Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin regions, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at the single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences on TF binding. Notably, cFOOT-seq, combined with FootTrack analysis, enables de novo prediction of TF binding sites and tracking of TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.
Transcription Factors/genetics* ; Humans ; Chromatin/genetics* ; Animals ; Binding Sites ; Mice ; DNA Footprinting/methods*

Objective To explore the value of combining apparent diffusion coefficient(ADC)heterogeneity with morphological indicators in assessing the pathological grading of non-muscle invasive bladder cancer(NMIBC).Methods The MRI images of 86 patients confirmed with NMIBC by surgical pathology were analyzed retrospectively.All patients underwent T2WI,diffusion weighted ima-ging(DWI),and dynamic contrast enhancement(DCE)examinations.Two radiologists independently measured tumor largest diam-eter(LD),actual tumor-wall contact length(ACTCL),ADCmean,ADCmin,and ADCmax values.ADC heterogeneity was calculated using the formula(ADCmax-ADCmin)/ADCmean.Differences in quantitative parameters between low-and high-grade NMIBC were compared using the Mann-Whitney U test,while differences in qualitative parameters were compared using the chi-square test.Univariate and multivariate logistic regression analyses were used to identify independent predictors of high-grade NMIBC,and receiver operating characteristic(ROC)curves were drawn to evaluate the performance of ADC heterogeneity combined with morphological indicators in assessing high-grade NMIBC.Results ADC heterogeneity and ACTCL were independent predictors for preoperative assessment of NMIBC pathological grading.The area under the curve(AUC)of ADC heterogeneity and ACTCL in assessing high-grade NMIBC were 0.843 and 0.744,respectively.The combined AUC was 0.902.The difference was statistically significant(P<0.05).Conclusion The combination of ADC heterogeneity with ACTCL can effectively improve the efficiency of preoperative assessment of NMIBC pathological grading,and providing more precise clinical decision-making and prognosis monitoring.

pAPN is a zinc-dependent metalloprotease,mediating the fusion between virus and host cell,and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV rep-lication,the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamH Ⅰ and Not Ⅰ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method,and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level,Western blot for N protein level,and TCID50.The results showed that the packaged lentivirus could infect Vero cells,and the monoclonal cell line Vero-pAPN(2C5)could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48 h(P＜0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(P＜0.05).In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.

Objective:To investigate the effect of CT-derived fractional flow reserve (CT-FFR) measurement sites on the values and the diagnostic performance, and to determine the optimal measurement site for CT-FFR using invasive FFR as the reference standard.Methods:This study was part of the CT-FFR CHINA clinical trial. Patients with suspected coronary artery disease who were scheduled for invasive coronary angiography (ICA) were prospectively recruited from five clinical centers across the country from November 2018 to March 2020. Each enrolled patient underwent coronary CT angiography (CCTA), CT-FFR, ICA, and invasive pressure wire-based FFR assessments sequentially within one week. Four groups of CT-FFR values were obtained on each enrolled target vessels according to different CT-FFR measurement locations: 1, 2, 3 cm distal to the target lesion, and terminal vessel groups. Spearman and Bland-Altman analyses were used to explore the correlation and consistency of CT-FFR values and FFR values at different measurement sites. The measurement deviation of CT-FFR was also compared. Diagnostic accuracy and performance of CT-FFR, including sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve (AUC), in discriminating myocardial ischemia were analyzed across all measurement site groups on a per-vessel level, using FFR as the reference standard.Results:A total of 289 patients with 345 target lesion vessels were included. According to CCTA, there were 51 target vessels (14.8%) with<50% stenosis, 106 vessels (30.7%) with 50%-69% stenosis, and 188 vessels (54.5%) with stenosis≥70%. At per-vessel level, CT-FFR and FFR values at each measurement position group were highly positively correlated: 1 cm distal to target lesion group, r=0.734 ( P<0.001); 2 cm distal to target lesion group, r=0.732 ( P<0.001); 3 cm distal to target lesion group, r=0.737 ( P<0.001); terminal vessel group was 0.719 ( P<0.001). At per-vessel level, CT-FFR and FFR values of all measurement sites were in good agreement (Bland-Altman analysis results): 1 cm distal to target lesion group, 0.014 (95% LoA 0.002-0.026); 2 cm distal to target lesion group, 0.026 (95% LoA 0.015-0.038); 3 cm distal to target lesion group, 0.040 (95% LoA 0.039-0.051); terminal vessel group, 0.075 (95% LoA 0.064-0.087). And at per-vessel level, the accuracy of diagnosing myocardial ischemia with CT-FFR at 1 cm was highest [84.6% (95% CI 80.4%-88.3%)], and the lowest accuracy in the terminal vessel group [67.0% (95% CI 61.7%-72.0%)]. However, there was no significant difference in the diagnostic accuracy of CT-FFR at 1 cm, 2 cm [80.6% (95% CI 76.1%-84.6%)] and 3 cm [77.5% (95% CI 72.6%-81.7%)]. AUC of CT-FFR at 1 cm distal to the lesion were both highest for global level and moderately stenosis (50%-69%) lesions [0.85 (95% CI 0.81-0.89), 0.84 (95% CI 0.77-0.90)]. And the differences were statistically significant among the four measurement location groups (all P<0.05). Conclusions:The deviation of CT-FFR increases with measurement site distance distal to target lesions. One centimeter distal to the target lesion is the optimal measurement site, and the CT-FFR value here shows the highest diagnostic performance for myocardial ischemic lesions, especially for moderate stenosis.

