Expression of Serum Vaspin and SFRP5 in Children with Idiopathic Nanosomia and Their Diagnostic Value / 现代检验医学杂志

Objective To explore the expression level of visceral adipose tissue-derived serine protease inhibitor(Vaspin)and secreted frizzled-related protein5(SFRP5)in the serum of children with idiopathic short stature(ISS)and its diagnostic value.Methods 70 children with ISS diagnosed in the First Hospital of Zhangjiakou from December 2021 to February 2023 were selected as the disease group,while 72 healthy volunteer children who underwent physical examination were collected as the control group.Immunoluminescence was applied to detect the expression level of VASPIN,Enzyme-linked immunosorbent assay(ELISA)was applied to detect the expression level of SFRP5 the clinical data of children in two groups were analyzed.Receiver operating characteristic(ROC)curve was applied to analyze the diagnostic value of serum Vaspin and SFRP5 for ISS,multivariate Logistic regression was used to analyze the influencing factors of ISS.Results Compared with the control group,the serum Vaspin level in the disease group was obviously increased(2.89±0.92 ng/ml vs 1.81±0.42 ng/ml),while the SFRP5 level was obviously reduced(10.22±2.84 pg/ml vs 13.21±3.53 pg/ml),the differences were statistically significant(t=9.040,5.552,all P＜0.05).The weight,height,body mass index(BMI)and proportion of sexual development stage II～V of children in the disease group were obviously lower than those in the control group,and the differences were statistically significant(t=7.687,6.330,5.559,7.024,all P＜0.05).The area under ROC curve showed that the AUC of Vaspin and SFRP5 and their combined detection in the diagnosis of ISS were 0.768,0.849 and 0.925,respectively,the combined diagnosis efficacy of Vaspin and SFRP5 was better than that of serum Vaspin and SFRP5 alone(Z =3.829,P＜0.001;Z =2.141,P=0.032).Multivariate Logistic regression analysis showed that BMI(OR=0.508,95%CI:0.260～0.991),Vaspin(OR=3.458,95%CI:1.125～10.631)and SFRP5(OR=0.378,95%CI:0.153～0.935)were the influencing factors for ISS(all P＜0.05).Conclusion The expression level of Vaspin in the serum of children with ISS is obviously increased,while the expression level of SFRP5 is obviously reduced.The two are influencing factors of ISS,and the combined detection of their expression levels has certain value in the diagnosis of ISS.

Genetic diversity in the partial sequence of the HIV-1 gag gene among people living with multidrug-resistant HIV-1 infection

ABSTRACT The group-specific antigen (gag) plays a crucial role in the assembly, release, and maturation of HIV. This study aimed to analyze the partial sequence of the HIV gag gene to classify HIV subtypes, identify recombination sites, and detect protease inhibitor (PI) resistance-associated mutations (RAMs). The cohort included 100 people living with HIV (PLH) who had experienced antiretroviral treatment failure with reverse transcriptase/protease inhibitors. Proviral HIV-DNA was successfully sequenced in 96 out of 100 samples for gag regions, specifically matrix (p17) and capsid (p24). Moreover, from these 96 sequences, 82 (85.42%) were classified as subtype B, six (6.25%) as subtype F1, one (1.04%) as subtype C, and seven (7.29%) exhibited a mosaic pattern between subtypes B and F1 (B/F1), with breakpoints at p24 protein. Insertions and deletions of amino acid at p17 were observed in 51 samples (53.13%). The prevalence of PI RAM in the partial gag gene was observed in 78 out of 96 PLH (81.25%). Among these cases, the most common mutations were R76K (53.13%), Y79F (31.25%), and H219Q (14.58%) at non-cleavage sites, as well as V128I (10.42%) and Y132F (11.46%) at cleavage sites. While B/F1 recombination was identified in the p24, the p17 coding region showed higher diversity, where insertions, deletions, and PI RAM, were observed at high prevalence. In PLH with virological failure, the analysis of the partial gag gene could contribute to more accurate predictions in genotypic resistance to PIs. This can aid guide more effective HIV treatment strategies.

