Objective: To establish a method for the genotyping of fetal M blood group antigen by extracting cell-free fetal DNA (cff-DNA) from maternal plasma, so as to guide the management of M antigen-negative pregnant women with IgG anti-M antibody during pregnancy. Methods: A realtime fluorescent quantitative PCR (realtime PCR) method was established. The specificity and sensitivity of the method were validated by dilution of genomic DNA. Subsequently, a total of 12 M antigen-negative pregnant women were enrolled. The cff-DNA was extracted from maternal plasma, and fetal M antigen genotyping was performed by realtime PCR. Fetuses were classified as M-positive or M-negative according to the presence or absence of amplification curve. The accuracy of the method was validated by comparing fetal M antigen genotyping results with the serological results using the cord or peripheral blood of the neonate at birth. Results: Among the 12 M antigen-negative pregnant women, anti-M was detected in five cases, of which four cases had IgG anti-M, and one case had fetal anemia. The results of fetal M antigen genotyping showed that 9 cases were M-positive (9/12, 75%) and 3 cases were M-negative (3/12, 25%). Serological results of blood samples collected after birth from four M-positive fetuses and one M-negative fetus were consistent with the genotyping results. Conclusion: We have, for the first time, established a non-invasive prenatal genotyping method for fetal M antigen using maternal plasma cff-DNA, and preliminarily demonstrated the feasibility of this method.

Objective To explore the biological functions and mechanisms of PIWI-interacting RNA(piRNA)in cervical cancer(CC).Methods RT-qPCR was used to detect the expression level of piRNA-2732 in CC tissue,CaSki cells and End1/E6E7 cells.EpiQuik m6A RNA methylation quantification kit was used to detect the methylation level of m6A RNA in CaSki cells.The expression levels of methyltransferases(METTL3,METTL14 and WTAP)and demethylases(FTO,ALKBH5)mRNA in CaSki cells were detected by RT-qPCR.After culturing CaSki cells to logarithmic growth stage,they were divided into six groups:piRNA-2732 mimic negative control group(mi-NC group),piRNA-2732 mimic group(mi-2732 group),piRNA-2732 inhibitor negative control group(in-NC group),piRNA-2732 inhibitor group(in-2732 group),piRNA-2732 mimic+METTL3 knockdown control group(mi-2732+si-NC group),and piRNA-2732 mimic+METTL3 knockdown group(mi-2732+si-METTL3 group).The viability of CaSki cells was detected by CCK8 assay.Colony formation assay was used to detect the proliferation ability of CaSki cells.Transwell experiment was used to detect the migration and invasion ability of CaSki cells.RT-qPCR and Western blot were used to detect the expression of methyltransferase like protein 3(METTL3).Transfected METTL3 wild-type(METTL3-WT)and METTL3 mutant(METTL3-MUT)in the mi-NC group,mi-2732 group,in-NC group,and in-2732 group respectively,and detected the effect of piRNA-2732 on METTL3 through dual luciferase reporter gene assay.Results Compared with the adjacent tissues,the expression of piRNA-2732(3.84±1.08 vs 1.32±0.53)was significantly higher in the cancer tissues of CC patients,and the difference was statistically significant(t=5.115,P<0.001).Compared with end1/E6E7 cells,the expression of piRNA-2732(1.00±0.13 vs 1.67±0.16)in CaSki cells was significantly higher,and the difference was statistically significant(t=5.632,P<0.01).Compared with mi-NC group,mi-2732 group promoted the viability,proliferation,migration and invasion of CaSki cells,and the differences were statistically significant(t=4.410～11.040,all P<0.01).Compared with mi-NC group,mi-2732 group increased m6A RNA methylation level and METTL3 mRNA and protein,the differences were statistically significant(t=6.176,9.211,12.550,all P<0.05).The results of dual luciferase reporter gene testing showed that compared with the mi-NC+METTL3-WT group,the relative luciferase activity of mi-2732+METTL3-WT group was significantly increased(t=11.850).Compared with mi-2732+METTL3-WT group,the relative luciferase activity of mi-2732+METTL3-MUT group was significantly lower(t=12.740),and the difference was statistically significant(all P<0.000 1).Compared with in NC+METTL3-WT group,the relative luciferase activity of in-2732+METTL3-WT group was significantly lower(t=7.828),compared with in-2732+METTL3-WT group,the relative luciferase activity of CaSki cells in in-2732+METTL3-MUT group was significantly increased(t=8.146),and the difference was statistically significant(all P<0.001).Compared with mi-2732+si-NC group,the expression level of m6A in mi-2732+si-METTL3 group was significantly lower,and the difference was statistically significant(t=7.630,P<0.01).Compared with mi-2732+si-NC group,the proliferation ability,colony number,cell migration and invasion ability of CaSki cells in mi-2732+si-METTL3 group were significantly decreased,and the differences were statistically significant(t=3.695～4.891,all P<0.001).Conclusion piRNA-2732 is overexpressed in CC tissues and cells,and piRNA-2732 promotes tumor development in CC through METTL3 mediated m6A methylation.

