Objective:To analyze the carriage status, subtype distribution and flanking gene sequence characteristics of oxacillinases (OXA enzyme) in 241 clinical strains of Pseudomonas aeruginosa, and assess their roles in the drug resistance of Pseudomonas aeruginosa and ability to horizontally transfer across species. Methods:Clinical P. aeruginosa isolates were collected from four hospitals in Sanya, Tangshan, Zhangjiakou, and Beijing. The prevalence of oxacillinases and their flanking gene sequences was analyzed by whole-genome sequencing (NGS) and bioinformatic approaches. Results:A total of 241 isolates of P. aeruginosa were gathered, and 35 blaOXA subtypes were identified through screening of 252 blaOXA genes. These genes were classified into three subfamilies: blaOXA-50-like (241, 95.6%), blaOXA-1-like (9, 3.6%) and blaOXA-10-like (2, 0.8%). Among these, 11 subtypes (11, 31.4%) were novel blaOXA subtypes. Nine of these belonged to the blaOXA-50-like subfamily and were designated as blaOXA-1244, blaOXA-1245, blaOXA-1246, blaOXA-1250, blaOXA-1252, blaOXA-1253, blaOXA-1254, blaOXA-1255, and blaOXA-1256. The remaining two belonged to the blaOXA-10-like subfamily and were named blaOXA-1247 and blaOXA-1248. Compared to the amino acid sequence of OXA-10, the newly identified subtype OXA-1247 exhibited a mutation at position 117, where a valine was replaced by a leucine. This change was thought to improve the enzyme′s ability to hydrolyze carbapenems. In the analysis of the flanking sequences of the blaOXA genes, Class I integrons were identified in four bacterial strains. The variable regions of these integrons carried three distinct patterns of resistance gene cassettes: aac( 6′) -Ib-blaOXA-1247-ant( 3′′) -Ia, aac( 6′) -Ib-blaOXA-1248 and aac( 6′) -Ib- blaIMP-45-blaOXA-1-catB3. Among these, the strain BJ2326 carried a class I integron that was connected to the downstream IS CR1 element to form a composite class I integron structure, additionally carrying the resistance gene blaPER-1. Out of the 223 non-wild-type P. aeruginosa strains, 127 strains exhibited non-wild-type profiles to the four beta-lactam antibiotics MEM, CAZ, FEP, and TZP, with the combination of MEM+CAZ+FEP being the most prevalent, representing 57.0% of the total. Conclusions:The blaOXA genes in 241 clinical P. aeruginosa strains showed diversity. Some blaOXA genes had a co-transfer risk with the metallo-β-lactamase resistance gene blaIMP-45. Among the 11 newly discovered blaOXA subtypes, the new subtype OXA-1247 may have carbapenemase activity and potential for horizontal transfer.

Objective:To analyze the carriage status, subtype distribution and flanking gene sequence characteristics of oxacillinases (OXA enzyme) in 241 clinical strains of Pseudomonas aeruginosa, and assess their roles in the drug resistance of Pseudomonas aeruginosa and ability to horizontally transfer across species. Methods:Clinical P. aeruginosa isolates were collected from four hospitals in Sanya, Tangshan, Zhangjiakou, and Beijing. The prevalence of oxacillinases and their flanking gene sequences was analyzed by whole-genome sequencing (NGS) and bioinformatic approaches. Results:A total of 241 isolates of P. aeruginosa were gathered, and 35 blaOXA subtypes were identified through screening of 252 blaOXA genes. These genes were classified into three subfamilies: blaOXA-50-like (241, 95.6%), blaOXA-1-like (9, 3.6%) and blaOXA-10-like (2, 0.8%). Among these, 11 subtypes (11, 31.4%) were novel blaOXA subtypes. Nine of these belonged to the blaOXA-50-like subfamily and were designated as blaOXA-1244, blaOXA-1245, blaOXA-1246, blaOXA-1250, blaOXA-1252, blaOXA-1253, blaOXA-1254, blaOXA-1255, and blaOXA-1256. The remaining two belonged to the blaOXA-10-like subfamily and were named blaOXA-1247 and blaOXA-1248. Compared to the amino acid sequence of OXA-10, the newly identified subtype OXA-1247 exhibited a mutation at position 117, where a valine was replaced by a leucine. This change was thought to improve the enzyme′s ability to hydrolyze carbapenems. In the analysis of the flanking sequences of the blaOXA genes, Class I integrons were identified in four bacterial strains. The variable regions of these integrons carried three distinct patterns of resistance gene cassettes: aac( 6′) -Ib-blaOXA-1247-ant( 3′′) -Ia, aac( 6′) -Ib-blaOXA-1248 and aac( 6′) -Ib- blaIMP-45-blaOXA-1-catB3. Among these, the strain BJ2326 carried a class I integron that was connected to the downstream IS CR1 element to form a composite class I integron structure, additionally carrying the resistance gene blaPER-1. Out of the 223 non-wild-type P. aeruginosa strains, 127 strains exhibited non-wild-type profiles to the four beta-lactam antibiotics MEM, CAZ, FEP, and TZP, with the combination of MEM+CAZ+FEP being the most prevalent, representing 57.0% of the total. Conclusions:The blaOXA genes in 241 clinical P. aeruginosa strains showed diversity. Some blaOXA genes had a co-transfer risk with the metallo-β-lactamase resistance gene blaIMP-45. Among the 11 newly discovered blaOXA subtypes, the new subtype OXA-1247 may have carbapenemase activity and potential for horizontal transfer.