Bioinformatics analysis and prokaryotic expression of Strongyloides stercoralis serine protease inhibitor 1 / 中国血吸虫病防治杂志

Objective To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein. Methods The amino acid sequence of the Ss-SRPN-1 protein was downloaded from the NCBI database, and the physicochemical properties, structure and antigenic epitopes of the Ss-SRPN-1 protein were predicted using bioinformatics tools, including ExPASy, SWISS-MODEL and Protean. Primers were designed according to the nucleotide sequences of Ss-SRPN-1, and the Ss-SRPN-1 gene was amplified, cloned and sequenced with genomic DNA extracted from the infective third-stage larvae of S. stercoralis as a template. The Ss-SRPN-1 protein sequence was cloned into the pET28a (+) expression vector and transformed into Escherichia coli BL21 (DE) cells for induction of the recombinant Ss-SRPN-1 protein expression. The recombinant Ss-SRPN-1 protein was then purified and identified using Western blotting and mass spectrometry. Results Bioinformatics analysis showed that the Ss-SRPN-1 protein, which was composed of 372 amino acids and had a molecular formula of C1948H3046N488O575S16, was a stable hydrophilic protein, and the subcellular localization of the protein was predicted to be extracellular. The Ss-SRPN-1 protein was predicted to contain 11 dominant B-cell antigenic epitopes and 20 T-cell antigenic epitopes. The Ss-SRPN-1 gene with a length of 1 119 bp was successfully amplified, and the recombinant plasmid pET28a (+)/Ss-SRPN-1 was constructed and transformed into E. coli BL21(DE) cells. The expressed recombinant Ss-SRPN-1 protein had a molecular weight of approximately 43 kDa, and was characterized as a Ss-SRPN-1 protein. Conclusions The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.

Hypoxia-induced ROS aggravate tumor progression through HIF-1α-SERPINE1 signaling in glioblastoma / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.

Mechanism of action and clinical significance of CST1 in the progression of gastric cancer / 国际肿瘤学杂志

Cysteine protease inhibitor 1 (CST1) is a member of type 2 Cystatins superfamily, plays a key role in targeted regulation. CST1 is highly expressed in gastric cancer, promotes tumor cell migration and invasion by activating the epithelial-mesenchymal transformation pathway and Wnt pathway, and regulates tumor growth and progression in combination with the corresponding target genes of homeobox C10 and glutathione peroxidase 4. A deeper understanding of the role and function of CST1 in gastric cancer will help to further develop potential therapeutic targets and diagnostic and prognostic markers for gastric cancer.

Relationship between preoperative serum cystatin C concentration and postoperative delirium / 中华麻醉学杂志

Objective:To evaluate the relationship between preoperative serum cystatin C (Cys C) concentration and postoperative delirium (POD) in the patients.Methods:Three hundred and ninety patients, aged >50 yr, of American Society of Anesthesiologists Physical Status classification Ⅰor Ⅱ, scheduled for elective knee and hip replacement under combined spinal-epidural anesthesia, with Mini-Mental State Examination scores >23 at 1 day before operation, were included in the study. Peripheral blood samples were collected before operation, and the serum Cys C concentration was measured by the latex-enhanced immunoturbidimetric assay. The cerebrospinal fluid (CSF) 2 ml was collected after successful spinal-epidural puncture for determination of amyloid-β 42 (Aβ42), total tau (T-tau) and phosphorylated tau (P-tau) concentrations by enzyme-linked immunosorbent assay. The patients were divided into POD group and non-POD group according to whether POD occurred. The logistic regression analysis was used to identify the risk and protective factors for POD, and the mediating effect of CSF biomarkers was analyzed. The receiver operating characteristic curve was drawn to evaluate the accuracy of serum Cys C concentration and combination of serum Cys C conceatration and CSF biomarker concentration in predicting POD.Results:Three hundred and twenty-seven patients were finally enrolled, and the incidence of POD was 13.5%. The results of logistic regression showed that increased serum Cys C concentration and increased concentrations of P-tau and T-tau in CSF were risk factors for POD, while increased concentration of Aβ42 and increased Aβ42/P-tau ratio and Aβ42/T-tau ratio in CSF were protective factors for POD ( P<0.05) after adjusting for multiple confounding variables such as age, sex, years of education, Mini-Mental State Examination score, smoking history, drinking history, hypertension and diabetes history. The mediation analysis showed that the relationship between serum Cys C concentration and POD was mediated by T-tau concentration in CSF (11.1%) and by Aβ42/T-tau ratio in CSF (18.0%). The area under the receiver operating characteristic curve of serum Cys C and CSF biomarker concentrations in predicting POD was 0.807 ( P<0.001). Conclusions:Increase in preoperative serum Cys C concentration is a risk factor for POD. T-tau concentration and Aβ42/T-tau ratio in CSF serve as the key mediators in the relationship between preoperative serum Cys C concentration and POD.