Objective:To explore the prognosis of patients with gastric cancer peritoneal metastasis (PM) receiving programmed cell death-1 (PD-1) antibody therapy, and investigate the biomarkers that affect the prognosis of anti-PD-1 therapy.Methods:This restrospecific study collected the clinic-pathological data of 56 patients with peritoneal metastasis of gastric cancer who received first-line treatment in the Nanjing Drum Town Hospital from March 2020 to September 2023, among which 41 had received anti-PD-1 immunotherapy and 15 hadn't. The relationship between overall survival (OS) and anti-PD-1 immunotherapy was evaluated by Kaplan-Meier analysis. The relationship between baseline peripheral blood indicators and treatment response of patients with anti-PD-1 treatment was analyzed using unpaired t-test. Subsequently, the Cox proportional risk regression model was used to explore the clinical prognostic factors that may affect anti-PD-1 immunotherapy by univariate and multivariate analysis. The clinical prognostic factors included baseline data and baseline peripheral blood indexes such as anti-PD-1 treatment lines, Eastern Cooperative Oncology Group performance status (ECOG PS), combined positive score (CPS), expression of human epidermal growth factor receptor 2 (Her-2), EBER status, pathological types, other metastatic lesions, ascites content before immunotherapy, with or without abdominal drainage during anti-PD-1 treatment, blood lipid indicators, inflammatory indicators, and tumor indicators. Results:Kaplan-Meier survival statistics showed similar OS (15.9 vs. 15.2 months, P=0.600) in patients with anti-PD-1 therapy compared to those without anti-PD-1 therapy. Patients with baseline high-density lipoprotein (HDL) ≥0.97 mmol/L ( n=22) demonstrated a significantly longer median OS compared to those with HDL＜0.97 mmol/L (15.2 vs. 13.5 months; P=0.018). Similarly, the cohort with apolipoprotein A1 (ApoA1) levels ≥0.86 g/L ( n=21) showed superior survival outcomes, with a median OS of 17.7 months versus 12.3 months in the ApoA1＜0.86 g/L group ( n=20; P=0.006). In contrast, elevated baseline alpha-fetoprotein (AFP) levels ( n=2) were associated with markedly reduced survival (median OS: 5.7 vs. 15.2 months in normal AFP group, n=37; P=0.005). Notably, elevated pretreatment ApoA1 levels correlated with enhanced immunotherapy response ( P=0.017). Multivariate Cox regression analysis revealed that ApoA1 deficiency (≥0.86 g/L) independently predicted better OS following PD-1 antibody therapy ( HR=0.35, 95% CI: 0.12-0.98, P=0.046) in gastric cancer patients with PM. Conclusions:In our study, it is first proposed that ApoA1 could be a significant predictor of the survival advantages of immunotherapy in gastric cancer patients with PM.

Objective To explore the value of combining apparent diffusion coefficient(ADC)heterogeneity with morphological indicators in assessing the pathological grading of non-muscle invasive bladder cancer(NMIBC).Methods The MRI images of 86 patients confirmed with NMIBC by surgical pathology were analyzed retrospectively.All patients underwent T2WI,diffusion weighted ima-ging(DWI),and dynamic contrast enhancement(DCE)examinations.Two radiologists independently measured tumor largest diam-eter(LD),actual tumor-wall contact length(ACTCL),ADCmean,ADCmin,and ADCmax values.ADC heterogeneity was calculated using the formula(ADCmax-ADCmin)/ADCmean.Differences in quantitative parameters between low-and high-grade NMIBC were compared using the Mann-Whitney U test,while differences in qualitative parameters were compared using the chi-square test.Univariate and multivariate logistic regression analyses were used to identify independent predictors of high-grade NMIBC,and receiver operating characteristic(ROC)curves were drawn to evaluate the performance of ADC heterogeneity combined with morphological indicators in assessing high-grade NMIBC.Results ADC heterogeneity and ACTCL were independent predictors for preoperative assessment of NMIBC pathological grading.The area under the curve(AUC)of ADC heterogeneity and ACTCL in assessing high-grade NMIBC were 0.843 and 0.744,respectively.The combined AUC was 0.902.The difference was statistically significant(P<0.05).Conclusion The combination of ADC heterogeneity with ACTCL can effectively improve the efficiency of preoperative assessment of NMIBC pathological grading,and providing more precise clinical decision-making and prognosis monitoring.