Objective To explore the biological functions and mechanisms of PIWI-interacting RNA(piRNA)in cervical cancer(CC).Methods RT-qPCR was used to detect the expression level of piRNA-2732 in CC tissue,CaSki cells and End1/E6E7 cells.EpiQuik m6A RNA methylation quantification kit was used to detect the methylation level of m6A RNA in CaSki cells.The expression levels of methyltransferases(METTL3,METTL14 and WTAP)and demethylases(FTO,ALKBH5)mRNA in CaSki cells were detected by RT-qPCR.After culturing CaSki cells to logarithmic growth stage,they were divided into six groups:piRNA-2732 mimic negative control group(mi-NC group),piRNA-2732 mimic group(mi-2732 group),piRNA-2732 inhibitor negative control group(in-NC group),piRNA-2732 inhibitor group(in-2732 group),piRNA-2732 mimic+METTL3 knockdown control group(mi-2732+si-NC group),and piRNA-2732 mimic+METTL3 knockdown group(mi-2732+si-METTL3 group).The viability of CaSki cells was detected by CCK8 assay.Colony formation assay was used to detect the proliferation ability of CaSki cells.Transwell experiment was used to detect the migration and invasion ability of CaSki cells.RT-qPCR and Western blot were used to detect the expression of methyltransferase like protein 3(METTL3).Transfected METTL3 wild-type(METTL3-WT)and METTL3 mutant(METTL3-MUT)in the mi-NC group,mi-2732 group,in-NC group,and in-2732 group respectively,and detected the effect of piRNA-2732 on METTL3 through dual luciferase reporter gene assay.Results Compared with the adjacent tissues,the expression of piRNA-2732(3.84±1.08 vs 1.32±0.53)was significantly higher in the cancer tissues of CC patients,and the difference was statistically significant(t=5.115,P<0.001).Compared with end1/E6E7 cells,the expression of piRNA-2732(1.00±0.13 vs 1.67±0.16)in CaSki cells was significantly higher,and the difference was statistically significant(t=5.632,P<0.01).Compared with mi-NC group,mi-2732 group promoted the viability,proliferation,migration and invasion of CaSki cells,and the differences were statistically significant(t=4.410～11.040,all P<0.01).Compared with mi-NC group,mi-2732 group increased m6A RNA methylation level and METTL3 mRNA and protein,the differences were statistically significant(t=6.176,9.211,12.550,all P<0.05).The results of dual luciferase reporter gene testing showed that compared with the mi-NC+METTL3-WT group,the relative luciferase activity of mi-2732+METTL3-WT group was significantly increased(t=11.850).Compared with mi-2732+METTL3-WT group,the relative luciferase activity of mi-2732+METTL3-MUT group was significantly lower(t=12.740),and the difference was statistically significant(all P<0.000 1).Compared with in NC+METTL3-WT group,the relative luciferase activity of in-2732+METTL3-WT group was significantly lower(t=7.828),compared with in-2732+METTL3-WT group,the relative luciferase activity of CaSki cells in in-2732+METTL3-MUT group was significantly increased(t=8.146),and the difference was statistically significant(all P<0.001).Compared with mi-2732+si-NC group,the expression level of m6A in mi-2732+si-METTL3 group was significantly lower,and the difference was statistically significant(t=7.630,P<0.01).Compared with mi-2732+si-NC group,the proliferation ability,colony number,cell migration and invasion ability of CaSki cells in mi-2732+si-METTL3 group were significantly decreased,and the differences were statistically significant(t=3.695～4.891,all P<0.001).Conclusion piRNA-2732 is overexpressed in CC tissues and cells,and piRNA-2732 promotes tumor development in CC through METTL3 mediated m6A methylation.

