Objective To synthetically evaluate the relationship between miR-31 and the prognosis of carcinoma and to investi-gate its related mechanism.Methods The correlative literatures of tumor prognosis were retrieved from the electronic databases PubMed,EMBASE and ISI Web of Science.The pooled hazard ratios (HRs)and 95% confidence interval(95%CI )were extracted. The prognostic data were performed the synthesis analysis.Results A total of 7 trials conformed to the inclusion criteria including accumulated 2 012 cases of carcinoma.Meta-analysis revealed that the decrease of miR-31 expression in the tumor patients had the poor prognosis (HR=0.784,95%CI :0.630-0.974);in the subgroup analysis,the synthesis results adopting the multivariable a-nalysis and China subjects were 3.512 (95%CI :1.797-6.865)and 1.574 (95%CI :1.062-2.333),which indicating that the in-crease of miR-31 expression predicted the poor prognosis;miR-31 had no statistical significance in the digestive system (P >0.05). Conclusion The prognostic role of miR-31 may possess the histological and regional specificity and has the potential as a novel marker.

Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity analysis could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibodies is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.
Amino Acid Sequence ; Antibodies, Monoclonal ; isolation & purification ; Chromatography, Ion Exchange ; Humans ; Isoelectric Focusing ; Mass Spectrometry ; Peptide Mapping ; Peptides ; Sequence Analysis, Protein ; methods

Objective: To examine the effects of perioperative intravenous administration of flurbiprofen axetil (FA) on pain associated with transrectal ultrasound-guided prostate biopsy.Methods: This was a randomized,controlled study.Eighty-one patients who underwent 12 core prostate biopsy were included in the study.The patients were randomly assigned to one of three groups (n=27 in each) by type of procedure during prostate biopsy.Group intrarectal local anesthesia (IRLA) received intrarectal 5% (0.05 g/L) lidocaine gel 60 mg, 5 minutes before the procedure alone;Group FA received intravenous flurbiprofen axetil (1 mg/kg) 1 hour before the procedure;Group IRLA+FA received intrarectal 5% lidocaine gel 60 mg, 5 minutes before the procedure and intravenous flurbiprofen axetil (1 mg/kg) 1 hour before the procedure.The patients were asked to score the pain by using visual analogue scale (VAS) in 4 situations,including when the probe was inserted (VASⅠ),during anesthesia (VASⅡ),during biopsy (VASⅢ) and 20 minutes after biopsy (VASⅣ).The findings were evaluated with analysis of variance,and the Tukey post hoc test was followed with an overall 2-tailed significance level at α =0.05.P1, P value between Group IRLA and Group FA;P2, P value between Group FA and Group IRLA +FA,P3, P value between Group IRLA and Group IRLA +FA.The bonferroni method was used to adjust the test level, α=0.017,a P value of less than 0.017 was accepted as the threshold for statistical significance.Results: No major complications,including sepsis and severe rectal bleeding,were noted in any patient.There were no differences in general condition of the patients before procedure among the 3 groups.There were statistically significant differences in VAS scores among the 3 groups in VASⅡ (5.7±2.2, 3.0±1.5,3.3±1.9,respectively,P=0.012) and VASⅢ (6.7±2.3,3.0±2.1,2.9±1.6,respectively,P=0.001).There were no differences in the pain scores among the 3 groups during probe insertion (VASⅠ, 3.2±1.0,4.1±2.1,4.2±1.7, respectively,P=5.752) and 20 minutes after biopsy (VASⅣ, 1.4±2.1,1.0±0.9,1.1±0.7,respectively,P=3.772).Between-column differences among the 3 groups were VASⅡ (P1=0.007,P2=5.655,P3=0.001,respectively) and VASⅢ(P1=0.008,P2=7.517,P3=0.001,respectively),the differences between Group IRLA and Group FA,Group IRLA and Group IRLA +FA in VASⅡ and VASⅢ were statistically significant.Conclusion:The intravenous flurbiprofen axetil was found to be more effective than intrarectal lidocaine gel alone.

Objective To analyze the differences of size and charge heterogeneities between origi-nal humanized anti-TNF-αantibody and four similar biotherapeutic products ( SBP ) .Methods The size exclusion chromatography ( SEC-HPLC ) and weak cation exchange chromatography ( WCX-HPLC ) were used to analyze the size and charge heterogeneities , respectively.Carboxypeptidase B (CpB) treatment was employed to analyze the source of charge heterogeneity of the antibody products .Results Four SBPs showed the same pattern with the originator in SEC-HPLC, and no significant difference with the percentage of mono-mer was observed .The percentages of the aggregates of SBP-3 and SBP-4 were a little higher than those of the originator .The charge distribution of SBPs was significantly different from the originator ′s, especially in the basic region .The results from the samples treated with CpB indicated that the difference of charge distri -bution in the basic region might be caused by the C-terminal lysine variants .Conclusion Four SBPs showed similar size heterogeneity with the originator , but significant differences with charge heterogeneity were observed among them .The study suggested that more attention should be paid to the charge heterogene -ity analysis of the biosimilar products .

