Fluorescent probes are potential fluorophores that display signals based on the changes in tissue microenvironment, interactions with analytes or specific biochemical reactions. Metabolic enzymes are the most important protein involved in bacteria activities. Complex dynamics of biological processes in bacteria are elucidated by these metabolic enzymes-based fluorescent probes with high spatial resolution and sensitivity. Here, we review recent advances in metabolic enzyme-responsive fluorescent probes for bacteria imaging. It was organized according to enzyme classification systems, focused on fluorescence masking strategies, molecular mechanisms of enzyme activation, and bio-related applications.

Objective To explore the inhibition of different immunosuppressive drugs or immunosuppressive combination regiments on the development of de novo donor specific antibodies (dnDSA).Methods We used BABL/c mice and C57BL/6 mice alloimmunized using donor splenocytes for establishing model of producing dnDSA.After that,mice were divided into 10 groups,and were administrated with mycophenolate mofetil (MMF,300 mg· kg-1 · d-1);sirolimus (0.3 mg·kg-1 ·d-1);tacrolimus(0.9 mg· kg-1 · d-1);cyclosporine A (45 mg· kg-1 · d-1);MMF (150 mg· kg-1· d-1) + tacrolimus(0.45 mg · kg-1 · d-1);MMF (150 mg · kg-1 · d-1) + cyclosporine A (22.5 mg·kg-1 ·d-1);MMF (150 mg·kg-1 ·d-1) + sirolimus (0.15 mg·kg-1 ·d-1);sirolimus (0.15 mg· kg-1 · d-1) + tacrolimus (0.45 mg· kg-1 · d-1);sirolimus (0.15 mg· kg-1 · d-1) + cyclosporine A (22.5 mg· kg-1 · d-1);and placebo;respectively.Anti-serum was harvested and tested using flow cytometry.Results (1) Four weeks after sensitization,the dnDSA level in BALB/c and C57BL/6 group was 88.86％ and 25.58％.Comparing with control group(25.33％),there was a significant increase in BALB/c group(89.23％ versus 25.33％;P＜0.001),whereas no change was found in C57BL/6 group (25.58％ versus 25.33％;P =0.259),that we chose BABL/c mice as the model for sensitization.(2)After 4 weeks of sensitization and immunosupression,dnDSA level was 29.31％,46.33％,57.10％ and 66.35％ in MMF,sirolimus,cyclosporine A and tacrolimus monotherapy group respectively.In comparison to sensitization group,dnDSA level in all monotherapy arm reduced significantly.Intra-group analysis found MMF monotherapy had the most effective inhibition of dnDSA production,sirolimus came the second,and tacrolimus and cyelosporine A had equivalent effectiveness.dnDSA level was 31.00％,33.12％,45.18％,44.62％ and 61.60％ in MMF+ sirolimus,MMF+ tacrolimus,MMF+ cyclosporine A,sirolimus + cyclosporine A and sirolimus + tacrolimus combination treatment arm,respectively,which was much lower than that of sensitization group (P＜0.001,P＜0.001,P＜0.001,P＜0.001,P =0.015).MMF + tacrolimus and MMF+ sirolimus combination treatment had comparable effectiveness in dnDSA inhibition,which is greater than that of the rest of three combination.Conclusion Our data show that the effects of single immunosuppressive drugs on the level of dnDSA is MMF＞sirolimus＞cyclosporine A =tacrolimus,and that in combination regimens is MMF + tacrolimus =MMF + sirolimus＞MMF + cyclosporine A =sirolimus + cyclosporine A =sirolimus + tacrolimus.In conclusion,this study demonstrates that MMF+ tacrolimus combination treatment arm can minimize the development of dnDSA.But,this conclusion remains to be confirmed by future clinical studies.

Objective:To explore the change of serum interleukin 16 (IL-16) level and the effects of highly-active antiretroviral therapy (HAART) on IL-16 in patients with HIV infection.Methods:77 patients with HIV infection were studied,with fifteen normal subjects studied as controls.The patients were subdivided into stages according to the standards by US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO).Of all the individuals,the CD4+T cells and CD8+T cells and the amout of IL-16 were measured at each stage to find the difference among normal and groups of the patests,and between the groups with and without HAART.Results:The level of CD4+ T cells in the experimental group were lower than that of the control group in of the patients (P

The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS). Firstly, the molecular mass of deglycosylated protein was measured. Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications. Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with -80% occupancy; N103, N448 and N184 (-15% occupancy) were glycosylated with complex-type oligosaccharides. LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.

Objective To investigate the anti tumor effects of cytotoxic T lymphocytes induced by tumor antigen specific dendritic cells. Methods Soluble tumor antigen was prepared by four freeze thaw cycles of gastric tumor cell line SCG7901. Anti gastric tumor vaccine was acquired by co incubation of tumor antigen and mouse bone marrow derived dendritic cells and cultured with granulocyte/macrophage colony stimulating factor and interleukin 4. Antigen specific cytotoxic T lymphocytes were induced by this tumor vaccine from the spleen and were applied to tumor bearing nude mice. Results Tumor growth was significantly inhibited and the apoptosis of tumor cells was promoted extensively. Conclusions Antigen specific dendritic cell tumor vaccine may play an important role in future immunotherapy of gastric cancer.

