AIM:To construct nine novel L-asparaginase mutants and study their enzyme-activity.METHODS:The mutants were constructed using overlap extension PCR according to the principle of alanine-scanning mutagenesis. The enzyme-activity was detected by Nessler's method. RESULTS:The DNA sequencing showed that the mutagenesis was consistent with the theoretical prediction. The enzyme-activity assay demonstrated that each mutant possessed enzyme activity equal to the original enzyme. CONCLUSION:Through gene modification,epitop of L-asparaginase was changed without activity loss.These results provide foundation for further study of the structure-function relationship of L-asparaginase.

Objective:To explore the correlation and mechanism between thalamic network abnormality and cognitive decline in patients with temporal lobe epilepsy (TLE).Methods:A total of 53 patients with unilateral TLE were consecutively enrolled through the epilepsy clinic of the First Affiliated Hospital of Guangxi Medical University from December 2018 to February 2020. During the same recruitment interval, 37 health controls(HC) with matching demographic characteristic were recruited. All subjects were received the Montreal cognitive assessment(MoCA) test and multimodal MRI scanning. Voxel-based morphometry method was used to study the changes of thalamic gray matter volume in patients with unilateral TLE. The structural covariance network and functional connectivity network based on seed points were used to analyze the changes of thalamic network in TLE patients. In addition, the correlations among abnormal thalamic structure, thalamic network and cognitive function score were analyzed. SPSS 22.0 software was used for statistical analysis. Independent sample t-test and Mann Whitney U test were used for inter group comparison. In order to explore the relationship between thalamus and thalamic network and cognitive performance in TLE patients, thalamic volume and gray matter volume and functional connection value of brain areas with abnormal synergistic changes were extracted and correlated with MoCA score. Results:The total score of MoCA in TLE patients (27.0(25.0, 29.0)) was significantly decreased compared with HC (29.0(28.0, 30.0))( Z=-4.601, P<0.001). Whole brain gray matter volume analysis showed that compared with HCTLE patients showed significant volume reduction in left cerebellum, right temporal pole, right fusiform gyrus, straight gyrus, bilateral middle temporal gyrus, thalamus, medial and paracingulate gyrus (GRF adjusted, voxel-level P<0.001 and cluster-level P<0.05). The thalamus-associated structural covariance network analysis revealed that compared with healthy controls, TLE patients exhibited decreased connectivity in right fusiform gyrus (MNI: x=28.5, y=-15.0, z=-34.5), left insula (MNI: x=-33.0, y=-18.0, z=-1.5), right middle temporal gyrus (MNI: x=55.5, y=-51.0, z=9.0), left complementary motor area (MNI: x=-10.5, y=1.5, z=57.0) and right posterior central gyrus (MNI: x=31.5, y=-33.0, z=51.0) ( P<0.001, cluster > 100). The thalamus-associated functional connectivity network analysis revealed that TLE patients exhibited decreased connectivity in left insula (MNI: x=-38, y=-7, z=-7), left lingual gyrus (MNI: x=-6, y=-81, z=-12), right lingual gyrus (MNI: x=15, y=-105, z=0) and left triangular inferior frontal gyrus (MNI: x=-39, y=36, z=-6) (GRF correction, voxel-level P<0.001 and cluster-level P<0.05). Volume of left insula which had decreased structural connectivity with thalamus were positively correlated with the MoCA score in TLE patients( r=0.279, P=0.043). Volume of left complementary motor area which had decreased structural connectivity with thalamus was positively correalated with the MoCA score and language score in TLE patients( r=0.323, P=0.018; r=0.334, P=0.015). Volume of left lingual gyrus which had decreased functional connectivity with thalamus was negatively correalated with the memory score in TLE patients ( r=-0.331, P=0.016). Conclusion:Thalamic volume, thalamic structural covariant network and functional connection network are changed in TLE patients. The abnormality of thalamic network is associated with cognitive performance in TLE patients, which may be the neural mechanism of thalamus participating in the cognitive impairment of TLE patients.