Objective To compare the capability of capillary electrophoresis-sodium dodecyl sul-fate ( CE-SDS) and size exclusion-high performance liquid chromatography ( SE-HPLC) for analysis of size heterogeneity of monoclonal antibody products .Methods The size heterogeneity of one humanized anti-VEGF monoclonal antibody was analyzed by using non-reduced and reduced CE-SDS, and conventional , de-natured and denatured reduced SE-HPLC.Results The percentage of aggregates detected by non-reduced CE-SDS (0.82%±0.01%) was equal to that by using denatured SE-HPLC (1.05%±0.02%), but it was significantly lower than that by using conventional SE-HPLC analysis (5.08%±0.10%).With regard to fragments analyzed with non-reduced antibodies, its percentage was (7.12±0.04)% measured by non-re-duced CE-SDS analysis that was significantly higher than that by conventional SE -HPLC analysis (0.02%± 0.01%) and denatured SE-HPLC analysis (0.62%±0.01%).Using reduced antibodies , the percentage of fragments was (3.19±0.50)%tested by reduced CE-SDS analysis that was significantly higher than that by using denatured reduced SE-HPLC analysis (0.07%±0.01%).Conclusion Conventional SE-HPLC was more objective than CE-SDS for content analysis of aggregates , as both the covalent and non-covalent forms of aggregates could be detected .Non-reduced CE-SDS could demonstrate the content of clips , while reduced CE-SDS showed the degraded fragments .Therefore, CE-SDS had an advantage over conventional SE-HPLC for content analysis of fragments .The use of the two analytical methods in combination provided solid techni-cal supports for the quality control of size heterogeneity of monoclonal antibodies .

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.

Objective: To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system.Methods: The expression of CMTM2 in human testis and sperm was confirmed by Western blot.Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction.Results: CMTM2 was presented in both human testis and sperm.In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells.In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction.CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports.The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages.TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction.CMTM2 was localized at the posterior head of sperm before and after acrosome reaction.The localization and expression of CMTM2 were not affected by sperm acrosome reaction.Conclusion: Expression of CMTM2 in the male reproductive system of the adult human exhibits cell-and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion.The expression of CMTM2 in the male reproductive system of the adult human exhibits cell-and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion.However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis.And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.

Objective To investigate the CT and MRI manifestations of intra-abdominal aggressive fibromatosis and correlation with pathology.Methods The CT and MRI manifestations of 26 cases with intra-abdominal aggressive fibromatosis confirmed by pathological examination were analyzed retrospectively.Results 26 cases showed single solid mass,13 cases showed well-circumscribed and round-like,9 cases wrapped around the common bile duct,intestine or ureter,4 cases were lobulated which had unclear margin with surrounding tissues in pelvic.All the lesions displayed isodensity or slightly low density non-enhanced CT appearance,heterogenous high intensity FS T2WI,some larger tumors showed mixed signal.CT/MRI enhanced scan showed gradual enhancement in 26 cases.Conclusion Intra-abdominal aggressive fibromatosis have some certain imaging features,MRI can offer the histo logical features of tumors and have some correlation with pathology.

Objective To investigate the effect of gefitinib, an inhibitor of epidermal growth factor receptor ( EGFR ) , on the proliferation and osteogenic differentiation of endosteum-derived stem cells ( EDSCs ) in rats. Methods Femoral fracture models were established in healthy male 4-week old SD rats. They were randomly divided into 2 groups. The experimental group was subjected to intragastric lavage with gefitinib, an EGFR signaling inhibitor ( 100 mg/kg·d ) while the control group to intragastric lavage with an isodose of methyl cellulose. Bilateral femurs and tibias were harvested one week after lavage for separation of EDSCs and bone marrow mesenchymal stem cells ( BMSCs ) respectively using density gradient centrifuga-tion. After proliferative cloning in vitro, expression of the cell surface antigens ( CD29, CD34, CD44 and CD45) of the third passage cells was detected by flow cytometry (FCM). Proliferation of the cells was detected by BrdU, cell cycle was measured by FCM, and expression of the genes related to cell cycle inhibitory factors (p15, p16, p21 and p27) was determined by PCR. ALP staining was performed 14 days after osteogenesis induction. After 21 days of chondrogenic induction, von Kossa staining was conducted. qRT-PCR of the mRNA obtained was used to detect expression of osteogenic differentiation of related genes ( osteocalcin, bsp, runx2 and osterix ). Results CD29 and CD44 were positively expressed while CD34 and CD45 negatively expressed in EDSCs and BMSCs. After the EGFR signaling pathway was blocked by gefitinib, BrdU detection found that gefitinib inhibited BMSCs ( 11.15%) much more than EDSCs ( 0.25%). Cell cycle detection showed that the volume of EDSCs was increased in phases G0/G1 and S but decreased significantly in phase G2-M. ALP staining showed that the increase of EDSCs ALP+ cells (53.31% ) was significantly higher than that of BMSCs (25.04% ) . The increased expression percentages of the genes related to cell cycle inhibitors in EDSCs (103.9%, 58.0%, 117.3% and 105.1%, respectively) were significantly higher than those in BMSCs (39.3%, 38.4%, 24.5% and 83.4%, respectively) ( P ＜0.05). The increased expression percentages of the genes related to osteogenic differentiation in EDSCs (247.0%, 289.9%, 66.1% and 233.2%, respectively) were significantly higher than those in BMSCs (106.5%, 186.4%, 41.7% and 190.8%, respectively). All the above differences were statistically significant ( P ＜0.05) . Conclusions Gefitinib, an EGFR inhibitor, can inhibit proliferation of EDSCs and BMSCs but promote their osteogenic differentiation. It inhibits proliferation of BMSCs more significantly as it promotes osteogenic differentiation of EDSCs.