Circular RNA(circRNA),as a kind of non-coding RNA,regulates a variety of biological functions.To explore the effect of circRNA on PEDV replication in the host porcine intestinal epi-thelial cells,this study screened and analyzed the differentially expressed circRNAs by bioinforma-tic software in African Green Monkey renal cells(Vero-E6 cells)infected by porcine epidemic di-arrhea virus(PEDV),the differentially expressed circRNA ssc_circ_PIK3C2A was identified and the secondary structure was analyzed.PCR was used to identify the ssc_circ_PIK3C2A circRNA structure,the model of PEDV-infected IPEC-J2 cells was constructed,the TCID50 test was used to validate the viral titer of PEDV.The expression of circ_PIK3C2A was detected by qRT-PCR in IPEC-J2 infected by PEDV.circ_PIK3C2A qRT-PCR was performed to detect the expression of N gene of PEDV when ssc_circ_PIK3C2A was over-expressed in IPEC-J2 cells.The results showed that ssc_circ_PIK3C2 A is a porcine circular RNA with a typical circular structure,the virus titer of PEDV reached 10-6/mL after PEDV infected IPEC-J2 cells for 48 h,the expression of circ_PIK3C2A increased extremely(P＜0.01)at 6 h after PEDV-infection,with the extension of infec-tion time,its expression gradually decreased,and the expression was the lowest at 24 h,but there was no time-dependent trend.The expression of PEDV N gene decreased significantly when ssc_circ_PIK3C2A was over-expressed in IPEC-J2 cells.In conclusion,when PEDV infects IPEC-J2 cells,the expression of porcine circ_PIK3C2A decreases,and replication of PEDV increases signifi-cantly in IPEC-J2 cells.our result provides a basis for further study of the mechanism of circular RNA on PEDV replication and its physiological activities in host cells in the future.

Objective To investigate the correlation between the expression of human leukocyte antigen class I(HLA-I)and programmed cell death ligand 1(PD-L1)with clinicopathological features and cellular immune infiltration.Methods A total of 150 patients with bladder cancer diagnosed and treated in Anhui Provincial Hospital from May 2020 to April 2023 were retrospectively selected as the study objects.The positive expression rates and positive scores of HLA-I and PD-L1 were compared between cancerous tissues and adjacent tissues.The positive scores of HLA-I and PD-L1 in cancer tissues of patients with different clinical characteristics were compared,and the correlation between HLA-I,PD-L1 and clinical characteristics of patients with bladder cancer was analyzed by Kendall's tau-b method.Logistic regression model was used to establish the combined model parameters of HLA-I and PD-L1 positive scores,and receiver operating characteristic(ROC)curve was drawn to analyze the HLA-I and PD-L1 positive scores and the area under the curve(AUC),sensitivity and specificity of the combined diagnosis of bladder cancer.Results The positive expression rate of HLA-I in cancer tissues was lower than that in paracancer tissues[38.67%(58/150)vs 81.33%(122/150)],while the positive expression rate of PD-L1 was higher than that in paracancer tissues[57.33%(86/150)vs 14.00%(21/150)],and the differences were statistically siginficant(χ2=56.889,61.377,all P<0.05).The HLA-I positive score of cancer tissues was lower than that of paracancer tissues[2.00(1.00,3.00)vs 3.00(3.00,5.00)],while the PD-L1 positive score was higher than that of paracancer tissues[3.00(2.00,5.00)vs 2.00(1.00,2.00)],and the fifferences were statistically significant(Z=-8.409,-6.346,all P<0.05).There was no significant difference in HLA-I and PD-L1 positive scores among different sex,age and tumor diameter(ZHLA-1=-1.834,-0.622,-0.543;ZPD-L1=0.811,0.812,0.919,all P＞0.05).The difference of HLA-I and PD-L1 positive scores among different pathological stages,lymph node metastasis,differentiation degree,CD4+,CD8+and CD68+were statistically significant(ZHLA-1=-7.034～3.814;ZPD-L1=-4.479～3.257,all P＜0.05).Kendall's tau-b correlation analysis showed that HLA-I was negatively correlated with pathological stage,lymph node metastasis,degree of differentiation,and positively correlated with negative infiltration of CD4+,CD8+and CD68+(r=-0.528～-0.286,all P<0.05).PD-L1 was positively correlated with pathological stage,lymph node metastasis,degree of differentiation and negatively correlated with negative infiltration of CD4+,CD8+and CD68+(r=-0.243～0.334,all P<0.05).ROC curve analysis showed that the positive scores of HLA-I and PD-L1 and the AUC values of the combined diagnosis of bladder cancer were 0.773,0.702 and 0.856,respectively.Sensitivity was 61.30%,57.30%and 82.00%.The specificity was 81.30%,86.00%and 73.30%.Conclusion The expression of HLA-I and PD-L1 is abnormal in patients with bladder cancer,and their expression is affected by the positive infiltration of immune cells.Observing the positive expression of HLA-I and PD-L1 is beneficial to provide guidance for clinical diagnosis and treatment.

