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Objective:To evaluate the potential of a previously identified CDR3 only single-domain antibodies (sdAbs) fragment, NBL42, as a general framework for affinity transfer.Methods:The H3 loops of VHH-A4(A4), VHH-H5(H5), cAb-Lys3(L3) and B6H12 which bind with alliinase, PD-1, lysozyme and CD47, respectively, were grafted into the corresponding loop of NBL42. The genes of the reconstituted CDR3 only sdAbs were synthesized, expressed in E. coliand purified with Ni 2+ column affinity chromatography. The antigen binding and stability of the recombinant CDR3 only sdAbs were assayed by ELISA. Results:The recombinant NBL42-A4CDR3, NBL42-H5CDR3, NBL42-L3CDR3 and NBL42-B6H12CDR3 ran as a single peak at 15, 15, 28 and 16 kDa, respectively, in SDS-PAGE as expected molecular weight. Grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 expressed in a soluble form and specifically bind with alliinase and PD-1, respectively, but lost about 50% of their binding activity. In contrast, the grafted sdAbs NBL42-Lys3CDR3 and NBL42-B6H12CDR3 completely lost their antigen binding capacity. NBL42 sdAbs and grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 retain roughly half of their binding activity after 90 ℃ heat treatment, indicating high stability. The C88Y mutation in NBL42 and the Swiss Mode 3D model predicted that the C88Y residue in FR3 may play a key role in NBL42 stability and CDR3 affinity transfer.Conclusions:The structure of NBL42 has potential as a framework for CDR3 transplantation and affinity transfer.
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Objective To preliminarily explore the effects of human microRNA-181a on migration of gastric cancer cells and its mechanism.Methods The expression of miRNA-181a-5p in gastric cancer cell line GC9811 and peritoneal high metastasis gastric cancer cell line GC9811-P were tested by quantitative real-time polymerase chain reaction (qRT-PCR ). GC9811 cell line was transfected byendogenous synthetic analog mimic and its homologous negative control of miRNA-181a-5p,which were considered as up-regulated group and its control group respectively;GC9811-P were transfected by miRNA inhibitor and its homologous negative control of miRNA-181a-5p,which were considered as down-regulated group and its control group respectively.After miRNA-181a-5p was up or down-regulated,cell proliferation,migration and apoptosis capabilities of gastric cells were detected by matrix thiazolyl tetrazollium (MTT)assay,cloning-forming assay,Transwell migration test,wound healing assays and apoptosis test.After miRNA-181a-5p was up or down regulated,the changes of matrix metalloproteinase (MMP)14 expression were determined by Western blot.Independent sample t test was performed for mean comparison between samples and chi square test was used for rate comparison.Results The results of qRT-PCR showed the relative expression quantity of miRNA-181a-5p in GC9811 was 1 .000 00 ± 0.021 26 and in GC9811-P was 3.175 61 ±0.106 76,and the difference was statistically significant (t =34.620,P <0.01 ).The results of MTT assay indicated that the cell proliferation rate of up-regulated group was higher than that of up-regulated control group,and that of down-regulated group was lower than down-regulated control group.The cloning-forming assay demonstrated that the number of clone forming and clone forming rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 234.00±10.12 and 46.8%,93.00±9.61 and 18.6%,51 .00 ±7.96 and 10.2%,99.00±8.05 and 19.8%,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t = 17.500,7.344,χ2 = 12.27, 9.51 ,all P <0.01).The results of Transwell migration experiment showed the number of cells migrated through membrane hole of up-regulated group was 164.00±19.31 ,and which was higher than that of up-regulated control group (87.00±23.04,t=4.436,P <0.05);that of down-regulated group was 157.00± 11 .50,and which was lower than that of down-regulated control group (234.00 ±12.12,t =7.982,P <0.05).The result of wound healing assays indicated that the rate of migration distance of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 2.09 ± 0.18,1 .27 ±0.23,1 .15 ±0.15 and 1 .67 ±0.12,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t =4.863 and 4.689, both P <0.05).The results of apoptosis experiments demonstrated that the apoptosis rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were (6.10± 1 .02 )%,(9.10 ± 2.13 )%,(12.70 ± 1 .23 )%,(8.70 ± 2.54 )%,respectively,and there was no statistically significant difference between up-regulated group,down-regulated group and their control group (both P *0.05).The results of Western blot showed that the grey value of up-regulated group was 561 .881 ±35 .740,which was higher than that of up-regulated control group (275 .784±23.520);that of down-regulated group was 579.565 ±37.950,which was lower than that of down-regulated control group (1 312.760±51 .270),and the differnces were statistically significant (t =11 .580 and 19.910,both P <0.01).Conclusion miRNA-181a-5p highly expresses in peritoneal high metastasis gastric cancer cell line GC9811-P and promoted the proliferation and migration of gastric cancer cell line GC9811 and GC9811-P with a tendency to suppress apoptosis.
