, P < 0.05). CONCLUSIONS The cGAS/STING pathway is activated in oleic acid-induced ALI, leading to exacerbated inflammatory responses and increased lung damage. RU.521 can inhibit cGAS, thereby down-regulating the expression of pathway proteins and cytokines, and providing protection to lung tissue.
Animals ; Acute Lung Injury/chemically induced* ; Male ; Nucleotidyltransferases/metabolism* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Membrane Proteins/metabolism* ; Oleic Acid/adverse effects* ; Transcription Factor RelA/metabolism* ; Lung/pathology* ; Interferon Regulatory Factor-3/metabolism* ; Disease Models, Animal

Objective:To explore the effects of G protein-coupled receptor 55 (GPR55) antagonist CID16020046 on renal fibrosis in mice, and provide a new method and idea for the treatment of renal fibrosis.Methods:(1) GPR55 overexpression and GPR55 antagonist CID16020046 were used in renal fibroblasts (NRK-49F) of rats, respectively. Meanwhile,transforming growth factor-β1 (TGF-β1) was applied in the NRK-49F cells to observe the expression of fibrosis-related factors and inflammatory factors. (2) A mouse model of renal fibrosis with unilateral ureteral obstruction (UUO) was established in vivo. Eight-week-old male C57BL/6J mice (20-25 g) were randomly divided into three groups according to the random number table method: sham group ( n=6), model group (UUO group, n=7), model + CID16020046 drug (UUO+CID group, n=8). The drug CID16020046 (10 mg/kg) was intraperitoneally injected 1 day before modeling, on the day of modeling and every day after surgery in UUO+CID group, and the corresponding dose of 0.9% normal saline was injected intraperitoneally in sham and UUO groups.The mice were sacrificed for sampling 7 days after UUO surgery, and their renal function indicators, liver transaminase, and cardiac markers were examined. Western blotting and quantitative real-time PCR were used to detect the expression of renal fibrosis-related factors and inflammatory factors. Immunohistochemistry staining, Sirius red staining and Masson trichrome staining were used to detect the pathological changes of renal tissues. Results:(1) After NRK-49F cells were stimulated by TGF-β1, the mRNA and protein expression levels of GPR55 were significantly increased (both P<0.05). There was no statistically significant difference in the mRNA expression of fibrosis-related factors fibronectin and collagen Ⅰ, and inflammatory factors interleukin-1β and tumor necrosis factor-α between TGF-β1 group and TGF-β1 + GPR55 overexpression group (all P>0.05). Compared with the TGF-β1 group, the protein expression levels of fibrosis-related factors alpha-smooth muscle actin (α-SMA) and vimentin, and the mRNA expression levels of collagen Ⅰ and α-SMA were lower in the TGF-β1 + CID group (all P<0.05). (2) Compared with sham group, the mRNA and protein expression levels of GPR55 in UUO group were higher (both P<0.05). The serum creatinine in the UUO+CID group was lower compared to the UUO group ( P<0.05). There was no statistically significant difference in blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine kinase isoenzyme between UUO group and UUO+CID group (all P>0.05). Compared with the UUO group, the protein expression levels of renal fibrosis-related factors fibronectin, collagen Ⅰ and vimentin, and the mRNA expression levels of fibronectin, collagen Ⅰ, collagen Ⅲ and α-SMA were lower in the UUO+CID group (all P<0.05). The degree of renal tubular dilation and interstitial collagen fiber deposition in the UUO+CID group was significantly reduced compared to the UUO group (all P<0.05). Conclusions:CID16020046 can reduce serum creatinine in UUO mice, protect renal function, and simultaneously decrease the expression of fibrosis-related factors in renal fibroblasts and mouse kidney tissues, thereby alleviating renal fibrosis.

