Human carboxylesterase 2A (hCES2A) plays pivotal roles in prodrug activation and hydrolytic metabolism of ester-bearing chemicals. Targeted inhibition of intestinal hCES2A represents a feasible strategy to mitigate irinotecan-triggered gut toxicity (ITGT), but the orally active, selective, and efficacious hCES2A inhibitors are rarely reported. Here, a novel drug-like hCES2A inhibitor was developed via three rounds of structure-based drug design (SBDD) and structural optimization. Initially, donepezil was identified as a moderate hCES2A inhibitor from 2000 US Food and Drug Administration (FDA)-approved drugs. Following two rounds of SBDD and structural optimization, a donepezil derivative (B7) was identified as a strong reversible hCES2A inhibitor. Subsequently, nine B7 carbamates were rationally designed, synthesized and biologically assayed. Among all synthesized carbamates, C3 showed the most potent time-dependent inhibition on hCES2A (IC₅₀ = 0.56 nmol/L), excellent specificity and favorable drug-like properties. C3 could covalently modify the catalytic serine of hCES2A with high selectivity, while this agent also showed favorable safety profiles, high intestinal exposure, and impressive effects for ameliorating ITGT in both human intestinal organoids and tumor-bearing mice. Collectively, this study showcases a rational strategy for developing drug-like and serine-targeting covalent inhibitors against target serine hydrolase(s), while C3 emerges as a promising orally active drug candidate for ameliorating ITGT.

Iron metabolism is a critical factor in tumorigenesis and development. Although TP53 mutations are prevalent in glioblastoma (GBM), the mechanisms by which TP53 regulates iron metabolism remain elusive. We reveal an imbalance iron homeostasis in GBM via TCGA database analysis. TP53 mutations disrupted iron homeostasis in GBM, characterized by elevated total iron levels and reduced ferritin (FTH). The gain-of-function effect triggered by TP53 mutations upregulates itchy E3 ubiquitin-protein ligase (ITCH) protein expression in astrocytes, leading to FTH degradation and an increase in free iron levels. TP53-mut astrocytes were more tolerant to the high iron environment induced by exogenous ferric ammonium citrate (FAC), but the increase in intracellular free iron made them more sensitive to Erastin-induced ferroptosis. Interestingly, we found that Erastin combined with FAC treatment significantly increased ferroptosis. These findings provide new insights for drug development and therapeutic modalities for GBM patients with TP53 mutations from iron metabolism perspectives.
Ferroptosis/drug effects* ; Humans ; Iron/metabolism* ; Glioblastoma/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; Homeostasis/physiology* ; Ferritins/metabolism* ; Brain Neoplasms/genetics* ; Mutation ; Astrocytes/drug effects* ; Cell Line, Tumor ; Piperazines/pharmacology* ; Quaternary Ammonium Compounds/pharmacology* ; Ferric Compounds