Objective:To investigate the effect of CT-derived fractional flow reserve (CT-FFR) measurement sites on the values and the diagnostic performance, and to determine the optimal measurement site for CT-FFR using invasive FFR as the reference standard.Methods:This study was part of the CT-FFR CHINA clinical trial. Patients with suspected coronary artery disease who were scheduled for invasive coronary angiography (ICA) were prospectively recruited from five clinical centers across the country from November 2018 to March 2020. Each enrolled patient underwent coronary CT angiography (CCTA), CT-FFR, ICA, and invasive pressure wire-based FFR assessments sequentially within one week. Four groups of CT-FFR values were obtained on each enrolled target vessels according to different CT-FFR measurement locations: 1, 2, 3 cm distal to the target lesion, and terminal vessel groups. Spearman and Bland-Altman analyses were used to explore the correlation and consistency of CT-FFR values and FFR values at different measurement sites. The measurement deviation of CT-FFR was also compared. Diagnostic accuracy and performance of CT-FFR, including sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve (AUC), in discriminating myocardial ischemia were analyzed across all measurement site groups on a per-vessel level, using FFR as the reference standard.Results:A total of 289 patients with 345 target lesion vessels were included. According to CCTA, there were 51 target vessels (14.8%) with<50% stenosis, 106 vessels (30.7%) with 50%-69% stenosis, and 188 vessels (54.5%) with stenosis≥70%. At per-vessel level, CT-FFR and FFR values at each measurement position group were highly positively correlated: 1 cm distal to target lesion group, r=0.734 ( P<0.001); 2 cm distal to target lesion group, r=0.732 ( P<0.001); 3 cm distal to target lesion group, r=0.737 ( P<0.001); terminal vessel group was 0.719 ( P<0.001). At per-vessel level, CT-FFR and FFR values of all measurement sites were in good agreement (Bland-Altman analysis results): 1 cm distal to target lesion group, 0.014 (95% LoA 0.002-0.026); 2 cm distal to target lesion group, 0.026 (95% LoA 0.015-0.038); 3 cm distal to target lesion group, 0.040 (95% LoA 0.039-0.051); terminal vessel group, 0.075 (95% LoA 0.064-0.087). And at per-vessel level, the accuracy of diagnosing myocardial ischemia with CT-FFR at 1 cm was highest [84.6% (95% CI 80.4%-88.3%)], and the lowest accuracy in the terminal vessel group [67.0% (95% CI 61.7%-72.0%)]. However, there was no significant difference in the diagnostic accuracy of CT-FFR at 1 cm, 2 cm [80.6% (95% CI 76.1%-84.6%)] and 3 cm [77.5% (95% CI 72.6%-81.7%)]. AUC of CT-FFR at 1 cm distal to the lesion were both highest for global level and moderately stenosis (50%-69%) lesions [0.85 (95% CI 0.81-0.89), 0.84 (95% CI 0.77-0.90)]. And the differences were statistically significant among the four measurement location groups (all P<0.05). Conclusions:The deviation of CT-FFR increases with measurement site distance distal to target lesions. One centimeter distal to the target lesion is the optimal measurement site, and the CT-FFR value here shows the highest diagnostic performance for myocardial ischemic lesions, especially for moderate stenosis.

pAPN is a zinc-dependent metalloprotease,mediating the fusion between virus and host cell,and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV rep-lication,the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamH Ⅰ and Not Ⅰ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method,and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level,Western blot for N protein level,and TCID50.The results showed that the packaged lentivirus could infect Vero cells,and the monoclonal cell line Vero-pAPN(2C5)could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48 h(P＜0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(P＜0.05).In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.

