The amino-terminal pro-C-type natriuretic peptide(NT-proCNP)is a diagnostic inflam-matory marker clinically used for diagnosing bacterial infections.This study aims to establish a phage display library of single-chain variable fragment(scFv)antibodies against canine NT-proC-NP and to screen for scFvs with high binding affinity to NT-proCNP.Initially,NT-proCNP was prepared using prokaryotic expression system and was used to immunize New Zealand White rab-bits.Upon achieving the desired serum titer,total RNA was extracted from the splenocytes of rab-bits and reverse transcribed into cDNA.Using this cDNA as a template,degenerate primers were employed to amplify the genes of the rabbit antibody light chain variable region(VL)and heavy chain variable region(VH).The VL and VH regions were spliced together to form a complete scFv fragment via overlap extension PCR.The scFv was then ligated into the phagemid pComb3XSS and electroporated into competent E.coli TG1 cells to construct a rabbit-derived anti-NT-proCNP scFv immunological library.This library underwent four rounds of enrichment and screening to isolate specific single-chain antibodies.The selected antibody was subsequently ex-pressed in a soluble form within a prokaryotic system,and its immunological activity was evalua-ted.Using phage display technology,this study successfully identified a single-chain antibody scFv-1-CNP with strong antigen-binding activity and genetic sequence characteristics of scFvs,providing a research direction for further exploration of scFv applications in the detection of NT-proCNP.

The amino-terminal pro-C-type natriuretic peptide(NT-proCNP)is a diagnostic inflam-matory marker clinically used for diagnosing bacterial infections.This study aims to establish a phage display library of single-chain variable fragment(scFv)antibodies against canine NT-proC-NP and to screen for scFvs with high binding affinity to NT-proCNP.Initially,NT-proCNP was prepared using prokaryotic expression system and was used to immunize New Zealand White rab-bits.Upon achieving the desired serum titer,total RNA was extracted from the splenocytes of rab-bits and reverse transcribed into cDNA.Using this cDNA as a template,degenerate primers were employed to amplify the genes of the rabbit antibody light chain variable region(VL)and heavy chain variable region(VH).The VL and VH regions were spliced together to form a complete scFv fragment via overlap extension PCR.The scFv was then ligated into the phagemid pComb3XSS and electroporated into competent E.coli TG1 cells to construct a rabbit-derived anti-NT-proCNP scFv immunological library.This library underwent four rounds of enrichment and screening to isolate specific single-chain antibodies.The selected antibody was subsequently ex-pressed in a soluble form within a prokaryotic system,and its immunological activity was evalua-ted.Using phage display technology,this study successfully identified a single-chain antibody scFv-1-CNP with strong antigen-binding activity and genetic sequence characteristics of scFvs,providing a research direction for further exploration of scFv applications in the detection of NT-proCNP.

Objective:To analyze the clinical features of MDA5 antibody positive dermatomyositis (MDA5-DM) and to provide evidence for early diagnosis and treatment.Methods:From March 2019 to June 2021, 272 patients with anti-MDA5-DM from the Nanjing Medical University myositis-associated interstitial lung disease cohort were enrolled, with 76 patients with anti-synthetase syndrome (ASS) as the control group. The clinical characteristics and the occurrence of interstitial lung disease were analyzed. T-test was used for normally distributed and variance-homogeneous independent samples, Mann-Whitney U test for non-normally distributed data, and chi-square test or Fisher′s exact test for dichotomous variables. Results:Among the 272 anti-MDA5-DM patients, 88.6% (241/272) developed interstitial lung disease (ILD), and 33.8% (92/272) developed rapidly progressive ILD (RP-ILD). The six-month all-cause mortality rate of anti-MDA5-DM patients was 16.9% (46/272), and it was as high as 47.8% (44/92) for those with RP-ILD. Compared with ASS patients, anti-MDA5-DM patients had a significantly higher proportion of males, arthritis, Gottron's sign, heliotrope rash, V-sign, periungual erythema, and skin ulcers ( P<0.05). The levels of ALT, AST, and ferritin were significantly increased ( P<0.05). Compared with non-RP-ILD patients, RP-ILD patients had a significantly higher proportion of males [35.9%(33/92) vs. 23.3%(42/180), χ2=4.79, P=0.029], higher levels of LDH [387 (276, 547) U/L vs. 310 (245, 400) U/L, Z=-3.67, P<0.001], ESR [45.5 (29.25, 63.25) mm/1 h vs. 31.2 (20, 51) mm/1 h, Z=-3.71, P<0.001], CRP [10.9 (4.1, 25.2) mg/L vs. 4.54 (2.58, 9.08) mg/L, Z=-4.97, P<0.001], ferritin [1 340 (650, 2 000) ng/ml vs. 556 (203, 1 186) ng/ml, Z=-4.40, P<0.001], and a higher proportion of anti-Ro52 antibody and anti-MDA5 antibody co-positivity [87.0%(80/92) vs. 52.2%(94/180), χ2=31.87, P<0.001]. Conclusion:Anti-MDA5-DM patients are prone to develop RP-ILD and have poor prognosis.

