The dynamic metabolic profile of Qi-Yu-San-Long decoction in rat urine using UPLC-QTOF-MS^(E) coupled with a post-targeted screening strategy

Qi-Yu-San-Long decoction(QYSLD)is a traditional Chinese medicine that has been clinically used in the treatment of non-small-cell lung cancer(NSCLC)for more than 20 years.However,to date,metabolicrelated studies on QYSLD have not been performed.In this study,a post-targeted screening strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight full information tandem mass spectrometry(UPLC-QTOF-MS^(E))was developed to identify QYSLD-related xenobiotics in rat urine.The chemical compound database of QYSLD constituents was established from previous research,and metabolites related to these compounds were predicted in combination with their possible metabolic pathways.The metabolites were identified by extracted ion chromatograms using predicted m/z values as well as retention time,excimer ions,and fragmentation behavior.Overall,85 QYSLD-related xenobiotics(20 prototype compounds and 65 metabolites)were characterized from rat urine.The main metabolic reactions and elimination features of QYSLD included oxidation,reduction,decarboxylation,hydrolysis,demethylation,glucuronidation,sulfation,methylation,deglycosylation,acetylation,and associated combination reactions.Of the identified molecules,14 prototype compounds and 58 metabolites were slowly eliminated,thus accumulating in vivo over an extended period,while five prototypes and two metabolites were present in vivo for a short duration.Furthermore,one prototype and five metabolites underwent the process of“appearing-disappearing-reappearing”in vivo.Overall,the metabolic profile and characteristics of QYSLD in rat urine were determined,which is useful in elucidating the active components of the decoction in vivo,thus providing the basis for studying its mechanism of action.

Residue behaviors and dietary risk of cyazofamid in turnip,onion and romaine lettuce assessed by a QuEChERS-LC-MS/MS methods

In this study, the dissipation, terminal residues and dietary risk of the cyazofamid residue in three globally consumed minor crops, turnip(Brassica rapa L.), onion(Allium cepa L.)and romaine lettuce(Lactuca sativa L.)were assessed by supervised field trials at 6 locations of China in 2020. A reliable QuEChERS-LC-MS/MS method was developed for simultaneous analysis of cyazofamid and its primary metabolite, 4-chloro-5-p-tolylimidazole-2-carbonitrile(CCIM). The average recoveries of cyazofamid and CCIM in turnip, onion and romaine lettuce were 79%–97% and 77%–99%, respectively. The half-lives of cyazofamid in these crops were 5.7–6.5, 5.3–8.7 and 5.8–6.5 days in turnip, onion and romaine lettuce, respectively, with the final cyazofamid residues at harvest all lower than limit of quantitation(LOQ, 0.01 mg/kg). The turnip roots, onion bulbs and romaine lettuce leaves grown in China under Good Agricultural Practices conditions and harvested 10, 14 and 5 days, respectively, after the cyazofamid SC(100 g/L)application, would be reasonably safe for consumption by the general population. The maximum residue limit(MRL)values of cyazofamid could be tentatively set as 10 mg/kg for turnip leaves, 0.3 mg/kg for turnip roots, 0.05 mg/kg for onion bulbs, and 10 mg/kg for romaine lettuce leaves.

A recombineering system for Bacillus subtilis based on the native phage recombinase pair YqaJ/YqaK

Bacillus subtilis plays an important role in fundamental and applied research,and it has been widely used as a cell factory for the production of enzymes,antimicrobial materials,and chemicals for agriculture,medicine,and industry.However,genetic manipulation tools for B.subtilis have low efficiency.In this work,our goal was to develop a simple recombineering system for B.subtilis.We showed that genome editing can be achieved in B.subtiliis 1A751 through co-expression of YqaJ/YqaK,a native phage recombinase pair found in B.sub-tilis 168,and the competence master regulator ComK using a double-stranded DNA substrate with short ho-mology arms(100 bp)and a phosphorothioate modification at the 5′-end.Efficient gene knockouts and large DNA insertions were achieved using this new recombineering system in B.subtilis 1A751.As far as we know,this is the first recombineering system using the native phage recombinase pair YqaJ/YqaK in B.subtilis.In conclusion,this new recombineering system provides a simple and fast tool for genetic manipulation of B.sub-tilis,and it will promote studies of genome function,construction of production strains,and genome mining in B.subtilis.