AIM:To investigate the expression profile and biological significance of long noncoding RNA(lncRNA)zinc finger antisense 1(ZFAS1)in the brains of mice with diabetic encephalopathy(DE).METHODS:Ten db/m mice served as the normal control group,while twenty 22-week-old db/db mice were used to establish the DE model and randomly divided into two subgroups:ten as the db/db model control and the remaining ten receiving ZFAS1 gene knockdown(db/db+sh-ZFAS1)via lentiviral transfection.Weekly measurements of body weight and blood glucose levels were performed.Brain tissues were collected for Nissl staining to evaluate neuronal damage,TUNEL assay to detect apop-tosis,and immunofluorescence staining to examine neural biomarker expression.Serum levels of tumor necrosis factor-α(TNF-α)and oxidative stress markers,including reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px),were determined.Western blot was conducted to quantify the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),p38 mitogen-activated protein kinase and phosphorylated p38(p-p38)in brain tissues.The expression levels of ZFAS1 and caspase-3 mRNA were determined by RT-qPCR.RESULTS:Knockdown of ZFAS1 in db/db mice significantly improved cognitive function,alleviated hippocampal neuronal damage,and reduced body weight and blood glucose levels(P<0.01).More-over,oxidative stress was mitigated,as evidenced by decreased MDA and ROS levels(P<0.01)and increased activity of antioxidant enzymes,GSH-Px,SOD and CAT(P<0.01 or P<0.05).Meanwhile,ZFAS1 silencing down-regulated Bax and p-p38/p38 protein expression(P<0.01 or P<0.05)while up-regulating Bcl-2(P<0.01).Consistently,RT-qPCR confirmed significant down-regulation of ZFAS1 and caspase-3 mRNA levels(P<0.01).CONCLUSION:lncRNA ZFAS1 is highly expressed in the hippocampus of DE mice.Down-regulation of ZFAS1 expression enhances cognitive func-tion,suppresses oxidative stress,and inhibits neuronal apoptosis,thereby attenuating neural damage in DE.