Background::Although overnight fasting is recommended prior to endoscopic retrograde cholangiopancreatography (ERCP), the benefits and safety of high-carbohydrate fluid diet (CFD) intake 2 h before ERCP remain unclear. This study aimed to analyze whether high-CFD intake 2 h before ERCP can be safe and accelerate patients’ recovery.Methods::This prospective, multicenter, randomized controlled trial involved 15 tertiary ERCP centers. A total of 1330 patients were randomized into CFD group ( n = 665) and fasting group ( n = 665). The CFD group received 400 mL of maltodextrin orally 2 h before ERCP, while the control group abstained from food/water overnight (>6 h) before ERCP. All ERCP procedures were performed using deep sedation with intravenous propofol. The investigators were blinded but not the patients. The primary outcomes included postoperative fatigue and abdominal pain score, and the secondary outcomes included complications and changes in metabolic indicators. The outcomes were analyzed according to a modified intention-to-treat principle. Results::The post-ERCP fatigue scores were significantly lower at 4 h (4.1 ± 2.6 vs. 4.8 ± 2.8, t = 4.23, P <0.001) and 20 h (2.4 ± 2.1 vs. 3.4 ± 2.4, t= 7.94, P <0.001) in the CFD group, with least-squares mean differences of 0.48 (95% confidence interval [CI]: 0.26–0.71, P <0.001) and 0.76 (95% CI: 0.57–0.95, P <0.001), respectively. The 4-h pain scores (2.1 ± 1.7 vs. 2.2 ± 1.7, t = 2.60, P = 0.009, with a least-squares mean difference of 0.21 [95% CI: 0.05–0.37]) and positive urine ketone levels (7.7% [39/509] vs. 15.4% [82/533], χ2 = 15.13, P <0.001) were lower in the CFD group. The CFD group had significantly less cholangitis (2.1% [13/634] vs. 4.0% [26/658], χ2 = 3.99, P = 0.046) but not pancreatitis (5.5% [35/634] vs. 6.5% [43/658], χ2 = 0.59, P = 0.444). Subgroup analysis revealed that CFD reduced the incidence of complications in patients with native papilla (odds ratio [OR]: 0.61, 95% CI: 0.39–0.95, P = 0.028) in the multivariable models. Conclusion::Ingesting 400 mL of CFD 2 h before ERCP is safe, with a reduction in post-ERCP fatigue, abdominal pain, and cholangitis during recovery.Trail Registration::ClinicalTrials.gov, No. NCT03075280.

Objective To explore the role of indole-3-propionic acid(IPA)in the pathogenesis of metabolic associated fatty liver disease(MAFLD)induced by high-fat diet(HFD)in order to reveal the role and related mechanism of adipose tissue metabolism in the process.Methods A mouse model of MAFLD was induced by HFD.Male C57BL/6J mice(6～7 weeks old)were randomly divided into control group(CON),HFD group,and HFD+IPA intervention group(HFD+IPA).The CON group was fed with control diet,and the HFD group and HFD+IPA group were fed with 60％of high-fat diet.The experiment period was 12 weeks,and IPA was administered at 20 mg/(kg·d)for 6 weeks starting from the 7th week.The body weight and food intake of each group were monitored weekly.After the intervention,the body composition of mice was detected by animal body composition analyzer.After the mice were euthanized,the morphological and structural changes in the liver and adipose tissues were observed by HE staining,the indicators relevant to lipid metabolism in the serum,l iver and adipose tissues were detected by automatic blood biochemical analyzer and biochemical kits,and the mRNA expression changes of lipid metabolism and inflammation related genes were detected by qRT-PCR.Results Compared with the CON group,the HFD group had significantly increased body weight and body fat percentage,obvious lipid deposition in the liver,obviously elevated serum alanine aminotransferase,aspartate aminotransferase,liver triglyceride and total cholesterol levels(P＜0.05),and raised mRNA levels of liver fatty acid transporter CD36(P＜0.05),while IPA intervention significantly reversed the above changes(P＜0.05).IPA intervention significantly inhibited the HFD-induced enlargement of visceral and brown fat cells,reduced the content of visceral adipose tissue(VAT)and serum level of free fatty acids(P＜0.05),and increased the mRNA expression levels of VAT lipolysis(HSL,CGI58),browning genes(Cidea,ND5,UCP1,Prdm16)(P＜0.05),as well as those of brown adipose tissue(BAT)lipolysis(HSL,ATGL)and fatty acid beta oxidation(Cpt1a,PPARα)genes(P＜0.05).Meanwhile,the mRNA levels of TNF-α,IL-1β,CXCL1 and CCL2 in VAT and BAT were decreased after IPA intervention(P＜0.05).Conclusion IPA can improve the occurrence of MAFLD induced by HFD,and its mechanism may be closely associated with its regulation of BAT and VAT morphology,and the mRNA expression of metabolic function and inflammation related genes.