AIM: To study the fingerprint of Danggui Injection (Radix Angelicae Sinensis). METHODS: HPLC with UV detector was used in analyzing the patterns of fingerprint of Danggui Injection. It was separated on C 18 column. The mobile phase consisted of (A) water ( 0.25% HAC) and (B) methanol using a linear gradient, and the detection wavelength at 246nm. The flow rate was at 1mL?min -1 . RESULTS: The fingerprint of Danggui Injection was set up and the fingerprint of Angelica sinensis showed a correlation. CONCLUSION: The research is helpful to the quality control of Danggui Injection.

Objective:To measure the effective optical zone (EOZ) after small incision lenticule extraction (SMILE) by using corneal topographic map and modulation transfer function (MTF), and to analyze the related factors affecting the EOZ.Methods:A retrospective observational case series study was performed.Sixty-two myopic patients (62 eyes) who underwent SMILE between December 2015 and July 2017 in Jinan Mingshui Eye Hospital were enrolled.The EOZ was measured by using tangential corneal curvature topographic map and MTF pre- and 6-month post-operatively.The repeatability of data was determined by intraclass correlation coefficient (ICC) and Cronbach Alpha coefficients; the agreement of data was identified by using Bland-Altman plot; the correlations between EOZ and surgical parameters were analyzed by utilizing Pearson correlation coefficient.This study protocol was approved by the Ethics Committee of Jinan Mingshui Eye Hospital(No.2015[013]). Written informed consent was obtained from each patient before surgery.Results:The ICC and Cronbach Alpha coefficients of EOZ measured by corneal topographic map were greater than 0.9, which showed high intraobserver repeatability.The Bland-Altman plots displayed relatively good agreement between the two methods.The 95% limits of agreement was -0.49 to 0.89 μm.Six months after SMILE, the EOZ measured by tangential corneal curvature gradient topographic map was (5.32±0.25)mm, which was (1.18±0.25)mm smaller than predicted optical zone, and the EOZ measured by MTF method was (5.07±0.32)mm, which was (0.20±0.35)mm smaller than corneal tomographic EOZ, and the difference was significant ( t=-4.487, P<0.01). A negative correlation was found between the EOZ and attempted refractive correction ( r=-0.364, P=0.004). The positive correlations were found between the EOZ and ΔKm or ΔQ 6 months after SMILE ( r=0.367, 0.514; both at P<0.01). Conclusions:The EOZ measured by tangential corneal curvature topographic map after SMILE is of high repeatability and is consistent with the result calculated by MTF method.The EOZ is significantly reduced after SMILE.

OBJECTIVETo obtain the Escherichia coli strains expressing N-Acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase).

METHODSThe gene (nanA) coding Neu5Ac aldolase was cloned from Escherichia coli C600, and the recombinant plasmid was sequenced and expressed in Escherichia coli.

RESULTSSequencing data revealed that the open reading frame was 894 bp and predicted to encode a protein consisting of 298 amino acids. The patterns of SDS-PAGE showed that the purified enzyme protein as a single protein band with a molecular weight of 33 kD, which was consistent with those reported in the reference. In the recombinant plasmid pRY1, the expression of nanA gene was controlled by the lac promoter with the induction of IPTG or lactose. The plasmid pRY3 was constructed, in which the nanA gene ws controlled by the tac promoter. The protein of Neu5Ac aldolase was constitutively expressed using the recombinant strain, E.coli DH5 alpha/pRY3 without induction of IPTG or lactose. The crystal was finally obtained with the efficiency of 90.2% of Neu5Ac. The HPLC indicated that the Neu5Ac crystal prepared in this experiment was same as Simga product.

CONCLUSIONThe protein products expressed by two recombinant strains E.coli BL21(DE3)/pRY1 and DH5 alpha/pRY3 has the characteristics of Neu5Ac.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Open Reading Frames ; Oxo-Acid-Lyases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Recombination, Genetic

The glycosylation heterogeneity of recombinant human pro-urokinase (pro-UK) was assessed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Firstly, the source of heterogeneity was determined by measuring the M_r of intact protein before and after N-deglycosylation. Glycosylation sites and the proportion of O-glycopeptides then were determined at the peptide level. Finally, the N-glycans were confirmed and quantified using the N-glycan profile. Results show that the structural heterogeneity of pro-UK is mainly caused by glycosylation. All T₁₈ were fucosylated, and 6.4% of S_138/139 was O-glycosylated with two kinds of oligosaccharides with a ratio of 6.0% and 0.4% respectively. All N₃₀₂ positions were N-glycosylated by more than ten types of glycans, among which A2F and A3F accounted for 80% of the total. The assessment of glycosylation heterogeneity of pro-UK will provide a reference for quality standardization.

The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample. UPLC-MS can be used to characterize and compare interferon reference standards from different manufacturers.
Amino Acid Sequence ; Chromatography, High Pressure Liquid ; methods ; Interferons ; standards ; Mass Spectrometry ; methods ; Molecular Weight ; Oxidation-Reduction ; Peptide Mapping ; Protein Processing, Post-Translational ; Reference Standards