Objective To investigate the correlation between the expression of human leukocyte antigen class I(HLA-I)and programmed cell death ligand 1(PD-L1)with clinicopathological features and cellular immune infiltration.Methods A total of 150 patients with bladder cancer diagnosed and treated in Anhui Provincial Hospital from May 2020 to April 2023 were retrospectively selected as the study objects.The positive expression rates and positive scores of HLA-I and PD-L1 were compared between cancerous tissues and adjacent tissues.The positive scores of HLA-I and PD-L1 in cancer tissues of patients with different clinical characteristics were compared,and the correlation between HLA-I,PD-L1 and clinical characteristics of patients with bladder cancer was analyzed by Kendall's tau-b method.Logistic regression model was used to establish the combined model parameters of HLA-I and PD-L1 positive scores,and receiver operating characteristic(ROC)curve was drawn to analyze the HLA-I and PD-L1 positive scores and the area under the curve(AUC),sensitivity and specificity of the combined diagnosis of bladder cancer.Results The positive expression rate of HLA-I in cancer tissues was lower than that in paracancer tissues[38.67%(58/150)vs 81.33%(122/150)],while the positive expression rate of PD-L1 was higher than that in paracancer tissues[57.33%(86/150)vs 14.00%(21/150)],and the differences were statistically siginficant(χ2=56.889,61.377,all P<0.05).The HLA-I positive score of cancer tissues was lower than that of paracancer tissues[2.00(1.00,3.00)vs 3.00(3.00,5.00)],while the PD-L1 positive score was higher than that of paracancer tissues[3.00(2.00,5.00)vs 2.00(1.00,2.00)],and the fifferences were statistically significant(Z=-8.409,-6.346,all P<0.05).There was no significant difference in HLA-I and PD-L1 positive scores among different sex,age and tumor diameter(ZHLA-1=-1.834,-0.622,-0.543;ZPD-L1=0.811,0.812,0.919,all P＞0.05).The difference of HLA-I and PD-L1 positive scores among different pathological stages,lymph node metastasis,differentiation degree,CD4+,CD8+and CD68+were statistically significant(ZHLA-1=-7.034～3.814;ZPD-L1=-4.479～3.257,all P＜0.05).Kendall's tau-b correlation analysis showed that HLA-I was negatively correlated with pathological stage,lymph node metastasis,degree of differentiation,and positively correlated with negative infiltration of CD4+,CD8+and CD68+(r=-0.528～-0.286,all P<0.05).PD-L1 was positively correlated with pathological stage,lymph node metastasis,degree of differentiation and negatively correlated with negative infiltration of CD4+,CD8+and CD68+(r=-0.243～0.334,all P<0.05).ROC curve analysis showed that the positive scores of HLA-I and PD-L1 and the AUC values of the combined diagnosis of bladder cancer were 0.773,0.702 and 0.856,respectively.Sensitivity was 61.30%,57.30%and 82.00%.The specificity was 81.30%,86.00%and 73.30%.Conclusion The expression of HLA-I and PD-L1 is abnormal in patients with bladder cancer,and their expression is affected by the positive infiltration of immune cells.Observing the positive expression of HLA-I and PD-L1 is beneficial to provide guidance for clinical diagnosis and treatment.

AIM:To investigate the expression profile and biological significance of long noncoding RNA(lncRNA)zinc finger antisense 1(ZFAS1)in the brains of mice with diabetic encephalopathy(DE).METHODS:Ten db/m mice served as the normal control group,while twenty 22-week-old db/db mice were used to establish the DE model and randomly divided into two subgroups:ten as the db/db model control and the remaining ten receiving ZFAS1 gene knockdown(db/db+sh-ZFAS1)via lentiviral transfection.Weekly measurements of body weight and blood glucose levels were performed.Brain tissues were collected for Nissl staining to evaluate neuronal damage,TUNEL assay to detect apop-tosis,and immunofluorescence staining to examine neural biomarker expression.Serum levels of tumor necrosis factor-α(TNF-α)and oxidative stress markers,including reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px),were determined.Western blot was conducted to quantify the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),p38 mitogen-activated protein kinase and phosphorylated p38(p-p38)in brain tissues.The expression levels of ZFAS1 and caspase-3 mRNA were determined by RT-qPCR.RESULTS:Knockdown of ZFAS1 in db/db mice significantly improved cognitive function,alleviated hippocampal neuronal damage,and reduced body weight and blood glucose levels(P<0.01).More-over,oxidative stress was mitigated,as evidenced by decreased MDA and ROS levels(P<0.01)and increased activity of antioxidant enzymes,GSH-Px,SOD and CAT(P<0.01 or P<0.05).Meanwhile,ZFAS1 silencing down-regulated Bax and p-p38/p38 protein expression(P<0.01 or P<0.05)while up-regulating Bcl-2(P<0.01).Consistently,RT-qPCR confirmed significant down-regulation of ZFAS1 and caspase-3 mRNA levels(P<0.01).CONCLUSION:lncRNA ZFAS1 is highly expressed in the hippocampus of DE mice.Down-regulation of ZFAS1 expression enhances cognitive func-tion,suppresses oxidative stress,and inhibits neuronal apoptosis,thereby attenuating neural damage in DE.