Objective To explore the effect of chenodeoxycholic acid(CDCA)on the expression of glucagon-like peptide-1(GLP-1)in the intestine of mice induced by high-fat diet(HFD)through farnesoid X receptor(FXR),and investigate the related mechanism.Methods Forty C57BL/6 mice were divided into control group,HFD group,HFD+CDCA group,HFD+Z-Gug(FXR antagonist)group,and HFD+CDCA+Z-Gug group,with 8 animals in each group.During intervention for 8 weeks,body weight and 24-hour food intake were measured every week.At the 8th week,oral glucose tolerance test(OGTT)and intraperitoneal glucose tolerance test(IPGTT)were conducted.After the mice were sacrificed,the serum levels of GLu,TG,CHO,LDL-C and HDL-C were detected;the expression levels of GLP-1 and FXR in intestinal tissues were detected by immunofluorescence assay;and the mRNA levels of TNF-α,IL-6,IL-1β,Gcg and FXR were detected by RT-qPCR;the serum level of GLP-1 was detected by ELISA,and the proportion of intraepithelial lymphocytes(IELs)subsets and the expression of CD26/DPP4 were detected by flow cytometry.Results Compared with the control group,the HFD group had increased body weight,abnormal serum glucose and lipid metabolism,impaired oral glucose tolerance,and weakened secretion of gastrointestinal hormones(P＜0.05),enhanced FXR expression at mRNA and protein levels,declined Gcg mRNA level and GLP-1 secretion level(P＜0.05),increased mRNA levels of intestinal inflammatory factors TNF-α,IL-6 and IL-1β(P＜0.05),raised proportions of TCRαβ+IELs,TCRαβ+CD8αα+IELs,and TCRαβ+CD8αβ+IELs but reduced proportion of TCRγδ+IELs,and increased total CD26/DPP4 expression in IELs(P＜0.05).Compared with the HFD group,HFD+CDCA treatment resulted in significantly increased body weight,impaired oral glucose tolerance,decreased secretion of gastrointestinal hormones,increased FXR mRNA and protein expression,and decreased Gcg mRNA expression and GLP-1 secretion(P＜0.05);decreased proportions of TCRαβ+IELs,TCRαβ+CD8αα+IELs and TCRααβ+CD8αβ+IELs but increased proportion of TCRγδ+ cells in IELs,and increased expression of total CD26/DPP4 in IELs(P＜0.05),which were significantly improved after Z-Gug intervention(P＜0.05).Conclusion CDCA may inhibit the expression and secretion of GLP-1 in intestinal tissue by activating FXR,and reduce the secretion of GLP-1.At the same time,CDCA may inhibit the expression of related inflammatory factors,regulate the proportions of IELs subsets,up-regulate the expression level of CD26/DPP4,promote the degradation of GLP-1 and aggravate insulin resistance.

Objective To develop a nutritional formula on enhancing the endurance of heavy load exercise,and evaluate its efficacy comprehensively.Methods Sixty C57BL/6J male mice were randomly divided into control group(CON group)and low-,medium-and high-dose nutritional formula groups(LDF,MDF and HDF groups),with 15 mice in each group.Each group received intervention with nutritional formula at corresponding dose for 2 weeks,and underwent adaptive training and heavy load exercise in the 1 st and 2nd weeks,respectively.Exhaustion exercise time,skeletal muscle antioxidant indicators(SOD,MDA,PC and GSH),fatigue related indicators(serum URA,LDH and LA),muscle glycogen,and serum exercise injury related indicators(ALT,AST,CK and CK-MB)were measured and detected in the mice,and comprehensive evaluation was conducted according to relevant evaluation standards.Results The LDF group,MDF group and HDF group had significantly prolonged running exhaustion time than the CON group(P＜0.05),with the HDF group showing the greatest improvement(P＜0.05).Compared with the CON group,the activities of SOD and GSH in the skeletal muscles were significantly increased(P＜0.05),while the levels of MDA and PC in skeletal muscles were obviously decreased in the 3 doses of nutritional formula groups(P＜0.05).PAS staining of the skeletal muscles displayed that the glycogen content was significantly increased in the MDF group and the HDF group than the CON group(P＜0.05),and the highest increase was observed in the HDF group(P＜0.05).Biochemical test revealed that the levels of LDH,LA,ALT,AST,CK,and CK-MB were remarkably lower in the 3 doses of nutritional formula groups than the CON group(P＜0.05).Conclusion The nutritional formula can significantly improve the endurance and skeletal muscle antioxidant capacity in mice under heavy load exercise,and has anti-fatigue and-injury protection effects.This nutritional formula can be used to support physical fitness during heavy load endurance exercise.

Irritable bowel syndrome(IBS)is a common functional gastrointestinal disorder characterized by symptoms such as abdominal pain,diarrhea,constipation,and indigestion.Given its unclear etiology and pathogenesis,and the absence of specific biomarkers,clinical diagnosis and treatment of IBS continue to pose significant challenges.In recent years,metabolomics technology,known for its non-invasive,high-throughput,high-precision,and highly reproducible features,has been widely applied in the diagnosis,treatment,and prognosis of various diseases.Therefore,metabolomics technology is expected to offer novel insights and methodologies for the biological mechanism research,diagnosis,and treatment of IBS.This article reviews recent advancements in the application of metabolomics to IBS,exploring its potential value in the clinical diagnosis and treatment of children with this condition.

Biliary fibrosis (BF) is the result of pathological repair of bile tract injury, characterized by thickening and sclerosis of the bile duct wall and progressive stricture of the lumen, which may ultimately lead to serious adverse outcomes such as biliary obstruction, biliary cirrhosis, liver failure, and hepatobiliary malignancies. Current research describes BF as a pathological feature of certain bile tract diseases, lacking a systematic summary of its etiology, pathophysiology, molecular mechanisms, and treatment. BF is a common but easily neglected disease state in biliary system, which may promote the development and progression of hepatobiliary diseases through abnormal repair mechanism after pathological biliary tract injury. Based on the latest research progress from both domestic and international perspectives, the authors review the concept, clinical manifestation, etiology, pathogenesis, and therapeutic strategies of BF to provide a reference for clinical physicians.