Transplantation of placenta-derived mesenchymal stem cells reduces hypoxic-ischemic brain damage in rats by ameliorating the inflammatory response

Hypoxic-ischemic brain damage(HIBD)is a common cause of infant death.The purpose of our research was to explore the immunoregulatory mechanism of placenta-derived mesenchymal stem cells(PD-MSCs)in HIBD treatment.Seven-day-old rat pups were randomly divided into HIBD,PD-MSC,fibroblast,and control groups.Forty-eight hours after HIBD induction,cells at a density of 5×104 cells/10μl were injected into the cerebral tissue in the PD-MSC and fibroblast groups.The TNF-α,interleukin-17(IL-17),interferon-γ(IFN-γ),and IL-10 levels were detected through quantitative real-time polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).Regulatory T cell(Tregs)populations were detected through flow cytometry,and forkhead box P3(Foxp3)was measured through western blot analysis.Behavioral tests and gross and pathological examinations showed that PD-MSC treatment exerted significantly stronger neuroprotective effects than the other treatments.The expression levels of pro-inflammatory cytokines were substantially upregulated after HI injury.Compared with fibroblast treatment,PD-MSC treatment inhibited the production of pro-inflammatory cytokines and increased the production of IL-10 in the ischemic hemispheres and peripheral blood serum(all P<0.01).Flow cytometry results showed a notable increase in the number of Tregs within the spleen of the HIBD group.Moreover,the number of Tregs and the Foxp3 expression levels were higher in the PD-MSC treatment group than in the HIBD and fibroblast groups(all P<0.01).Our research suggests that the mechanism of PD-MSC treatment for HIBD partially involves inflammatory response suppression.

Cascade-Cas3 facilitates high-accuracy genome engineering in Pseudomona s using phage-encoded homologous recombination

Phage-encoded homologous recombination(PEHR)is an efficient tool for bacterial genome editing.We previously developed and utilized a Pseudomonas-specific PEHR system.However,when using the PEHR system for Pseu-domonas genome editing,false positives can be a problem.In this study,we combined a compact Cascade-Cas3 system from P.aeruginosa(PaeCas3c)with a Pseudomonas-specific PEHR system,and the results of our recom-bineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy.

The emerging role of recombineering in microbiology

Recombineering is a valuable technique for generating recombinant DNA in vivo,primarily in bacterial cells,and is based on homologous recombination using phage-encoded homologous recombinases,such as Red βγ from the lambda phage and RecET from the Rac prophage.The recombineering technique can efficiently mediate homol-ogous recombination using short homologous arms(∼50 bp)and is unlimited by the size of the DNA molecules or positions of restriction sites.In this review,we summarize characteristics of recombinases,mechanism of recombineering,and advances in recombineering for DNA manipulation in Escherichia coli and other bacteria.Furthermore,the broad applications of recombineering for mining new bioactive microbial natural products,and for viral mutagenesis,phage genome engineering,and understanding bacterial metabolism are also reviewed.

Targeting lactate dehydrogenase A (LDHA) exerts antileukemic effects on T-cell acute lymphoblastic leukemia

Background:T-cell acute lymphoblastic leukemia(T-ALL)is an uncommon and aggressive subtype of acute lymphoblastic leukemia(ALL).In the serum of T-ALL patients,the activity of lactate dehydrogenase A(LDHA)is increased.We proposed that targeting LDHA may be a potential strategy to improve T-ALL outcomes.The current study was conducted to investigate the antileukemic effect of LDHA gene-targeting treatment on T-ALL and the underlying molecular mechanism.Methods:Primary T-ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate.MTT,colony formation,apoptosis,and cell cycle assays were performed to investigate the effects of oxamate on T-ALL cells.Quantitative real-time PCR(qPCR)and Western blotting analyses were applied to determine the related signaling pathways.A mitochondrial reactive oxygen species(ROS)assay was performed to evaluate ROS production after T-ALL cells were treated with oxamate.A T-ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene-editing technology,and then TUNEL,Western blotting,and T-ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T-ALL transgenic zebrafish.Results:Oxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells.It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production(all P<0.001).Blocking LDHA significantly decreased the gene and protein expression of c-Myc,as well as the levels of phosphorylated serine/threonine kinase(AKT)and glycogen synthase kinase 3 beta(GSK-3β)in the phosphatidylinositol 3′-kinase(PI3K)signaling pathway.LDHA gene knockdown delayed disease progression and down-regulated c-Myc mRNA and protein expression in T-ALL transgenic zebrafish.Conclusion:Targeting LDHA exerted an antileukemic effect on T-ALL,representing a potential strategy for T-ALL treatment.

Biochemical characterization of a polyethylene terephthalate hydrolase and design of high-throughput screening for its directed evolution

Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.