Research progress of SARS-CoV-2 main protease inhibitors / 药学学报

As a common protease with high similarity among coronavirus species, the main protease (Mpro) of SARS-CoV-2 is responsible for the catalytic hydrolysis of viral precursor proteins into functional proteins, which is essential for coronavirus replication and is one of the ideal targets for the development of broad-spectrum antiviral drugs. This paper reviews the main protease inhibitors of SARS-CoV-2, including their molecular structures, potencies and drug-like profiles, binding modes and structure-activity relationships, etc.

Effect of serine protease inhibitor Kazal-type 1 on the proliferation of hepatocellular carcinoma cell and its mechanism / 解剖学报

[Abstract] Objective To explore the effect of serine protease inhibitor Kazal-type 1(SPINK1) on the proliferation of hepatocellular carcinoma cells RH-35 and its underling molecular mechanism. Methods Spink1 gene expression in liver cancer and rat liver cancer models were analyzed by Gene Expression Omnibus (GEO) data, RH-35 cells were treated with rrSPINK1 protein, the effect of rrSPINK1 on the proliferation and apoptosis of RH-35 cells was explored by MTT, 2’-deoxy-5-ethynyluridine(EdU) and flow cytometry, the molecular mechanism of SPINK1 regulating liver cancer were detected by Real-time PCR and Western blotting. Results The results showed that Spink1 gene was over-expressed significantly in liver cancer and rat liver cancer models, rrSPINK1-treated RH-35 cells showed increased viability, EdUpositive cell rate, and the proportion of cells in S phase and G

Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity / 生物工程学报

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His6-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His6-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

Expressions of matrix metalloproteinase 7 and secretory leukocyte protease inhibitor in papillary thyroid carcinoma and their significances / 中国现代普通外科进展

Objective:To investigate the expressions of matrix metalloproteinase 7(MMP7)and secretory leukocyte protease inhibitor(SLPI)in papillary thyroid carcinoma(PTC)and their signifi-cances.Methods:Based on the gene expression data of thyroid cancer in the tumor Genome Atlas(TCGA)database,a total of 567 samples were collected,including 509 cancer tissues and 58 normal tissues.The gene matrix data were extracted and sorted out.Two groups of differentially expressed genes were screened by using the R language edger package,and the potential key genes were screened by the mcode plug-in in Cytoscape.Select a key gene and mine closely related genes through the UALCAN database.Immunohistochemical SABC method was used to detect the ex-pressions of MMP7 and SLPI proteins in PTC tissues and their paracancerous tissues collected from 69 patients in Binzhou People's Hospital Affiliated to Shandong First Medical University from January 2020 to June 2021,and the association of expression levels of MMP7 and SLPI with the clinico-pathological factors of PTC patients was also analyzed.Results:Based on the data of TCGA database,1471 genes were obtained,of which 1000 were up-regulated and 471 were down-regu-lated.Through the mcode plug-in in Cytoscape,20 key genes were screened(MMP7,CCL18,CYR61,SPECC1,CRABP2,PLXNA3,KRT17,TMEM59L,RETN,SRF,ITGB4,PPL,PLEKHN1,RMI2,LCN6,SPX,NRIP1,ARHGEF28,SLC39A14,SNCA).Through the UALCAN database,the correlation between MMP7 and SLPI was retrieved(Pearson correlation coefficient was 0.5,P＜0.05).The results of immunohistochemistry showed that the positive expression rates of MMP7 and SLPI proteins in PTC tissues were significantly higher than those in paracancerous tissues[82.6％(57/69)vs 29.0％(20/69),71.0％(49/69)vs 15.9％(11/69)],and the differences were statistically significant(x2 val-ues were 40.222 and 42.579,both P＜0.01).The expressions of MMP7 and SLPI in PTC tissues were correlated with TNM stage,differentiation,extramembranous invasion and lymph node metastasis(all P＜0.05).There was a positive correlation between MMP7 and SLPI proteins expressions in PTC(r=0.381,P=0.001).Conclusions:The interaction between MMP7 and SLPI proteins can be in-volved in the development and progression of PTC.