Objective To analyze the clinical characteristics and short-term prognostic factors in acute cerebral infarction patients who underwent recanalization. Methods This retrospective study enrolled 94 cases of acute cerebral ischemic patients in the First Affiliated Hospital of Nanjing Medical University between October 2014 and August 2016. Based on the clinical characteristics of the enrolled patients, a multivariate Logistic regression model was established to analyze the risk factors of unfavorable prognosis. Besides, patients were further divided into good collateral circulation group (1-2) and poor collateral circulation group (3-5) according to the Pial Collateral score, and the prognosis improvement rates between patients recanalized within 4 h and over 4 h were analyzed in each group. Chi-square test or Fisher's exact test was used to analyze statistical difference as indicated. Results By multivariate Logistic regression analysis, age older than 70 years old (OR=2.651, 95％CI: 1.013-6.937)and poor collateral circulation (OR=3.160, 95％CI: 1.113-8.977) were independent risk factors of short-term poor prognosis. In the poor collateral circulation subgroup, patients recanalized within 4 h exerted a relatively better prognosis than patients recanalized over 4 h (42.9％ vs.10.5％, P=0.047). However, the effect of recanalization duration on the prognosis in the good collateral circulation subgroups was not statistically significant (42.9％ vs. 10.5％, P=0.047), however, the effect of recanalization duration on prognosis in patients with good collateral circulation was not statistically significant (58.3％ vs. 37.8％, P=0.117). Conclusions For patients with acute cerebral infarction, age and collateral circulation status may influence the prognosis of recanalization therapy. The treatment time had a significant influence on the prognosis in patients with poor collateral, while it had minimal significance on patients with good collateral.

Objective The goal of this study is to compare the prognosis of recombinant tissue plasminogen activator (rt-PA) thrombolysis for middle cerebral artery (MCA) occlusion with patients with good and poor cerebral collateral circulation.Methods This retrospective study included 49 patients diagnosed with acute MCA occlusion and treated with rt-PA in the First Affiliated Hospital of Nanjing Medical University between October 1,2014 and February 1,2016.Patients were divided into good collaterals group (n =31) and poor collaterals group (n =18) according to their distribution of leptomeningeal arteries with CTA.Thirty day mortality rate,the incidence of symptomatic intracranial hemorrhage,24h and 30 day Stroke scores with National Institute of Health Stroke Scale (NIHSS) were compared between the two groups.Corrected chi-squared test,Fisher's exact test,or t test was used to statistical analysis as appropriate.Results The 30 day mortality rate of good collaterals group was significantly lower than that of poor collaterals group (0％ vs.16.7％,P ＜ 0.05).There were no significant differences in the incidence of symptomatic intracranial hemorrhage and 24h NIHSS score between the two groups (P ＞ 0.05),however,30 day NIHSS score of good collaterals group was significantly lower than that of poor collaterals group (7.2 ± 3.1 vs.9.6 ± 2.7,P ＜ O.05).Conclusion For patients with MCA occlusion and receiving intravenous thrombolysis,good cerebral collateral circulation may reduce their mortality and improve their clinical outcome after thrombolysis.However,good cerebral collateral circulation does not reduce the risk of symptomatic intracranial hemorrhage in those patients.