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MTANN [Massive Training Artificial Neural Network] is a promising tool, which applied to eliminate falsepositive for thoracic CT in recent years. In order to evaluate whether this method is feasible to eliminate false-positive of different CAD schemes, especially, when it is applied to commercial CAD software, this paper evaluate the performance of the method for eliminating false-positives produced by three different versions of commercial CAD software for lung nodules detection in chest radiographs. Experimental results demonstrate that the approach is useful in reducing FPs for different computer aided lung nodules detection software in chest radiographs
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Objective To investigate the expression of microRNA-29b (miR-29b) in gastric cancer cell line GC9811 and high peritoneal metastatic gastric cancer cell line GC9811-P and its effect on invasion,proliferation and apoptosis.Methods The relative quantitative expression of miR-29b was detected by quantitative realtime polymerase chain reaction(qRT PCR).GC9811 cells were divided into three groups,miRNA down-regulated and transfected with lentiviruses LV-miR-29b inhibitor group,negative control group with negative transfection,and untransfected blank control group.GC9811-P cells were divided into three groups,miR-29b up regulated and transfected with lentiviruses LV miR 29b group,negative control with negative transfection group,and untransfected blank control group.The cell invasion ability was detected with Transwell assay,the cell proliferation ability was measured by methyl-thiazolyl tetrazolium (MTT) test,the colony forming ability was determined by plate colony formation assay,and the apoptosis was tested by flow cytometry.The expressions of miR 29b and secreted protein,acidic and rich in cystenie (SPARC) in gastric cell line GC9811-P,GC9811,MKN28M,MKN28NM and normal gastric cell line GES were determined by qRT-PCR,and the correlation was analyzed.Two independent samples t test or SNK-q test was performed for mean comparison between two groups,and one way analysis of variance was used for mean comparison among three groups.Results The relative quantitative expression of miR-29b inGC9811-P (0.21±-0.04) was significantly lower than that of GC9811 (1.00±0.03,t 28.140,P＜ 0.01).After GC9811 cells transfected with lentiviruses LV-miR-29b inhibitor,the expression of miR-29b (0.21±0.04) was significantly lower than that of control group (0.89±0.07) and blank control group (1.00±0.04,q 12.76,14.73,both P＜0.01).Compared with negative group,the transmembrane cell number and the clonality of miRNA up-regulated group raised(274.33± 9.03 vs 110.67 ± 13.69,t=9.981,P＜0.01;131.33±4.91 vs69.67±2.33,t 11.340,P＜0.01),and the results of MTT test also shows the proliferation was increased.After GC9811-P cells transfected with LV-miR-29b,the expression of miR 29b (4.08±0.20) was significantly higher than that of negative control group (1.15±0.05) and blank control group (1.00±0.10,q=21.73,22.81,both P＜0.01).Compared with negative group,the transmembrane cell number and the clonality of miRNA up regulated group reduced (51.33±5.55 vs 104.00±6.24,t 6.305,P＜0.01; 48.00±5.51 vs 113.33±5.17,t 11.340,P＜0.01),and the results of MTT test also shows the proliferation was weakened.Apoptosis assays demonstrated miR-29b promoted apoptosis; however,the difference was not statistically significant.The expression of miR-29b was negatively correlated with SPARC mRNA in gastric cancer cells (r=-0.97,P=0.03).Conclusions The low expression of miR-29b in high peritoneal metastatic gastric cancer cell inhibited the ability of invasion and proliferation.MiR-29b might be a new target of inhibiting peritoneal metastasis in gastric cancer.