Objective:To explore the reform practice and teaching effect of flipped classroom combined with case-based learning (CBL) in the undergraduate teaching of International Classification of Diseases.Methods:The undergraduates of Chongqing Medical University majoring in information management and information system from the classes of 2018 and 2019 were selected as the control group and the experimental group, respectively, and the undergraduates in the control group received lecture-based learning, while those in the experimental group received flipped classroom combined with CBL. After teaching, theoretical assessment and practical skill assessment were performed to evaluate learning effect. R3.6.3 was used to perform statistical analyses; the t-test or the rank-sum test (Mann-Whitney U test) was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data. Results:There were no significant differences between the two groups in the general information such as age and sex distribution ( t=-1.22, P=0.227; χ2=1.77, P=0.183). There was no significant difference in theoretical assessment score between the two groups [(78.84±8.97) vs. (76.01±8.65), P=0.140]. Compared with the control group, the experimental group had significantly better results in the correct rate of ICD coding [(94.34±3.22)% vs. (91.36±2.79)%, P=0.006] and the number of coded copies per person per day [15.41 (7.90, 40.97) vs. 7.22 (2.33, 8.83), P=0.006], as well as a better level of practical skills. Conclusions:Flipped classroom combined with CBL can help to enhance the hands-on ability to solve problems among students, thereby improving the overall teaching effect.

Glyceryl phenylbutyrate(GPB)serves as a long-term management medication for Ornithine transcarbamylase deficiency(OTCD),effectively controlling hyperammonemia,but there is a lack of experience in using this medicine in China.This article retrospectively analyzes the case of a child diagnosed with OTCD at Shanghai Children's Medical Center,Shanghai Jiao Tong University School of Medicine,including a review of related literature.After diagnosis,the patient was treated with GPB,followed by efficacy follow-up and pharmacological monitoring.The 6-year and 6-month-old male patient exhibited poor speech development,disobedience,temper tantrums,and aggressive behavior.Blood ammonia levels peaked at 327 μmol/L;urine organic acid analysis indicated elevated uracil levels;cranial MRI showed extensive abnormal signals in both cerebral hemispheres.Genetic testing revealed de novo mutation in the OTC gene(c.241T＞C,p.S81P).Blood ammonia levels were approximately 43,80,and 56 μmol/L at 1,2,and 3 months after starting GPB treatment,respectively.During treatment,blood ammonia was well-controlled without drug-related adverse effects.The patient showed improvement in developmental delays,obedience,temperament,and absence of aggressive behavior.

Diabetic kidney disease(DKD)is one of the serious microvascular complications of diabetes.Immune disorder-mediated chronic inflammation is an essential mechanism underlying the occurrence of DKD.Macrophages,as important participants in the immune-inflammatory response,can polarize into the pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype under different microenvironments.The two are functionally antagonistic to each other.They can be transformed into each other in different developmental stages of DKD,which is similar to the connotation of yin and yang in the theory of"yin-yang theory"in TCM,such as restriction of opposites,mutual root and mutual use,balance of growth and decline,and mutual transformation.The imbalance of M1 and M2 macrophages is an important factor leading to DKD inflammatory response and renal fibrosis.Therefore,based on the yin-yang theory in TCM,this article clarified the effects of macrophage polarization on DKD,and proposed that the basic treatment method is to"tonify deficiency and damage excess"to adjust the polarization of macrophages and restore the balance of yin and yang,in order to provide ideas for the clinical treatment of DKD.