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.

Objective To compare the biomechanical characteristics of new assembly of locking compression plate (NALCP) and locking compression plate (LCP) in internal fixation of femoral shaft comminuted fractures.Methods The preparation of a femoral shaft wedge fracture model (AO type 32-C2.1),six pairs of (12) femoral specimens were collected and divided into two groups randomly,with six in each group.The Group A was made up of the new assembly of locking compression plate fixation model (NALCP),and Group B the locking compression plate fixation model (LCP).The biomechanical properties of steel plates in two groups were tested by axial loading and torsional loading tests.The relative maximum displacement of fracture blocks in two groups on the X,Y and Z axes (the coronal axis was set as X axis,through the medial and lateral femur;the transverse axis was set as Z axis,through the femoral intercondylar fossa,perpendicular to the X axis;the sagittal axis was set as Y axis,perpendicular to the X and Z axis),the maximum strain,and the average strain of the steel plate were recorded.Strain distribution nephogram was produced,and the axial loading fatigue test results of Group A were recorded.Results Axial loading test:the relative maximum displacement of fracture in Group A on X,Y and Z axis were smaller than those in Group B (P ＜0.05 or 0.01);the main strain of Group A was greater than that of Group B (P ＜0.01);there was no significant difference in the average strain between Group A and Group B (P ＞ 0.05).Torsional loading test:The relative maximum displacement of fracture in Group A on X and Z axis was smaller than that of Group B (P ＜ 0.01);there was no statistically significant difference between the two groups on the Y axis (P ＞ 0.05);the main strain of plate in Group A was greater than that in Group B (P ＜ 0.01);there was no statistically significant difference in the average strain between Group A and Group B (P ＞ 0.05).There was no obvious difference in strain distribution between the two groups.In Group A,the fatigue test of axial cyclic loading was performed for 1 million times,and the NALCP was intact without deformation,loosening,or rupture.Conclusion NALCP can provide strong mechanical stability for comminuted femoral fracture.The design of bridge steel plate is reasonable,which can effectively avoid stress concentration,reduce the stress shielding of steel plate,and facilitate bone healing.

Objective To compare the stress and its distribution between our self-designed new assembly of locking compression plate (NALCP) of low elastic modulus versus conventional locking compression plate (LCP) in fixation of femoral comminuted fractures.Methods Six pairs of cadaveric femur were used to create models of middle femoral comminuted fracture.The femoral fracture models were fixated respectively by NALCP of Ti2448 with low elastic modulus (E =30 Gpa) (NALCP group) and conventional LCP of Ti-6Al-4V with high elastic modulus (E =110 Gpa) (LCP group).Axial and torsion loads were applied on the models in the 2 groups to simulate those on one leg when a person slowly walks.The relative maximum displacements on the X,Y and Z axes of fracture fragments,and the maximum and average strains of the plate were recorded in the 2 groups.Nephograms of strain distribution were made for the 2 groups.The results of fatigue test under axial loads were recorded for NALCP group.Results Both the axial and torsion loading tests showed significantly larger principal and average strains in NACLP group than in LCP group (P ＜ 0.05).However,there were no significant differences between the 2 groups in the relative maximum displacements of fracture fragments on X,Y or Z axis (P ＞ 0.05).The plate strain nephograms for the 2 groups showed consistent strain distributions.The plates in NALCP group survived 1,000,000 fatigue tests under axial loads,without any deformation,loosening or breakage.Conclusion As our NALCP of low elastic modulus may be better in stress transmission and distribution,it can effectively reduce the effect of stress-shielding and promote bone healing.

Objective To investigate the molecular mechanism of Twist induced-drug resistance in human lung cell line A549 cells.Methods The sensibility of A549 cells over-expressed Twist to cisplatin was measured by CCK8 assay. The expressions of Twist,MRP1,MRP2 and MRP3 were detected by RT-PCR. Western blotting was used to detect the expressions of Twist and MRP3 in A549 cells over-expressed Twist.The correlation between Twist and MRP3 expressions in 30 cases of lung cancer tissues was detected with immunohistochemistry. Results The viability of A549 cells over-expressed Twist was significant better than the control group under cispla-tin(32,64,128 μmol/L)treatment(0.79 ± 0.039,0.63 ± 0.048,0.46 ± 0.039 vs.0.53 ± 0.039,0.41 ± 0.043, 0.27 ± 0.063,respectively). After knockdown of MRP3 expression,it reversed drug resistance induced by Twist. Clinically,the expressions of Twist and MRP3 in lung cancer tissues were significantly relevant(P < 0.05). Conclusions Twist induces the drug resistance of human lung cell line A549 to cisplatin via up-regulating the expression of MRP3.

Objective:To investigate the role of pleiotrophin (PTN) gene in carcino genesis using cDNA microarray and in situ hybridization. Methods:The expression of PTN gene in 5 cases of glioma, 10 laryngeal squamous cell carcinoma, 6 cases of hepatocarcinoma, and normal controls were detected by BioDoor 4096 type cDNA microarray and in situ hybridization. Results: The expression of PTN gene in carcinoma samples were significantly higher than in normal controls by cDNA microarray, the results was the same as by in situ hybridization. Conclusion: cDNA microarray is an effective technique in analysis of functional study of associated genes in carcinoma. High expression of PTN gene might be correlated with mechanism of multiple carcinoma. [