Objective: To investigate the distribution of GP (B-A-B) hybrid glycophorins in several Chinese minority populations from southern regions of China (Guangdong & Guizhou). Methods: Whole blood samples were collected from 536 blood donors representing 15 different Chinese ethnic minority groups, including She, Bouyei, Yi and Miao, as well as Chuanqing populations. Genomic DNA was extracted and GYP (B-A-B) genotyping was conducted by high resolution melting (HRM) minority method using the GYPB pseudoexon 3-specific primers. Direct sequencing of GYPB pseudoexon 3 was performed in the samples with variant curves. Results: Only one genotype of GP (B-A-B) hybrid glycophorins (GYP Mur/GYPB) was identified among these 536 samples. In total, 15 She (15/162, 9.26%), 18 Bouyei (18/113, 15.93%), 3 Yi (3/79, 3.80%), 3 Chuanqing (3/45, 6.67%), 2 Bai (2/42, 4.76%), 3 Miao (3/40, 7.50%), 1 Shui (1/12, 8.33%), 2 Gelao (2/12, 16.67%), 1 Tujia (1/8, 12.50%) and 1 Dong (1/6, 16.67%) blood donors with heterozygous GYP Mur allele were identified. Among 8 Hui, 5 Manchu, 2 Mongolian, 1 Yao and 1 Li donors, no GYP (B-A-B) hybrid gene carrier was found. In addition, four nucleotide polymorphisms (SNPs) were identified in 6 samples with a variant melting curve detected by HRM. Conclusion: GP. Mur is the most common type of GP (B-A-B) hybrid glycophorins among Chinese minority populations, with frequency varying across different populations. It is recommended to involve GP. Mur reagent cells in the antibody screening cells for populations with a high frequency of GYP Mur allele.

Objective:To evaluate the left ventricular（LV）function in heart transplant（HTx）patients with post-transplant diabetes（PTDM），and to examine the relevance of PTDM and LV function to the patient's prognosis.Methods:Two hundred and thirteen adult HTx patients who underwent echocardiography at Union Hospital，Tongji Medical College，Huazhong University of Science and Technology between January 2018 and January 2022 were prospectively included. The patients were divided into PTDM group（ n=86）and Non-PTDM group（ n=127）. LV function parameters were acquired using conventional and two-dimensional speckle-tracking echocardiography（2D-STE），and were compared between the two groups. The primary endpoints included all-cause mortality or transplant-related readmission. Results:Compared with Non-PTDM group，the LV mass of PTDM group was higher，the LV ejection fraction，LV global longitudinal strain（GLS），peak systolic global longitudinal strain rate，and early diastolic global longitudinal strain rate（dGLSr）were lower（all P＜0.05）. After a median follow-up period of 37.6（29.3）months，27 patients experienced clinical events. A multivariate analysis revealed that PTDM（ HR=2.198，95% CI=1.018-4.743， P=0.045）and low GLS（ HR=6.456，95% CI=2.889-14.426， P＜0.001）were independent predictors of adverse clinical events after adjustment for dGLSr，body mass index and age. After subdividing the two groups into 4 subgroups by the cutoff value of GLS（16.5%），the prognosis was worst for HTx patients with PTDM and low GLS. Conclusions:HTx patients with PTDM have worse LV systolic and diastolic function than those without PTDM. Management of HTx patients with PTDM may be improved using GLS guidance.