Objective To investigate the effects of salidroside (Sal) on mitochondrial autophagy in ischemic cardiomyopathy (ICM) rats by modulating the PTEN-induced kinase 1 (PINK1)/E3 ubiquitin ligase (Parkin) signaling pathway. Methods Thirty SD rats were randomly divided into control group, model group, low-dose Sal group, high-dose Sal group, mitochondrial autophagy inhibitor (Mdivi-1) group and high-dose Sal + Mdivi-1 group, with five rats in each group. The ICM rat model was established. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameters (LVIDs) were analyzed. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine kinase (CK) and N-terminal pro-brain natriuretic peptide (NT-proBNP). Hematoxylin and eosin (HE) staining was performed to observe pathological changes in myocardial tissue, and transmission electron microscopy was used to examine mitochondrial structure. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. Western blot analysis was conducted to determine the expression levels of microtubule-associated protein light chain 3 (LC3), Beclin1, p62, PINK1 and Parkin in myocardial tissue. Results Compared with the control group, the model group showed more severe myocardial tissue and mitochondrial damage. The LVEF, LVFS, SOD and p62 levels in the model group were significantly lower than those in the control group, while the LVIDd, LVIDs, LDH, cTnI, CK, NT-proBNP, ROS, MDA, LC3II/LC3I, Beclin1, PINK1 and Parkin levels were significantly higher (P < 0.05). Compared with the model group, the low-dose and high-dose Sal groups exhibited reduced myocardial and mitochondrial damage. The LVEF, LVFS, SOD, LC3II/LC3I, Beclin1, PINK1 and Parkin levels in the low-dose and high-dose Sal groups were significantly higher than those in the model group, while the LVIDd, LVIDs, LDH, cTnI, CK, NT-proBNP, ROS, MDA and p62 levels were significantly lower (P < 0.05). Compared with the model group, the Mdivi-1 group showed more severe myocardial and mitochondrial damage. The LVEF, LVFS, SOD, LC3II/LC3I, Beclin1, PINK1 and Parkin levels in the Mdivi-1 group were significantly lower than those in the model group, while the LVIDd, LVIDs, LDH, cTnI, CK, NT-proBNP, ROS, MDA and p62 levels were significantly higher (P < 0.05). Compared with the low-dose Sal group, the high-dose Sal group demonstrated further improvement in myocardial and mitochondrial damage. The LVEF, LVFS, SOD, LC3II/LC3I, Beclin1, PINK1 and Parkin levels in the high-dose Sal group were significantly higher than those in the low-dose Sal group, while the LVIDd, LVIDs, LDH, cTnI, CK, NT-proBNP, ROS, MDA and p62 levels were significantly lower (P < 0.05). Mdivi-1 significantly inhibited mitochondrial autophagy and suppressed the activation of the PINK1/Parkin signaling pathway and the improvement in cardiac function induced by Sal (P < 0.05). Conclusion Sal may improve myocardial injury in ICM rats by activating the PINK1/Parkin signaling pathway and promoting mitochondrial autophagy.

Toxoplasma gondii, an intracellular protozoan parasite that infects one-third of the world’s population, has been reported to hijack host cell apoptotic machinery and promote either an anti- or proapoptotic program depending on the parasite virulence and load and the host cell type. However, little is known about the regulation of human FHs 74 small intestinal epithelial cell viability in response to T. gondii infection. Here we show that T. gondii RH strain tachyzoite infection or ESP treatment of FHs 74 Int cells induced apoptosis, mitochondrial dysfunction and ER stress in host cells. Pretreatment with 4-PBA inhibited the expression or activation of key molecules involved in ER stress. In addition, both T. gondii and ESP challenge-induced mitochondrial dysfunction and cell death were dramatically suppressed in 4-PBA pretreated cells. Our study indicates that T. gondii infection induced ER stress in FHs 74 Int cells, which induced mitochondrial dysfunction followed by apoptosis. This may constitute a potential molecular mechanism responsible for the foodborne parasitic disease caused by T. gondii.

OBJECTIVETo clone calmodulin (CaM) gene in Eleutherococcus senticosus, and study the effect of endophytic fungi on expression amount of CaM gene.

METHODThe CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends (RACE). The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods. The expression amount of CaM gene affected of endophytic fungus P116-1a, P116-1b, P1094 and P312-1 was detected by RT-PCR.

RESULTThe full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids. The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota. RT-PCR results showed that endophytic fungus improved CaM expression amount significantly (P<0.05). The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P1094, up to 2.96 times of the control.

CONCLUSIONThe CaM gene of E. senticosus was successfully cloned for the first time. The results demonstrated that endophytic fungus of E. senticosus improved CaM expression amount significantly.

Calmodulin ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Eleutherococcus ; classification ; genetics ; metabolism ; microbiology ; Endophytes ; physiology ; Fungi ; physiology ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus.

METHODTotal RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy.

RESULTSequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices.

CONCLUSIONThe two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.

Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Eleutherococcus ; enzymology ; genetics ; Isoenzymes ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Squalene Monooxygenase ; classification ; genetics

OBJECTIVETo analyze the effect of endophytic fungi on expression amount of key enzyme genes SS (squalene synthase gene), SE (squalene epoxidase gene) and bAS (beta-amyrin synthase gene) in saponin biosynthesis and saponins content in Eleutherococcus senticosus.

METHODWound method was used for back meeting the endophytic fungi to E. senticosus. With GAPDH as internal control gene, the expression of key enzyme genes was detected by real time PCR method. E. senticosus saponins content was measured by spectrophotometry method.

RESULTWhen wound method back meeting P116-1a and P116-1b after 30 d, the expression content of SS improved significantly (P < 0.05), however the back meeting of P109-4 and P312-1 didnt change the expression of SS. After that SS expression showed reduction-equality-reduction varying trend. Thirty days after back meeting P312-1, the expression content of SE improved significantly (P < 0.05). Ninty days after back meeting P116-1b and P312-1, the expression content of SE improved significantly to 130%,161%, respectively (P < 0.05). After 120 d, back meeting four endophytic fungi, the expression of SE were significantly higher than the control (P < 0.05). Back meeting four endophytic fungi form 60 d to 120 d, the expression of bAS was significantly higher than the control (P < 0.05). The back meeting four endophytic fungi improved E. senticosus saponins content significantly (P < 0.05).

CONCLUSIONEndophytic fungi P116-1a, P116-1b, P1094 and P312-1 significantly effected the expression of key enzyme genes SS, SE and bAS and then affected E. senticosus saponins content. Among the genes, bAS was key target gene.

Eleutherococcus ; chemistry ; metabolism ; microbiology ; Endophytes ; physiology ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; Fungi ; physiology ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Intramolecular Transferases ; genetics ; Saponins ; analysis ; biosynthesis ; Squalene Monooxygenase ; genetics

OBJECTIVETo clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene.

METHODThe FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR.

RESULTThe full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05).

CONCLUSIONThe FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.

Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; Conserved Sequence ; Eleutherococcus ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Geranyltranstransferase ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Conformation

BACKGROUND: There are many in vitro selection method of sperm, and swim-up and density gradient centrifugation are commonly used. It remains unclear which method minimizes bad stimulation to the sperm and select sperm with high fertilization potential. OBJECTIVE: To evaluate the effectiveness of swim-up and gradient centrifugation preparation techniques on intrauterine insemination (IUI).METHODS: A computer-based online search of Cochrane Library, PubMed, EMBASE databases was performed, and some related journals were manually searched for related articles published between January 1966 and February 2009. The quality of included randomized controlled trials (RCT) and q-randomized trials (Q-RCT) was evaluated and Meta-analysis was conducted by the Cochrane Collaboration's software RevMan5.0. Experts.RESULTS AND CONCLUSION: A total of 6 studies were included, involving 4 RCTs and 2 Q-RCTs. A total of 486 patients (1 099 IUI cycles) were enrolled. The Meta-analysis indicated that there was no difference between swim-up and gradient centrifugation preparation techniques for the IUI in terms of cycle pregnancy rates [OR = 1.11, 95%CI(0.8,1.55)], miscarriage rates [OR = 0.31, 95%CI(0.09,1.04)], sperm count [the weight mean difference (WMD) =-0.89, 95%CI(-14.17,12.38)], sperm motility [WMD = -2.31, 95%CI(-7.27,2.65)]. There is insufficient evidence to confirm which is the best method in the two specific preparation techniques. The quality of study methods should be improved. And more measure parameters should be included when comparing it before or after treatment, such as sperm motility, sperm count, sperm function.

Magnetic resonance spectroscopy(MRS)is an advanced medical imaging technique that can provide more accurate physiological and biochemical information than an MRI.MRS has more advantages in displaying abnormal physiological signs than the conventional MRI.A key problem in radiation therapy is the definition of the tumor target volume.MRS can define the tumor margin and range as well as predict the immediate response or side effects to radiotherapy.This paper summarized the role of MRS in tumor volume definition and clinical evaluation in radiation therapy.