Protein phosphorylation is one of the most common post-translational modifications (PTMs)in various organisms,which plays critical roles in the regulation of intracellular biological processes,such as cell prolifera-tion,signal transduction,metabolismis and tumorigenesis.However,the low abundance of phosphoprotein in the biological systems poses significant challenges of current analytical techniques.In order to further understand the phosphoproteomics,the roles of phosphorylated proteins in life process,discovery of biomarkers,diagnosis and treatment of disease,enrichment strategies of high efficiency have been developed,including the design of new nanomaterials and combination of a variety of analytical methods,et al.In this paper,we reviewed the develop-ment of enrichment strategies for phosphoproteomics and application of phosphoproteomics in disease.

OBJECTIVETo study the clinical efficacy of three kinds of hybrid bioartificial liver support systems (HBLSS) in treating chronic severe hepatitis.

METHODSA bioartificial liver support system (BAL), comprising porcine hepatocytes and fiber tube style bioreactor, was constructed. Then three kinds of HBLSS were constructed: Molecular absorbent recirculating system (MARS) plus BAL; slow plasma exchange (SPE) plus continuous hemodiafiltration (CHDF) and BAL; and SPE plus hemoperfusion (HP) and BAL. One hundred-twenty patients in middle or late stages of chronic severe hepatitis were enrolled in this study. They were randomly divided into 6 groups: H1 group was treated with BAL+MARS, H2 with BAL+SPE+CHDF and H3 with BAL+SPE+HP (as treatment groups); C1 group was treated with MARS, C2 with SPE+CHDF and C3 with SPE+HP (as control groups). The changes in the clinical symptoms, in the hepatic encephalopathy stages, and in the serum total bilirubin (TBIL), the serum albumin (ALB), the prothrombin activities (PTA), endotoxin, ammonia, creatinine and a-fetal protein (AFP) were all observed before the treatment, right after it and 72 hours later. The improving and curing rates and the rates of side effect occurrences in each group were observed.

RESULTSIn all 6 groups, the patients' clinical symptoms ameliorated; their TBIL, endotoxin and ammonia levels decreased (P<0.05), and their PTA and AFP levels lowered significantly (P<0.05). But in the H1, H2 and H3 groups they were more distinctive than in the control groups. In H1 and H2 groups creatinine and ammonia levels were decreased more significantly than in the H3 group (P<0.05). The improving and curing rates of each group were 65 % (13/20), 60% (12/20), 45% (9/20), 45% (9/20), 40% (8/20) and 20% (4/20) respectively. No serious side effects were observed during the treatment.

CONCLUSIONIn treating middle and late stage chronic severe hepatitis, the measures used in H1, H2 and H3 are better than those in C1, C2 and C3. Furthermore, H1 and H2 treatments can ameliorate hepatic and renal functions, prevent the development of multiple organ dysfunction syndrome, and are better than those used in H3.

Adult ; Aged ; Animals ; Bioreactors ; Critical Illness ; Female ; Hemodiafiltration ; Hepatic Encephalopathy ; blood ; therapy ; Hepatitis, Viral, Human ; therapy ; Humans ; Liver ; cytology ; Liver Failure, Acute ; therapy ; Liver, Artificial ; Male ; Middle Aged ; Plasma Exchange ; Swine

OBJECTIVETo study the survival and growth of Vibrio cholerae inside the Acanthamoeba polyphage.

METHODSSurvival and growth of Vibro cholerae O139, co-cultured with Acanthamoeba polyphaga, was observed inside the trophozoites and cysts, using Gram stain and electron microscope.

RESULTSViable O139 was observed inside the amoebal vacuoles in 24 hours. Vacuoles were filled with more bacteria along with the longer period of co-culture. The process of O139 infection with Amoebae would include uptake, formation of O139 vacuole, multiplication, trophozoites lysed and expel under electron microscopy. Some infected trophozoites could subsequently encyst and the surviving O139 could locate in the vesicles inside the cysts.

CONCLUSIONO139 might survive and multiply in the trophozoites and reside inside the cysts of Amoebae, suggesting that Acanthamoebae might serve as one of the environmental hosts of Vibro cholerae.

Acanthamoeba ; growth & development ; microbiology ; ultrastructure ; Animals ; Coculture Techniques ; Colony Count, Microbial ; Culture Media ; Vibrio cholerae O139 ; growth & development ; ultrastructure ; Water ; parasitology

OBJECTIVETo determine whether Acanthamoeba polyphaga could affect the survival and growth of Vibrio cholerae O139 in low temperature.

METHODSV. cholerae O139 was co-cultured with the Acanthamoeba polyphaga to be examined on its intracellular growth and survival rate within cysts at low temperature, using methods as Gram-staining, electron microscope and passage culture.

RESULTSV. cholerae O139 were observed to enter into the trophozoites and grow the within the vacuoles after 8 hour incubation with Acanthamoeba polyphaga. The germs survived in the vacuole and/or endo-layer of wall and could be re-isolated from the excystment of Acanthamoeba polyphaga. At 30 degrees C, V. cholerae O139 could survive for 120 days with the amoeba while less than 45 days in PAS. At 4 degrees C, the number of viable bacteria decreased and reached undetectable levels for both study and control groups after a 30-day incubation. V. cholerae O139 could be re-isolated from the 30-, 45-, 60- and 75-day's infected cysts after excystment. However the ability of excystment for 90-day's infected cysts decreased and V. cholerae O139 within the cyst could not be isolated again because the amoebae had lysed.