Objective:To investigate the diagnostic and differential diagnostic values of midbrain morphological measurements at the mammillary body level on axial cranial MRI in progressive supranuclear palsy (PSP).Methods:This cross-sectional study included 50 patients with clinically diagnosed, probable or possible PSP, admitted to Department of Neurology, Xuanwu Hospital, Capital Medical University from January 2023 to December 2024. Additionally, 44 patients with Parkinson's disease (PD), 30 patients with multiple system atrophy-cerebellar type (MSA-C), and 35 gender- and age-matched healthy controls recruited from the community were chosen. The following midbrain morphological parameters on axial cranial MRI were measured in all participants: distance from the interpeduncular fossa to the aqueduct (IF-AQ), distance from the lateral mesencephalic sulcus to the interpeduncular cistern (LS-IC), distance between the bilateral lateral mesencephalic sulci (D-BMS), cerebral peduncle angle (CPA), distance between the medial aspects of the cerebral peduncles (D-MP), and distance from the line connecting the highest points of the cerebral peduncle to the interpeduncular fossa (PP-IF). Differences in these measurements among the 4 groups were compared, and the diagnostic and differential diagnostic performances of each parameter in PSP was evaluated using receiver operating characteristic (ROC) curve.Results:The PSP group exhibited significantly shorter IF-AQ, LS-IC, and D-BMS distances compared with the other 3 groups ( P<0.05). (1) ROC curve analysis of PSP group versus non-PSP group (MSA-C patients, PD patients, and healthy controls) showed that IF-AQ had an area under the curve (AUC) of 0.977 (95% CI: 0.959-0.995, P<0.001), and at a cutoff of 10.895 mm, IF-AQ demonstrated a sensitivity of 96.0% and a specificity of 89.9% for diagnosing PSP; LS-IC had an AUC of 0.917 (95% CI: 0.868-0.966, P<0.001), and at a cutoff of 10.82 mm, LS-IC demonstrated a sensitivity of 82.0% and a specificity of 89.0% for diagnosing PSP; D-BMS had an AUC of 0.785 (95% CI: 0.704-0.866, P<0.001), and at a cutoff of 20.01 mm, D-BMS demonstrated a sensitivity of 66.0% and a specificity of 83.5% for diagnosing PSP. (2) In distinguishing PSP from MSA-C, IF-AQ achieved an AUC of 0.939 (95% CI: 0.889-0.988, P<0.001), with a sensitivity of 84.0% and a specificity of 90.0% at a cutoff of 10.385 mm; LS-IC achieved an AUC of 0.846 (95% CI: 0.756-0.936, P<0.001), with a sensitivity of 80.0% and a specificity of 76.7% at a cutoff of 10.710 mm; D-BMS achieved an AUC of 0.696 (95% CI: 0.578-0.813, P<0.001), with a sensitivity of 60.0% and a specificity of 80.0% at a cutoff of 19.810 mm. (3) In discriminating PSP from PD, IF-AQ yielded an AUC of 0.986 (95% CI: 0.970-1.000, P<0.001), with a sensitivity of 96.0% and a specificity of 89.2% at a cutoff of 10.955 mm; LS-IC achieved an AUC of 0.937 (95% CI: 0.885-0.988, P<0.001), with a sensitivity of 82.0% and a specificity of 95.5% at a cutoff of 10.820 mm; D-BMS had an AUC of 0.825 (95% CI: 0.740-0.909, P<0.001), with a sensitivity of 60.0% and a specificity of 95.5% at a cutoff of 19.820 mm. Conclusion:IF-AQ, LS-IC, and D-BMS distances on axial cranial MRI at the mamillary body level can diagnose and differentiate PSP to a certain extent, with IF-AQ enjoying the best efficacy.

AIM:To evaluate the safety and toler-ability of single dose oral BTK inhibitor YZJ-3058 tablets under fasting conditions in healthy adults,as well as the pharmacokinetic and pharmacologi-cal characteristics of YZJ-3058 and its metabolites.METHODS:A total of 22 healthy subjects were en-rolled in this experiment and administered a single dose orally.They were divided into three groups:50 mg,100 mg,and 200 mg.Among them,2 sub-jects were enrolled in the 50 mg dose group,and 10 subjects were enrolled in the 100 mg and 200 mg dose groups,respectively.RESULTS:In healthy subjects,YZJ-3058 tablets were administered orally on an empty stomach at doses of 50,100,and 200 mg,with a median Tmax of 1.25 to 2.00 hours and an average Cmax of 62.85,89.44,and 99.20 ng/mL,re-spectively.The average AUC0-t was 183.87,297.72,and 453.98 h·ng-1·mL,respectively.The average AUC0-∞ was 189.30,321.33,and 551.44 h·ng-1·mL,and the median t1/2 was 1.16,5.06,and 7.97 hours,respectively.After a single oral administration of 50,100,and 200 mg YZJ-3058 tablets,the highest target occupancy rate was achieved at 4 hours.The average BTK occupancy rates at 24 hours after ad-ministration were 88.95％,96.73％,and 99.24％,re-spectively.The average BTK occupancy rates at 48 hours after administration were 75.65％,89.80％,and 96.68％,respectively.No serious adverse events or adverse events leading to withdrawal oc-curred,and all subjects had good tolerability.CON-CLUSION:YZJ-3058 tablets have good safety and tolerability for single oral administration on an empty stomach in healthy subjects within the dose range of 50-200 mg.Cmax and AUC increase with dose,with fast absorption and saturation.The ter-minal elimination rate gradually slows down with dose increase,and it has a significant and sus-tained occupying effect on BTK targets.

