Objective:To screen plasma exosomal protein molecular markers in patients with spinal cord injury (SCI) by applying Label-Free quantification and bioinformatics analysis.Methods:Fifty plasma specimens from the First Affiliated Hospital of Nanjing Medical University (from January 2021 to June 2022) were collected from SCI patients and healthy people, respectively. Plasma exosomes were isolated using ultracentrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. Plasma exosomal differentially expressed proteins (DEPs) were analyzed using Label-Free quantitative proteomics, and DEPs were characterized, annotated, and enriched based on Gene Ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) databases. The screened DEPs were validated by western blot and enzyme linked immunosorbent assay (ELISA) using plasma exosomal specimens.Results:According to the spinal cord injury classification of the American Spinal Injury Association, 14 cases were grade A, 19 cases were grade B, 12 cases were grade C, and 5 cases were grade D. Plasma exosomes of SCI patients and control groups showed typical cup-like morphology, with diameters mainly ranging from 30-200 nm. A total of 493 exosomal proteins were identified by Label-Free quantification, and 126 proteins were screened for differential expression, of which 38 were up-regulated and 88 were down-regulated. GO annotation revealed that DEPs were mainly involved in functions such as protein activation cascade, complement activation and immune response. KEGG pathway analysis revealed that DEPs were involved in biological pathways such as complement and coagulation cascade reactions, proteasome and neurodegenerative disease pathways. Two candidate proteins, APOB and S100A9, were initially screened based on quantitative results from proteomics and bioinformatics analyses. Western blot results showed that the relative expression of S100A9 protein in plasma exosomes of 30 SCI patients (1.62±0.19) was elevated compared with that of 30 control groups (0.86±0.24), and the difference was statistically significant ( t=8.55, P<0.001), while the relative expression of APOB protein (1.06±0.13 and 1.02±0.23) were not statistically significant ( t=0.46, P=0.653). The results of ELISA analysis showed that the expression of S100A9 in plasma exosomes of patients with different degrees of SCI (grade A 197.7±11.7 pg/ml, grade B 151.7±15.2 pg/ml, grade C 136.3±14.7 pg/ml) had statistical significance ( F=69.94, P<0.001), the higher the severity of SCI, the higher the expression of S100A9 in plasma exosomes (A vs. B, q=13.11, P<0.001; A vs. C, q=15.66, P<0.001; B vs. C, q=4.19, P=0.005). Conclusion:S100A9 is a potentially valid plasma exosomal molecular marker for assessing the severity of SCI.

Objective:To investigate the clinical characteristics and prognosis of patients with RUNX1-RUNX1T1 fusion gene-positive acute myeloid leukemia (AML) with ASXL2 gene mutation.Methods:The clinical data of 145 newly diagnosed RUNX1-RUNX1T1 fusion gene-positive AML patients treated at the Second Hospital Center of Shanxi Medical University from October 2010 to March 2021 were retrospectively analyzed. Sanger sequencing was used to detect the gene mutation. According to the presence or absence of ASXL2 gene mutation, the patients were divided into mutation group and non-mutation group. The clinical characteristics, gene mutations and prognosis were compared among the two groups.Results:Among 145 AML patients with positive RUNX1-RUNX1T1 fusion gene, we identified recurrent mutations of c-kit, ASXL2, N/KRAS, FLT3, ASXL1, TET2, NPM1 and DNMT3A genes, with mutation rates of 40.7% (59/145), 20.7% (30/145), 15.9% (23/145), 12.4% (18/145), 11.7% (17/145), 11.0% (16/145), 5.5% (8/145), and 2.1% (3/145), respectively. A total of 18 mutation sites were detected in 30 patients with ASXL2 gene mutations including 5 point mutations and 13 frameshift mutations, which mainly occured in the exons 12 and 13. Lactate dehydrogenase (LDH) at initial diagnosis of 30 AML patients with ASXL2 mutation was lower than that of those with ASXL2 non-mutation ( Z = 2.34, P = 0.020), while prothrombin time (PT) of AML patients with ASXL2 mutation was longer than that of those with ASXL2 non-mutation ( Z = 1.99, P = 0.047). A total of 21 (21/30, 70%) patients simultaneously had other gene mutations. The incidence of RAS mutations in patients with ASXL2 mutation was higher than that those with ASXL2 non-mutation, and the difference was statistically significant [30.0% (9/30) vs. 12.1% (14/115), χ2 = 4.41, P = 0.036]. There were no statistically significant differences in complete remission rate [86.7% (26/30) vs. 74.8% (86/115)] and recurrence rate [43.3% (13/30) vs.31.3% (36/115)] of patients with ASXL2 mutation and ASXL2 non-mutation ( χ2 = 0.39, P = 0.534; χ2 = 0.54, P = 0.432). The median overall survival (OS) time was 26 months (1-135 months) and 30 months (1-120 months), respectively in patients with ASXL2 mutation and ASXL2 non-mutation; the median disease-free survival (DFS) time was 14 months (0-60 months) and 13 months (0-94 months), respectively in patients with ASXL2 mutation and ASXL2 non-mutation; and the differences in OS and DFS were not statistically significant of both groups ( χ2 = 0.05, P = 0.822; χ2 = 0.34, P = 0.562). Compared with ASXL1 mutant patients, cases with ASXL2 mutation had higher OS and DFS rates, and the differences were statistically significant ( P = 0.003, P = 0.007). The differences in OS and DFS between patients with ASXL2 mutations and those with positive mutations of c-kit, RAS, FLT3, TET2, NPM1, DNMT3A were not statistically significant (all P > 0.05). Conclusions:RUNX1-RUNX1T1 positive AML patients with ASXL2 mutation tend to have low LDH and high PT, and often coexist with RAS mutations, and their prognosis is better than that in patients with ASXL1 positive mutation.