Circular RNA(circRNA),as a kind of non-coding RNA,regulates a variety of biological functions.To explore the effect of circRNA on PEDV replication in the host porcine intestinal epi-thelial cells,this study screened and analyzed the differentially expressed circRNAs by bioinforma-tic software in African Green Monkey renal cells(Vero-E6 cells)infected by porcine epidemic di-arrhea virus(PEDV),the differentially expressed circRNA ssc_circ_PIK3C2A was identified and the secondary structure was analyzed.PCR was used to identify the ssc_circ_PIK3C2A circRNA structure,the model of PEDV-infected IPEC-J2 cells was constructed,the TCID50 test was used to validate the viral titer of PEDV.The expression of circ_PIK3C2A was detected by qRT-PCR in IPEC-J2 infected by PEDV.circ_PIK3C2A qRT-PCR was performed to detect the expression of N gene of PEDV when ssc_circ_PIK3C2A was over-expressed in IPEC-J2 cells.The results showed that ssc_circ_PIK3C2 A is a porcine circular RNA with a typical circular structure,the virus titer of PEDV reached 10-6/mL after PEDV infected IPEC-J2 cells for 48 h,the expression of circ_PIK3C2A increased extremely(P＜0.01)at 6 h after PEDV-infection,with the extension of infec-tion time,its expression gradually decreased,and the expression was the lowest at 24 h,but there was no time-dependent trend.The expression of PEDV N gene decreased significantly when ssc_circ_PIK3C2A was over-expressed in IPEC-J2 cells.In conclusion,when PEDV infects IPEC-J2 cells,the expression of porcine circ_PIK3C2A decreases,and replication of PEDV increases signifi-cantly in IPEC-J2 cells.our result provides a basis for further study of the mechanism of circular RNA on PEDV replication and its physiological activities in host cells in the future.

Objective:To evaluate the efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with Ph-like acute lymphoblastic leukemia (Ph-ALL) .Methods:Patients with Ph-ALL who underwent CAR-T therapy followed by allo-HSCT from March 2018 to August 2023 at the First Affiliated Hospital of Soochow University were included, and their clinical data were retrospectively analyzed.Results:Of the 21 patients, 14 were male and 7 were female. The median age at the time of CAR-T therapy was 22 (6-50) years. Seven patients had ABL1-like rearrangements, and 14 had JAK-STAT rearrangements. Prior to CAR-T therapy, 12 patients experienced hematologic relapse; 7 were multiparameter flow cytometry minimal residual disease (MFC-MRD) -positive and 2 were MFC-MRD-negative. CAR-T cells were derived from patients’ autologous lymphocytes. Nine patients were treated with CD19 CAR-T cells, and 12 were treated with CD19/CD22 CAR-T cells. After assessment on day 28 after CAR-T therapy, 95.2% of the patients achieved complete remission, with an MRD-negative remission rate of 75%. Nineteen patients developed grade 0–2 cytokine release syndrome (CRS) and 2 patients suffered grade 3 CRS, all cases of which resolved after treatment. All patients underwent allo-HSCT after CAR-T therapy. The median time from CAR-T therapy to allo-HSCT was 63 (38-114) days. Five patients experienced relapse after CAR-T therapy, including four with hematologic relapse and one with molecular relapse. The 3-year overall survival (OS) rates in the ABL1 and JAK-STAT groups were (83.3±15.2) % and (66.6±17.2) %, respectively ( P=0.68) . The 3-year relapse-free survival (RFS) rates were (50.0±20.4) % and (55.6±15.4) % in the ABL1 and JAK-STAT groups, respectively. There was no significant difference in 3-year OS or RFS between the two groups. Conclusions:CAR-T therapy followed by allo-HSCT leads to rapid remission in most patients with Ph-ALL and prolongs leukemia-free survival.