CONCLUSIONThese findings indicated that V. cholerae O139 could grow within Acanthamoeba polyphaga and the survival time could be increased in the cysts at low temperature. It seemed that Acanthamoeba can provide an environmental reservoir for V. cholerae O139.

Acanthamoeba ; microbiology ; Bacterial Capsules ; Colony Count, Microbial ; Temperature ; Vibrio cholerae ; growth & development

NDM-1 (New Delhi metallo-beta-lactamase) gene encodes a metallo-beta-lactamase (MBL) with high carbapenemase activity, which makes the host bacterial strain easily dispatch the last-resort antibiotics known as carbapenems and cause global concern. Here we present the bioinformatics data showing an unexpected similarity between NDM-1 and beta-lactamase II from Erythrobacter litoralis, a marine microbial isolate. We have further expressed these two mature proteins in E. coli cells, both of which present as a monomer with a molecular mass of 25 kDa. Antimicrobial susceptibility assay reveals that they share similar substrate specificities and are sensitive to aztreonam and tigecycline. The conformational change accompanied with the zinc binding visualized by nuclear magnetic resonance, Zn(2+)-bound NDM-1, adopts at least some stable tertiary structure in contrast to the metal-free protein. Our work implies a close evolutionary relationship between antibiotic resistance genes in environmental reservoir and in the clinic, challenging the antimicrobial resistance monitoring.
Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; Aztreonam ; pharmacology ; Cephalosporinase ; chemistry ; genetics ; metabolism ; Computational Biology ; methods ; Drug Resistance, Bacterial ; genetics ; Enzyme Stability ; drug effects ; Evolution, Molecular ; Minocycline ; analogs & derivatives ; pharmacology ; Molecular Sequence Data ; Phylogeny ; Protein Structure, Tertiary ; drug effects ; Sequence Homology, Nucleic Acid ; Sphingomonadaceae ; drug effects ; enzymology ; genetics ; Tigecycline ; Zinc ; pharmacology ; beta-Lactamases ; chemistry ; genetics ; metabolism

Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.
Cells, Cultured ; Secondary Metabolism ; Sorbus ; Stress, Physiological

Objective: To establish a method for determining arabinose, mannose, fructopyranose and amylaceum in Shenxiong glucose injection by UPLC-MS/MS, so as to provide the basis for the scientific evaluation of the quality of Shenxiong glucose injection, and lay a foundation for the safe use of drugs in clinic. Method: Domestic GDX-403 solid-phase extraction column was used to purify Waters ACQUITY UPLC BEH Xbridge Amide column (2.1 mm×100 mm, 1.7 μm)at the column temperature of 35℃, and the mobile phase was 0.1% ammonia, 0.1% acetonitrile-0.1% ammonia water and water 85:15. The contents of arabinose, mannose, fructose and glucose in Shenxiong glucose injection were determined by UPLC-MS/MS with a flow rate of 0.3 mL·min^-1. Result: A method was established to determine arabinose, mannose, fructopyranose and amylaceum in Shenxiong glucose injection. The concentration range of arabinose, mannose, fructopyranose and amylaceum showed a good linear relationship with the peak area, with a good repeatability and precision. Recoveries were 98.43%, 102.13%, 100.72%, 101.75%, and RSD were 2.4%, 1.3%, 3.1%, 2.7%. Arabinose and mannose content were stable in five batches of Shenxiong glucose injection. Conclusion: The method is simple and specific. Compared with the determination of total sugar, the method is more scientific and stable, and can be used for the quality control of Shenxiong glucose injection.

Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: SaUGT1 and SaUGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame (ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54.27 kDa and theoretical isoelectric point (pI) of 5.50.SaUGT2 gene contained 1 431 bp ORF,encoding a polypeptide of 476 amino acids,with a relative molecular weight of 53.49 kDa and theoretical pI of 5.63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract (YE), with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni²⁺ affinity chromatography. Conclusion: The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 proprietary Chinese medicines (APCMs). Multi-type dosage forms (, tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

OBJECTIVETo systematically evaluate the effectiveness and safety of Sodium Tanshinone II A Sulfonate Injection (STS) as one adjuvant therapy for treating unstable angina pectoris (UAP).

METHODSRandomized controlled trials (RCTs) of UAP treated by STS were searched in the China National Knowledge Infrastructure Database (CNKI), VIP Database for Chinese Technical Periodicals (VIP), Wanfang Database, the Chinese Biomedical Literature Database (CBM), Web of Science, the Cochrane Library, Embase, and PubMed, which from inception to January, 2016. The Cochrane Risk Assessment Tool was used to evaluate the methodological quality of the RCTs. The Review Manager 5.3 software was used to conduct the metaanalysis.

RESULTSThe results showed that 17 RCTs involving 1,372 patients were included. The meta-analysis indicated that the combined use of STS and Western medicine (WM) in the treatment of UAP can obviously improve the total effective rate [risk ratio (RR)=1.31, 95% confidence interval (CI) (1.24,1.39), P<0.0001], and the total effective rate of electrocardiogram [RR=1.43, 95% CI (1.30,1.56), P<0.0001], decrease the level of CRP [mean difference (MD)=-3.06, 95%CI (-3.85,-2.27), P<0.00001], fibrinogen [MD=-1.03, 95% CI (-1.16,-0.89), P<0.00001], and whole blood high shear viscosity [MD=-0.70, 95% CI (-0.92,-0.49), P<0.00001]. Additionally, the occurrence of adverse drug reaction of the experimental group was significantly higher than that of the control group [RR=3.57, 95% CI (1.28, 9.94), P<0.05].

CONCLUSIONSCompared with WM, the combined use of STS was more effective.