AIM:To evaluate the safety and toler-ability of single dose oral BTK inhibitor YZJ-3058 tablets under fasting conditions in healthy adults,as well as the pharmacokinetic and pharmacologi-cal characteristics of YZJ-3058 and its metabolites.METHODS:A total of 22 healthy subjects were en-rolled in this experiment and administered a single dose orally.They were divided into three groups:50 mg,100 mg,and 200 mg.Among them,2 sub-jects were enrolled in the 50 mg dose group,and 10 subjects were enrolled in the 100 mg and 200 mg dose groups,respectively.RESULTS:In healthy subjects,YZJ-3058 tablets were administered orally on an empty stomach at doses of 50,100,and 200 mg,with a median Tmax of 1.25 to 2.00 hours and an average Cmax of 62.85,89.44,and 99.20 ng/mL,re-spectively.The average AUC0-t was 183.87,297.72,and 453.98 h·ng-1·mL,respectively.The average AUC0-∞ was 189.30,321.33,and 551.44 h·ng-1·mL,and the median t1/2 was 1.16,5.06,and 7.97 hours,respectively.After a single oral administration of 50,100,and 200 mg YZJ-3058 tablets,the highest target occupancy rate was achieved at 4 hours.The average BTK occupancy rates at 24 hours after ad-ministration were 88.95％,96.73％,and 99.24％,re-spectively.The average BTK occupancy rates at 48 hours after administration were 75.65％,89.80％,and 96.68％,respectively.No serious adverse events or adverse events leading to withdrawal oc-curred,and all subjects had good tolerability.CON-CLUSION:YZJ-3058 tablets have good safety and tolerability for single oral administration on an empty stomach in healthy subjects within the dose range of 50-200 mg.Cmax and AUC increase with dose,with fast absorption and saturation.The ter-minal elimination rate gradually slows down with dose increase,and it has a significant and sus-tained occupying effect on BTK targets.

Objective:To evaluate the left ventricular（LV）function in heart transplant（HTx）patients with post-transplant diabetes（PTDM），and to examine the relevance of PTDM and LV function to the patient's prognosis.Methods:Two hundred and thirteen adult HTx patients who underwent echocardiography at Union Hospital，Tongji Medical College，Huazhong University of Science and Technology between January 2018 and January 2022 were prospectively included. The patients were divided into PTDM group（ n=86）and Non-PTDM group（ n=127）. LV function parameters were acquired using conventional and two-dimensional speckle-tracking echocardiography（2D-STE），and were compared between the two groups. The primary endpoints included all-cause mortality or transplant-related readmission. Results:Compared with Non-PTDM group，the LV mass of PTDM group was higher，the LV ejection fraction，LV global longitudinal strain（GLS），peak systolic global longitudinal strain rate，and early diastolic global longitudinal strain rate（dGLSr）were lower（all P＜0.05）. After a median follow-up period of 37.6（29.3）months，27 patients experienced clinical events. A multivariate analysis revealed that PTDM（ HR=2.198，95% CI=1.018-4.743， P=0.045）and low GLS（ HR=6.456，95% CI=2.889-14.426， P＜0.001）were independent predictors of adverse clinical events after adjustment for dGLSr，body mass index and age. After subdividing the two groups into 4 subgroups by the cutoff value of GLS（16.5%），the prognosis was worst for HTx patients with PTDM and low GLS. Conclusions:HTx patients with PTDM have worse LV systolic and diastolic function than those without PTDM. Management of HTx patients with PTDM may be improved using GLS guidance.