Effect of phenylmethylsulfonyl fluoride, a protease inhibitor, on enamel surface remineralization

Phenylmethylsulfonyl fluoride (PMSF) is a protease inhibitor widely used in research, but fluoride is released during its action and this knowledge has been neglected in dental research. Aim: to evaluate if fluoride released by salivary protease action on PMSF affects enamel remineralization and fluoride uptake. Methods: Groups of 10 enamel slabs, with caries-like lesions and known surface hardness (SH), were subjected to one of the following treatment groups: Stimulated human saliva (SHS), negative control; SHS containing 1.0 µg F/mL (NaF), positive control; and SHS containing 10, 50 or 100 µM PMSF. The slabs were subjected to a pH-cycling regimen consisting of 22 h/day in each treatment solution and 2 h/day in a demineralizing solution. After 12 days, SH was again measured to calculate the percentage of surface hardness recovery (%SHR), followed by enamel fluoride uptake determination. The time-related fluoride release from 100.0 µM PMSF by SHS action was also determined. Data were analyzed by ANOVA followed by Newman-Keuls test. Results: The release of fluoride from PMSF by SHS was rapid, reaching a maximum value after 10 min. Fluoride released from PMSF was more effective in enhancing %SHR and increasing fluoride uptake in enamel compared with SHS alone (p < 0.05); furthermore, it was equivalent to the positive control (p > 0.05). Conclusion: In conclusion, fluoride released by saliva from PMSF is available to react with enamel and needs to be taken into account in research using this protease inhibitor

Pyramiding and evaluation of segregating lines containing lectin and protease inhibitor genes for aphid resistance in Brassica juncea-Supplementary Data

Aphids are one of the most devastating pests, affecting the potential yield and quality ofBrassica juncea. In the current study, we have attempted to pyramid two transgenic lines containing chickpea lectin (CHPL, P1) and urdbean protease inhibitor (UPI, P2) in each under the phloem specific rolC promoter, through conventional breeding approach. In the derived F2 population, both lectin and protease inhibitor genes were segregating in a 9:3:3:1 ratio (p-value: 0.81), indicative of a single copy of the transgenes in the parents. Furthermore, the parental, as well as pyramided progenies were evaluated for their potential resistance to aphids in terms of mortality and natality. The lines containing both the transgenes were found to be superior over single gene transgenics as a higher mortality rate (96%) was found in F2on the 9th day as compared to single gene transgenics (86% and 80% in P1 and P2 respectively). A significant decrease in the number of nymphs was observed in P1 and P2 but most in F2 plants as almost 43, 32.08, and 107.5 times decrease in the number of nymphs was found in P1, P2, and F2 individuals over control. Expression profiling was done to see if there was any impact of gene pyramiding on the expression pattern of both transgenes before and after aphid treatment, and no significant changes were observed, indicating constitutive expression of transgenes in pyramided lines also. In conclusion, pyramided lines were found to be promising and were superior for aphid resistance.

Pyramiding and evaluation of segregating lines containing lectin and protease inhibitor genes for aphid resistance in Brassica juncea

Aphids are one of the most devastating pests, affecting the potential yield and quality ofBrassica juncea. In the current study, we have attempted to pyramid two transgenic lines containing chickpea lectin (CHPL, P1) and urdbean protease inhibitor (UPI, P2) in each under the phloem specific rolC promoter, through conventional breeding approach. In the derived F2 population, both lectin and protease inhibitor genes were segregating in a 9:3:3:1 ratio (p-value: 0.81), indicative of a single copy of the transgenes in the parents. Furthermore, the parental, as well as pyramided progenies were evaluated for their potential resistance to aphids in terms of mortality and natality. The lines containing both the transgenes were found to be superior over single gene transgenics as a higher mortality rate (96%) was found in F2on the 9th day as compared to single gene transgenics (86% and 80% in P1 and P2 respectively). A significant decrease in the number of nymphs was observed in P1 and P2 but most in F2 plants as almost 43, 32.08, and 107.5 times decrease in the number of nymphs was found in P1, P2, and F2 individuals over control. Expression profiling was done to see if there was any impact of gene pyramiding on the expression pattern of both transgenes before and after aphid treatment, and no significant changes were observed, indicating constitutive expression of transgenes in pyramided lines also. In conclusion, pyramided lines were found to be promising and were superior for aphid resistance.