Objective:To explore the prognosis of patients with gastric cancer peritoneal metastasis (PM) receiving programmed cell death-1 (PD-1) antibody therapy, and investigate the biomarkers that affect the prognosis of anti-PD-1 therapy.Methods:This restrospecific study collected the clinic-pathological data of 56 patients with peritoneal metastasis of gastric cancer who received first-line treatment in the Nanjing Drum Town Hospital from March 2020 to September 2023, among which 41 had received anti-PD-1 immunotherapy and 15 hadn't. The relationship between overall survival (OS) and anti-PD-1 immunotherapy was evaluated by Kaplan-Meier analysis. The relationship between baseline peripheral blood indicators and treatment response of patients with anti-PD-1 treatment was analyzed using unpaired t-test. Subsequently, the Cox proportional risk regression model was used to explore the clinical prognostic factors that may affect anti-PD-1 immunotherapy by univariate and multivariate analysis. The clinical prognostic factors included baseline data and baseline peripheral blood indexes such as anti-PD-1 treatment lines, Eastern Cooperative Oncology Group performance status (ECOG PS), combined positive score (CPS), expression of human epidermal growth factor receptor 2 (Her-2), EBER status, pathological types, other metastatic lesions, ascites content before immunotherapy, with or without abdominal drainage during anti-PD-1 treatment, blood lipid indicators, inflammatory indicators, and tumor indicators. Results:Kaplan-Meier survival statistics showed similar OS (15.9 vs. 15.2 months, P=0.600) in patients with anti-PD-1 therapy compared to those without anti-PD-1 therapy. Patients with baseline high-density lipoprotein (HDL) ≥0.97 mmol/L ( n=22) demonstrated a significantly longer median OS compared to those with HDL＜0.97 mmol/L (15.2 vs. 13.5 months; P=0.018). Similarly, the cohort with apolipoprotein A1 (ApoA1) levels ≥0.86 g/L ( n=21) showed superior survival outcomes, with a median OS of 17.7 months versus 12.3 months in the ApoA1＜0.86 g/L group ( n=20; P=0.006). In contrast, elevated baseline alpha-fetoprotein (AFP) levels ( n=2) were associated with markedly reduced survival (median OS: 5.7 vs. 15.2 months in normal AFP group, n=37; P=0.005). Notably, elevated pretreatment ApoA1 levels correlated with enhanced immunotherapy response ( P=0.017). Multivariate Cox regression analysis revealed that ApoA1 deficiency (≥0.86 g/L) independently predicted better OS following PD-1 antibody therapy ( HR=0.35, 95% CI: 0.12-0.98, P=0.046) in gastric cancer patients with PM. Conclusions:In our study, it is first proposed that ApoA1 could be a significant predictor of the survival advantages of immunotherapy in gastric cancer patients with PM.

ObjectiveTo explore the therapeutic effect and mechanism of Guipitang on rats with myocardial ischemia. MethodFifty SD rats were divided into five groups： a control group， a model group， low and high-dose Guipitang （7.52， 15.04 g·kg^-1） groups， and a trimetazidine group （0.002 g·kg^-1）. By intragastric administration of vitamin D₃ and feeding rats with high-fat forage and injecting isoproterenol， the rat model of myocardial ischemia was established. After drug treatment of 15 d， an electrocardiogram （ECG） was performed to analyze the degree of myocardial injury. A fully automatic biochemical analyzer was used to detect the changes in the serum levels of total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C）. Hematoxylin-eosin （HE） staining and Masson staining were used to observe myocardial histopathological changes. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect cardiomyocyte apoptosis. Western blot was adopted to detect the protein levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， phospho-ERK1/2 （p-ERK1/2）， p38 mitogen-activated protein kinase （p38 MAPK）， phospho-p38 MAPK （p-p38 MAPK）， B-cell lymphoma-2 （Bcl-2）-associated X （Bax）， Bcl-2， and cleaved cysteine aspartate proteolytic enzyme （cleaved Caspase-3）. ResultCompared with the control group， the ECG S-T segment decreased in the model group. The serum levels of TC， TG， and LDL-C were increased significantly （P<0.05）. The arrangement of myocardial tissue was disordered， and the proportion of cardiomyocyte apoptosis increased. The protein levels of cleaved Caspase-3， Bax， and p-p38 MAPK in the heart were increased， and the Bcl-2 expression was decreased （P<0.05）. Compared with the model group， the S-T segment downward shift was restored in the low and high-dose Guipitang groups and trimetazidine group， and the levels of TC， TG， and LDL-C were decreased. The protein expression of cleaved Caspase-3 and Bax in the heart dropped， and p-p38 MAPK and p-ERK1/2 protein expressions increased significantly （P<0.05）. The degree of myocardial injury was alleviated， and the proportion of cardiomyocyte apoptosis decreased. Bcl-2 protein expression was increased significantly in the low-dose Guipitang group （P<0.05）. ERK1/2 and p38 MAPK proteins had no significant difference among different groups. ConclusionGuipitang could alleviate myocardial injury and inhibit cardiomyocyte apoptosis in rats by activating the expression of ERK1/2 and p38 MAPK.