ObjectiveTo explore the syndromes and mechanisms of depression induced by maternal separation （MS） combined with chronic restraint stress （RS） in mice. MethodOn postnatal day 0 （PD0）， the offspring mice were randomized into a blank group （NC） and a modeling group. The mouse model of depression was established by MS+RS for 21 days. After removal of female mice on PD21， the modeled mice were randomized into model， Wenyang， Jieyu， Wenyang Jieyu， and fluoxetine groups， with 15 mice in each group. The sucrose preference， tail suspension， and open field tests were carried out to evaluate the anxiety and depression-like behavior in mice. Enzyme-linked immunosorbent assay was used to measure the adrenocorticotrophic hormone （ACTH） and corticosterone （CORT） levels in mouse plasma. High performance liquid chromatography-electrochemical detector was used to determine the content of monoamine neurotransmitters in the hippocampus. Real-time fluorescence quantitative polymerase chain reaction was employed to determine the mRNA levels of genes in the 5-hydroxytryptamine （5-HT） system， hypothalamic-pituitary-adrenal （HPA） axis， and brain-derived neurotrophic factor （BDNF） signaling pathway in the hippocampus. Immunohistochemistry was employed to determine the expression levels of proteins in the 5-HT system and HPA axis in the hippocampus. The Simple Western system was used to determine the protein levels of BDNF and tyrosine kinase receptor B （TrkB） in the hippocampus. ResultCompared with the NC group， the model group exhibited depression-like behavior， which was significantly relieved by Wenyang Jieyu prescription and fluoxetine. Compared with the NC group， the model group showed elevated levels of CORT and ACTH in the plasma （P<0.01）， which， however， were lowered by Wenyang Jieyu prescription and fluoxetine （P<0.05， P<0.01）. Compared with the NC group， the model group showed inhibited expression of neurotransmitters in the hippocampus （P<0.05， P<0.01）， while Wenyang Jieyu prescription and fluoxetine restored the expression of neurotransmitters （P<0.05， P<0.01）. Compared with NC group， the model group showed inhibition of the 5-HTergic nerve and abnormal activation of the HPA axis， and Wenyang Jieyu prescription and fluoxetine regulated the abnormal state of the 5-HTergic nerve and HPA axis. Compared with NC group， the modeling down-regulated the mRNA and protein levels of BDNF and TrkB in the hippocampus （P<0.05， P<0.01）， which， however， were recovered in Wenyang， Jieyu， Wenyang Jieyu， and fluoxetine groups （P<0.05， P<0.01）. ConclusionThe mouse model of depression induced by MS+RS may present the syndrome of Yang deficiency and liver depression. Wenyang Jieyu prescription may increase the content of hippocampal neurotransmitters by regulating the 5-HT system and the BDNF signaling pathway mediated by the HPA axis， thereby alleviating depression-like behavior in mice.

ObjectiveTo explore the effects of Wenyang Jieyu prescription （WJP） on neuroinflammation and synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomized into a control group and a modeling group. Maternal separation combined with restraint stress was employed to establish the mouse model of depression. After the removal of female mice， the modeled mice were randomized into model， Wenyang prescription （5.85 g·kg^-1）， Jieyu prescription （12.03 g·kg^-1）， WJP （16.71 g·kg^-1）， and fluoxetine （2.6 mg·kg^-1） groups on the weaning day （PD21）， with 15 mice in each group. The mice were administrated with corresponding drugs mixed with the diet from PD21 to PD111. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then carried out to evaluate the depression state， memory， and learning ability of the mice. Immunohistochemistry （IHC） was employed to observe the ionized calcium-binding adapter molecule-1 （Iba-1） in hippocampal microglia. High performance liquid chromatography （HPLC） was employed to measure the content of noradrenaline （NE） and epinephrine （E） in the hippocampus. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the content of interleukin （IL）-18 and IL-1β in the hippocampus. Western blot was employed to determine the protein levels of NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， cysteine aspartate-specific protease-1 （Caspase-1）， IL-1β， synaptophysin （Syn）， and postsynaptic density 95 （PSD95）. ResultCompared with control group， the model group showed decreased sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. The microglia in the model group presented amoeba-like appearance， the Iba1 increased. Moreover， the model group showed decreased content of NE and E （P<0.01）， elevated levels of IL-1β and IL-18 （P<0.01）， down-regulated protein levels of PSD95 and Syn （P<0.05， P<0.01）， and up-regulated protein levels of NLRP3， ASC， Caspase-1， and IL-1β （P<0.05， P<0.01）. Compared with model group， WJP and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. They recovered the microglia and the Iba1 decreased. Moreover， the drugs increased the content of NE and E （P<0.05， P<0.01）， lowered the levels of IL-1β and IL-18 （P<0.01）， up-regulated the protein levels of PSD95 and Syn （P<0.01）， down-regulated the protein levels of NLRP3， ASC， Caspase-1， and IL-1β （P<0.05， P<0.01）. ConclusionWJP can treat the depressive behavior induced by maternal separation combined with restraint stress in mice， with the performance outperforming Wenyang prescription and Jieyu prescription. It may alleviate the neuroinflammation induced by microglia and improve the synaptic plasticity by regulating the NLRP3 pathway and increasing neurotransmitters in the hippocampus.