Construction of multi-epitope gene of periodic Brugia malayi and its expression in eukaryotic cells / 中华地方病学杂志

Objective:To construct a eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi cysteine protease inhibitor/glyceraldehyde 3-phosphate dehydrogenase (CPI502/GAPDH720) multi-epitope gene and observe its protein expression in Hela cells. Methods:Primers were designed according to the predicted gene sequences of T cell epitopes. The target gene fragment was amplified by reverse transcription (RT)-PCR using plasmid pGEM-T-CPI621 as template. The fragment was cloned into prokaryotic expression plasmid pET-28a(+) to construct prokaryotic expression plasmid pET28a(+)-BmCPI502. The BmCPI502 gene and BmGAPDH720 gene were amplified by RT-PCR, respectively. The gene fragments of pcDNA3.1(+), BmCPI502 and BmGAPDH720 were digested by double enzyme digestion and ligated with the target gene. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was constructed. The recombinant plasmid was transfected into Hela cells and verified by RT-PCR to obtain the desired target bands. The expression product was detected by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).Results:The recombinant plasmid pET28a(+)-BmCPI502 was obtained, and the 502 bp specific fragment was identified by enzyme digestion, which was in line with the expected value; the eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of the multi-epitope gene was successfully constructed, and the fragment size was in line with the expected value. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was transfected into Hela cells and the recombinant protein was stable expressed. SDS-PAGE analysis showed that the relative molecular weight ( Mr × 10 3) of the recombinant protein was about 50. Conclusions:The eukaryotic expression plasmid of pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi multi-epitope gene is successfully constructed, and the corresponding recombinant protein is obtained in eukaryotic cells. This study has laid a foundation for further study of the purification and biological activity of the recombinant protein.

Progress of researches on serine protease inhibitors for zoonotic cestode family Taeniidae / 中国血吸虫病防治杂志

Serine protease inhibitor, a protein superfamily that inhibits the serine protease activity, protects hosts from parasitic infections. This review describes the spatial structure and classification of serine protease inhibitor, mechanisms underlying the interplay between serine protease inhibitor and host immune responses and current advances in serine protease inhibitor of zoonotic cestode family Taeniidae, so as to provide insights into the diagnosis of zoonotic tapeworm infections, discovery of therapeutic targets and screening of vaccine candidates.

Key active sites of proteases and protease inhibitors: a review / 生物工程学报

Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.

Recombinant human secretory leukocyte protease inhibitor ameliorated vessel preservation in experimentally isolated rat arteries

Vessel allograft preservation is essential for vascular reconstruction surgery. The preservation solution is crucial inextending the preservation period while maintaining vascular endothelial function. Currently, a 0.9% normal salinesolution (NSS) is widely used as a storage solution although its protective effect on endothelial preservation islimited. In this study, we determine the beneficial effect of recombinant human secretory leukocyte protease inhibitor(rhSLPI), supplemented to 0.9% NSS, for isolated aortic preservation. The thoracic and abdominal aorta were isolatedfrom Wistar rats (n = 6), and the aortic rings were subsequently cut and preserved in 0.9% NSS in the presenceand absence of 1 µg/ml rhSLPI for 0, 6, 24, and 48 hours. The preservative solution was collected to determine thereleased lactate dehydrogenase (LDH) activity and pro-inflammatory cytokine levels, including tumor necrosis factorα (TNF-α) and interleukin-6 (IL-6). Furthermore, the appearance and severity of vessel degeneration were subjected toblind histopathological assessment by pathologists. The results indicated that 0.9% NSS, supplemented with rhSLPI,significantly reduced the released LDH activity, TNF-α, and IL-6 levels and could attenuate endothelial detachment,endothelial degeneration, and endothelial necrosis. This study demonstrated for the first time that adding rhSLPI to asaline preservative solution could prevent vascular injury and possibly extend the graft storage duration before clinicalintervention.

Synthesis and synthetic process optimization of the enterovirus 71 3C protease inhibitor NK-1.8k / 国际药学研究杂志

Objective: To synthesize the enterovirus 71 3C protease inhibitor NK-1.8k and optimize the synthetic process. Methods: With N-Boc-L-glutamic acid dimethyl ester as the starting material, the target compound NK-1.8k was synthesized via the substitution, reductive amination, deprotection, amide condensation, hydrolysis, and reduction reactions. Compared with the original synthetic route, the tandem string type in the important intermediate 5 synthesis was changed to parallel type, thereby the total synthetic reactions were condensed from seven steps to six steps, and some post-processing methods were also optimized. Results: The structures of intermediates and the target compound were confirmed by MS and 1H NMR data, and the total yield of the target compound synthesis was increased to 13.3% from 10.7% of the original route. Conclusion: The synthetic route established in this article for NK-1.8k is reasonable and feasible, the raw materials used are cheap and easily available, the operation is simple, most of the reaction conditions are mild and controllable, the post-processing is simple, the intermediates are easy to separate and purify, the steps are short, and the yield is high. This method provides a valuable reference for the further synthesis of NK-1.8k and similar products.