Objective To evaluate the changes in motor function impairment and brain functional networks of patients with acute ischemic stroke(AIS) through parameters of spontaneous activities of both upper limbs and electroencephalogram graph theory analysis methods. Methods The data of 34 acute ischemic stroke patients(observation group) with upper limb motor disorders who were treated in the Department of Neurology of the Affiliated Hospital of Southwest Medical University from January 2022 to October 2023, and 40 healthy controls (HC group) were collected. The subjects completed the National Institutes of Health Stroke Scale (NIHSS) and Fugl-Meyer Assessment (FMA) within 7 days, and wore wrist activity recorders (Actiwatch) continuously for 24 hours to collect data on spontaneous activities of upper limbs and analyzed related parameters such as the coordination coefficient of both upper limbs (r), the activity ratio of the affected side to the healthy side upper limb (ULAR), etc. At the same time, all subjects completed approximately 2 hours of 19-channel electroencephalogram examination. After preprocessing the electroencephalogram data, 5 segments of 10-second resting-state electroencephalogram were extracted for graph theory. Results ① Compared to healthy individuals, AIS patients exhibited decreased functional connectivity edges in the δ and θ bands, with substantial reductions in network connections in the α band. In the β band, connections between the frontal, right parietal, and occipital regions weakened, while connections from the right temporal lobe to the left temporal lobe strengthened. In the γ band, there was a significant increase in connections throughout the brain. ② Graph theory analysis revealed significantly increased shortest path lengths (α band: t=2.228, P < 0.05, d=-0.52; β band: t=-3.641, P < 0.01, d=-0.878) and decreased global efficiency (α band: t=2.535, P < 0.05, d=0.591; β band: t=3.321, P < 0.01, d=0.803) in the observation group compared to the control group. In the γ band, local efficiency (t=3.279, P < 0.01, d=0.765) and clustering coefficients were significantly higher (t=3.358, P < 0.01, d=0.783). ③ In the γ band, the ULAR≤30% group showed significantly reduced shortest path length (t=-2.063, P < 0.05, d=-0.802) and increased global efficiency (t=2.226, P < 0.05, d=0.865), local efficiency (t=2.95, P < 0.05, d=1.147), and clustering coefficient (t=2.962, P < 0.05, d=1.148). ④ In the observation group, the bilateral upper limb coordination coefficient during sleep was negatively correlated with NIHSS scores (r=-0.389, P < 0.05) and ULAR (r=-0.395, P < 0.05), while FMA scores were positively correlated with ULAR (r=0.442, P < 0.05). Conclusion The parameters of spontaneous activities of the upper limbs can be used to determine the impairment of motor function in AIS patients. The combination of changes in brain functional networks and motor impairments can provide new ideas for the study of their neural network mechanisms.