ObjectiveTo explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomly assigned into a control group （n=10） and a modeling group （n=50）. Maternal separation combined with restraint stress was adopted to establish the mouse model of depression， and the modeled mice were randomized into model， Wenyang prescription， Jieyu prescription， Wenyang Jieyu prescription， and fluoxetine groups （n=10） on the weaning day （PD21）. From PD21 to PD111， the mice were fed with the diets mixed with corresponding medicines. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then conducted to evaluate the depression， memory， and learning abilities of mice. Immunohistochemistry （IHC） was employed to measure the atomic absorbance （AA） of postsynaptic density protein 95 （PSD95） in the hippocampus. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of hippocampal neurons. Western blot was employed to determine the protein levels of brain-derived neurotrophic factor （BDNF）， phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B （p-TrkB/TrkB）， phosphorylated protein kinase B/protein kinase B （p-Akt/Akt）， phosphorylated mammalian target of rapamycin/mammalian target of rapamycin （p-mTOR/mTOR）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cysteinyl aspartate-specific proteinase-3 （Caspase-3）， synaptophysin （Syn）， and PSD95. ResultCompared with the control group， the modeling decreased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.01）. Furthermore， it decreased the expression of PSD95， increased the neuron apoptosis in the hippocampus （P<0.01）， down-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and up-regulated the protein levels of Bax and Caspase-3 （P<0.05） in the hippocampus. Compared with the model group， Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. Moreover， the drugs increased the expression of PSD95， reduced the neuron apoptosis （P<0.01）， up-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and down-regulated the protein levels of Bax and Caspase-3 （P<0.01）. ConclusionWenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice. It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.

AIM To investigate the protective effects and the mechanism of the Liuwei Dihuang Pills on mouse brain microvascular endothelial(bEnd.3)cells damaged by β-Amyloid protein1-40(Aβ1-40).METHODS CCK8 method was used to detect the effects of Aβ1-40 and medicated serum of Liuwei Dihuang Pills(MSLDP)on cell activity,and to screen the appropriate concentration.bEnd.3 cells of the control group,the Aβ1-40 group,the MSLDP+Aβ1-40 group and the MSLDP group had their low density lipoprotein-associated protein 1(LRP1),receptor for advanced glycation end products(RAGE),matrix metalloproteinase-2(MMP-2),MMP-9,scaffold protein zonule protein-1(ZO-1)detected by Western blot.bEnd.3 cells assigned into the control group,the Aβ1-40 group,the FPS-ZM1(RAGE inhibitor)+Aβ1-40 group and the FPS-ZM1+Aβ1-40+MSLDP group had their expressions of RAGE,MMP-9,MMP-2 and ZO-1 detected by Western blot as well.RESULTS The cell activity of bEnd.3,was dose-dependently decreased by Aβ1-40(P<0.01),but was protected by MSLDP(P<0.05,P<0.01).And 10 μmol/L Aβ1-40 and 10%MSLDP were selected for subsequent experiments.Compared with the control group,the Aβ1-40 group displayed increased protein expressions of RAGE,MMP-2 and MMP-9(P<0.01),decreased protein expressions of LRP1,ZO-1 and BDNF(P<0.05,P<0.01),and decreased fluorescence intensities of LRP1 and ZO-1(P<0.01).Compared with the Aβ1-40 group,the MSLDP group shared decreased expressions of RAGE,MMP-2,MMP-9 proteins and RAGE fluorescence intensity(P<0.05,P<0.01),and increased expressions of LRP1,ZO-1 and BDNF proteins,and the fluorescence intensity of LRP1 and ZO-1(P<0.05,P<0.01);the Aβ1-40+FPS-ZM1 group displayed decreased protein expressions of MMP-2,MMP9 and RAGE(P<0.05,P<0.01),and increased ZO-1 protein expression(P<0.05);and the Aβ1-40+FPS-ZM1+ MSLDP group displayed an even more decreased protein expressions of MMP-2,MMP9 and RAGE(P<0.01),increased ZO-1 protein expression(P<0.01)due to the the combination use of FPS-ZM1 and MSLDP.CONCLUSION Liuwei Dihuang Pills can protect the tight junction of bEnd.3 injured by Aβ1-40 and neurovascular units from Alzheimer's disease by alleviating the dysfunction of the blood-brain barrier via RAGE-mediated MMP-2/MMP-9 pathway inhibition.

Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate (UDP)-glucose, which serves as a bioactive glycosyl donor for various metabolic processes. In this study, a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa, a traditional Chinese medicine known for its rich content of diverse glycosides, based on transcriptome analysis results. The open reading frame of CtSus was 2 418 bp long encoding 805 amino acids. Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein. Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order. Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1, a characterized glycosyltransferase enzyme. The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1. The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^TM TF expression vector. In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDP-glucose in the presence of sucrose and UDP. Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C. tubulosa and the suspension cell cultures of C. tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides, respectively. Furthermore, key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results. Overall, our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C. tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.
Biological Products/chemistry* ; Catalysis ; Alkaloids ; Biocatalysis

To develop antimicrobials against Staphylococcus aureus by high throughput screening of drug library. The type of this study is experimental research. The clinical isolates of S. aureus were collected from the sputum samples of respiratory inpatient department of the Third Xiangya Hospital of Central South University. The anti-planktonic cells growth inhibition activity of FDA-approved drugs library (including 1 573 molecules) was assessed by building a planktonic cells screening platform; The biofilm inhibitory effect of the FDA-approved drugs was detected by building a biofilm screening platform combined with crystal violet staining; Minimal inhibitory concentrations of the selected hits were determined by broth microdilution assay. Finally, the cytotoxicity of the selected hits was detected by CCK-8 assay. The results showed that 218 hits were exhibited effective growth inhibitory effects against S. aureus by setting the concentrations of the molecules in the FDA-approved library to 100 μmol/L. These selected molecules are mainly anti-infective drugs, accounting for 118 hits; Followed by anti-cancer drugs, anti-inflammatory/-immune drugs, neurological drugs, cardiovascular drugs, endocrine drugs, and metabolic disease drugs, which accounts for 40, 19, 12, 9, 8, and 3 hits; Other unclassified drugs accounts for 9 hits. The top 10 hits exhibiting anti-planktonic cells activity against S. aureus were mainly including antitumor drugs, followed by neurological drugs and unclassified drugs like vitamin K3 with the inhibition rate of 99.65%-100%. Similarly, the top 10 hits showing biofilm inhibitory effects against S. aureus were also mainly including antitumor drugs, followed by neurological drugs and anti-inflammatory/-immune drugs with the inhibition rate of 50.22%-92.95%. The minimal inhibitory concentration (MIC) of the 51 hits by second round screening was determined by micro-dilution assay, which mainly include the antitumor drugs, cardiovascular drugs, endocrine drugs, anti-inflammatory/-immune drugs, metabolic disease drugs, neurological drugs and other unclassified drugs accounted for 22, 5, 3, 9, 2, 5 and 5 hits, respectively, with the MICs of 1.56-50 μmol/L, 6.25-25 μmol/L, 6.25-25 μmol/L, 0.2-50 μmol/L, 25-50 μmol/L, 1.56-50 μmol/L and 0.1-12.5 μmol/L, respectively. In conclusion, the minimum inhibitory concentrations of small molecules screened through high-throughput assay are at the level of micromolar with strong drug development potential and high modifiability. The high effective anti-planktonic cells and anti-biofilm activity by these molecules are expected to provide new ideas for the development of new antimicrobials against S. aureus.
Humans ; Staphylococcus aureus ; Anti-Bacterial Agents/pharmacology* ; High-Throughput Screening Assays ; Staphylococcal Infections ; Anti-Infective Agents/pharmacology* ; Microbial Sensitivity Tests ; Biofilms ; Antineoplastic Agents/pharmacology* ; Anti-Inflammatory Agents/pharmacology* ; Cardiovascular Agents/pharmacology* ; Metabolic Diseases