Polarization of bone marrow-derived macrophages induced by recombinant Trichinella spiralis cysteine protease inhibitors in vitro / 中国血吸虫病防治杂志

Objective To investigate the regulatory role of recombinant Trichinella spiralis cysteine protease inhibitors (rTs-Cys) in induction of polarization of bone marrow-derived macrophages (BMDMs) in vitro. Methods BMDMs were captured and cultured in conditioned medium for 7 days. Then, mature BMDMs were harvested and assigned into four groups. Cells in Group A (negative control) were given 10 ng/mL IFN-γ combined with 100 ng/mL LPS, cells in Group B (positive control) were treated with IL-4 and IL-10 (at 10 ng/mL both), and cells in Group C (recombinant protein alone) were stimulated with 1 μg/mL rTs-Cys, while cells in Group D (protein co-culture) were simultaneously treated with 1 μg/mL rTs-Cys, 10 ng/mL IFN-γ and 100 ng/mL LPS. Cells and culture supernatant were collected 24 hour post-treatment, and the proportions of F4/80+, CD11b+, CD206+ and CD11c+ cells were detected by flow cytometry. The levels of interleukin IL-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10 and transforming growth factor-β (TGF-β) in the cell culture supernatant were measured by ELISA and the CD86+ and CD206+ phenotypes were identified by immunofluorescent staining. Results Flow cytometry detected no significant difference in the proportion of F4/80+ CD11b+ CD11c+ cells among the four groups (F = 46.184, P < 0.001), and a lower proportion of F4/80+ CD11b+ CD11c+ cells was seen in groups C and D than in group A (all P values < 0.001). There was a significant difference in the proportion of F4/80+ CD11b+ CD206+ cells among the four groups (F = 11.032, P < 0.001), and a greater proportion of F4/80+ CD11b+ CD206+ cells was seen in groups C and D than in group A (all P values < 0.01). Immunofluorescent staining showed higher CD206+ expression and lower CD86+ expression in groups C and D than in Group A. There were significant differences in the IL-6 and (F = 3.950, P < 0.001) and TNF-α (F = 205.827, P < 0.001) levels in the cell culture supernatants among the four groups, and significantly lower IL-6 and TNF-α levels were measured in groups C and D than in Group A (both P < 0.05). There were significant differences in the IL-10 and (F = 8.274, P < 0.001) and TGF-β (F = 13.559, P < 0.01) levels in the cell culture supernatants among the four groups, and greater IL-10 and TGF-β levels were measured in Group C than in Group A (both P values < 0.01). In addition, the TGF-β level was significantly higher in Group D than in Group A (P < 0.05); however, there was no significant difference in the IL-10 level between groups D and A (P > 0.05). Conclusion rTs-Cys may induce the polarization of BMDMs to antiinflammatory M2 macrophages in vitro and inhibit the activation of M1 macrophages.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective: To explore the effect of the protease inhibitor from Agaricus bisporus (J.E. Lange) Imbach (AbPI) on glucose uptake and oxidative stress in 3T3-L1 adipocytes. Methods: Adipocytes were differentiated and stained with Oil-Red-O staining to confirm adipogenesis. The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay, intracellular reactive oxygen species generation through flow cytometry, and morphologically through confocal microscopy using propidium iodide, 4,6-diamino-2-phenylindol dihydrochloride, and 2',7'-dichlorofluorescein diacetate dyes. The uptake of fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy. Results: MTT assay showed that the cell survival rate was (28.00±3.00)％, (92.33±2.60)％, and (71.34±2.10)％ in the presence of 2 mM H2O2, AbPI alone, and AbPI and H2O2 both, respectively, in comparison to the control. Oil-Red-O staining indicated that AbPI enhanced adipogenesis. AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone, and showed insulin-sensitizing effect in the presence of insulin, but failed to stimulate the uptake in the absence of insulin. Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon AbPI treatment. Confocal microscopy showed that the damaged cell population rose to 3.50％, 117.84％, and 261.50％ in the presence of AbPI alone, AbPI with H2O2, and H2O2 alone, respectively. Conclusions: The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.