Objective:To explore the effects of human chorionic gonadotropin (hCG), progesterone as well as the change of estradiol 12 h after hCG trigger on the outcomes of in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET). Methods:A retrospective study was conducted at the Center for Reproduction and Genetics of the Affiliated Suzhou Hospital of Nanjing Medical University. A total of 2 506 patients received IVF/ICSI-ET treatment with gonadotropin-releasing hormone agonist (GnRH-a) protocol from March 2015 to June 2020 were selected. With Spearman rank correlation analysis and path analysis, we explore the relationship among the changes of these hormones and the baseline characteristic of patients, as well as the relationship among the changes of these hormones and the outcomes of IVF treatment.Results:The increase of hCG was accompanied by the rise of progesterone and the decline of estradiol change rate ( r=0.094, P<0.001; r=-0.093, P<0.001). Meanwhile the rise of progesterone was accompanied by the decline of estradiol change rate ( r=-0.089, P<0.001). The dosage of hCG trigger was directly positively correlated to hCG level after hCG trigger, path coefficients (PC) was 0.307 ( P<0.001). Body mass index (BMI) was directly negatively correlated to hCG level and progesterone level after hCG trigger (PC=-0.434, P<0.001; PC=-0.154, P<0.001), whereas positively correlated to estradiol change rate (PC=0.097, P<0.001). Meanwhile the duration and dosage of gonadotropin (Gn) used were positively correlated to progesterone level after hCG trigger (PC=0.102, P<0.001; PC=0.080, P=0.030). hCG level and progesterone level after hCG trigger had positive correlation to oocyte retrieved rate (PC=0.098, P<0.001; PC=0.080, P<0.001). While estradiol change rate was not correlated to oocyte retrieved rate ( P>0.05). Progesterone level on hCG trigger day negatively related to normal fertilization rate (PC=-0.050, P=0.039). hCG level, progesterone level and estradiol change rate after hCG trigger had no correlation with high-quality embryo rate, clinical pregnancy rate and live birth rate (all P>0.05). Conclusion:Oocyte retrieved rate was positively affected by hCG level and progesterone level 12 h after hCG trigger. While normal fertilization rate, high-quality embryo rate, clinical pregnancy rate and live birth rate were not affected by the change of hormones level 12 h after hCG trigger. Therefore we should pay attention to hCG level and progesterone level 12 h after hCG trigger.

Objective:To explore the effects of human chorionic gonadotropin (hCG), progesterone as well as the change of estradiol 12 h after hCG trigger on the outcomes of in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET). Methods:A retrospective study was conducted at the Center for Reproduction and Genetics of the Affiliated Suzhou Hospital of Nanjing Medical University. A total of 2 506 patients received IVF/ICSI-ET treatment with gonadotropin-releasing hormone agonist (GnRH-a) protocol from March 2015 to June 2020 were selected. With Spearman rank correlation analysis and path analysis, we explore the relationship among the changes of these hormones and the baseline characteristic of patients, as well as the relationship among the changes of these hormones and the outcomes of IVF treatment.Results:The increase of hCG was accompanied by the rise of progesterone and the decline of estradiol change rate ( r=0.094, P<0.001; r=-0.093, P<0.001). Meanwhile the rise of progesterone was accompanied by the decline of estradiol change rate ( r=-0.089, P<0.001). The dosage of hCG trigger was directly positively correlated to hCG level after hCG trigger, path coefficients (PC) was 0.307 ( P<0.001). Body mass index (BMI) was directly negatively correlated to hCG level and progesterone level after hCG trigger (PC=-0.434, P<0.001; PC=-0.154, P<0.001), whereas positively correlated to estradiol change rate (PC=0.097, P<0.001). Meanwhile the duration and dosage of gonadotropin (Gn) used were positively correlated to progesterone level after hCG trigger (PC=0.102, P<0.001; PC=0.080, P=0.030). hCG level and progesterone level after hCG trigger had positive correlation to oocyte retrieved rate (PC=0.098, P<0.001; PC=0.080, P<0.001). While estradiol change rate was not correlated to oocyte retrieved rate ( P>0.05). Progesterone level on hCG trigger day negatively related to normal fertilization rate (PC=-0.050, P=0.039). hCG level, progesterone level and estradiol change rate after hCG trigger had no correlation with high-quality embryo rate, clinical pregnancy rate and live birth rate (all P>0.05). Conclusion:Oocyte retrieved rate was positively affected by hCG level and progesterone level 12 h after hCG trigger. While normal fertilization rate, high-quality embryo rate, clinical pregnancy rate and live birth rate were not affected by the change of hormones level 12 h after hCG trigger. Therefore we should pay attention to hCG level and progesterone level 12 h after hCG trigger.