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*

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.

Objective:In this study,we explored the application prospects and mechanisms of action of co-inhibiting fibroblast growth factor receptor 2(FGFR2)and Src homology region 2-containing protein tyrosine phosphatase 2(SHP2)in gastric cancer with the TACC2-FGFR2 fu-sion gene.Methods:We established human gastric cancer cell lines overexpressing the TACC2-FGFR2 fusion gene(MKN45TACC2-FGFR2 and NUGC4TACC2-FGFR2 cells)or a control lentiviral virus(MKN45NC and NUGC4NC cells).The cells were treated with the FGFR2 inhibitor AZD4547,the SHP2 inhibitor SHP099,or a combination of both.The proliferation and migration of tumor cells were detected using cell counting Kit-8(CCK-8)and scratch assays.After treating MKN45TACC2-FGFR2 and NUGC4TACC2-FGFR2 cells with different formulations for 1 or 48 h,Western blot was used to detect variations in the levels of FGFR2,SHP2,and proteins downstream of the RAS/ERK and PI3K/AKT signaling pathways.Results:Com-pared to monotherapy,the combination of AZD4547 and SHP099 significantly inhibited the proliferation and migration of MKN45TACC2-FGFFR2 and NUGC4TACC2-FGFFR2 cells.After 1 h of treatment,the combination therapy inhibited the RAS/ERK and PI3K/AKT pathways in MKN45TACC2-FGFFR2 cells to a greater extent than the AZD4547 monotherapy.Forty-eight hours of AZD4547 monotherapy resulted in feedback activation of p-FGFR and p-SHP2,but failed to inhibit the RAS/ERK pathway.However,combination therapy continuously suppressed upstream FGFR2 and SHP2 signaling,as well as downstream RAS/ERK and PI3K/AKT pathways.Conclusions:Co-inhibiting FGFR2 and SHP2 further inhibit gastric cancer with the TACC2-FGFR2 fusion gene by suppressing the RAS/ERK and PI3K/AKT pathways.These findings provide a new treatment mode for patients with gastric cancer with the TACC2-FGFR2 fusion gene.

Objective:To study the repair effects of bone marrow mesenchymal stem cells (BMSCs) injection via the caudal vein on the nerve cells in the spinal cord tissue of rats with acute spinal cord injury.Methods:Sixty Sprague-Dawley male rats were divided into sham operation group, model group and BMSCs group using the random number table method, with 20 rats in each group. The Allen's method was used in the model group and BMSCs group to construct the rat models of a spinal cord injury model. Rats in the sham operation group did not undergo spinal cord injury and only received surgical exposure. 24 hours after the establishment of the model, rats in the BMSCs group were received 0.2 ml BMSCs single cell suspension (2 ×10 6 cells) via tail vein injection. Rats in the sham operation group and model group were received the same volume of 0.2 ml Sodium chloride solution via tail vein injection. The motor function of the rats on the 1st, 4th, 7th, 15th and 30th day after modeling was recorded by Basso-Beattie-Bresnahan (BBB) scoring method. The contents of inflammatory factors tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-β) and Prostaglandin E 2 (PGE 2) in spinal cord tissue of rats were detected by enzyme-linked immunosorbent assay (ELASA) on the 30th day after modeling. Hematoxylin-eosin staining was used to observe the pathological changes of rat spinal cord tissue. Nissl staining was used to analyze the changes of Nissl bodies and neuron cells in rat spinal cord tissue. Result:Compared with the model group, the BBB scores of the BMSC group were significantly increased on the 7 (5.68±0.82 vs 1.82±0.84), 15 ( 10.25±1.55 vs 3.38±0.88) and 30 (13.25±2.36 vs 5.83±1.36) days after modeling, and the differences were statistically significant (all P<0.01). Compared with the model group, the levels of TNF-α, IL-1β and PGE 2 in the spinal cord tissue of the BMSCs group were significantly lower than those in the model group on the 30 days after modeling (all P<0.01). Besides, the spinal cord tissue injury was significantly reduced, and the number of neurons and Nissl bodies in the BMSCs group were also significantly higher than those in the model group (all P<0.01). Conclusions:BMSCs injection via the caudal vein can significantly ameliorate acute spinal cord injury in rats. BMSCs may accelerate the repair of nerve cells in acute spinal cord injury tissue and further promote the recovery of motor function in rats with acute spinal cord injury through the regulation of TNF-α, IL-1β, PGE 2 inflammatory factors.

Objective To investigate the clinical value of Fluorine-18-fluorodeoxyglucose (18 F-FDG)positron-emission temography/computed tomography (PET/CT) examination to predict microvascular invasion (MVI) of hepatocellular carcinoma (HCC).Methods The retrospective cohort study was conducted.The clinicopathological data of 51 HCC patients who were admitted to Changhai Hospital of the Second Military Medical University (32 patients) and Universal Medical Imaging Diagnostic Center (19 patients) from January 2013 to October 2017 were collected.Of 51 patients receiving postoperative pathological examination,21 diagnosed with positive MVI and 30 diagnosed with negative MVI were respectively allocated into the positive and negative MVI groups.All the patients received preoperative 1s F-FDG PET/CT examination and underwent surgery after related examinations.Two imaging doctors independently read films and made a semi-quantitative analysis.Observation indicators:(1) results of 18F-FDG PET/CT examination;(2) multivariate analysis and diagnostic value affecting MVI of HCC;(3) treatment and follow-up.Follow-up using outpatient examination and telephone interview was performed to detect the postoperative patients' survival up to November 2017.Measurement data with normal distribution were represented as (x)±s,and comparisons between groups were evaluated with the independent-sample t test.Measurement data with skewed distribution were described as M (Qn),and comparisons between groups were analyzed using the independent-sample rank sum test.Comparisons of count data were analyzed using the chisquare test.Logistic regression analysis was performed in variables with statistical significance.The inclusion criteria was 0.05 and exclusion criteria was 0.10 according to Backward (LR) method for screening variables.Receiver Operating Characeristic (ROC) curve analysis was used to evaluate the diagnostic value using MVI as a diagnostic standard.Results (1) Results of 18F-FDG PET/CT examination:of 51 HCC patients,tumors located in the right lobe,left lobe and caudate lobe of the liver were respectively detected in 37,12 and 2 patients.CT examinations of 51 HCC patients:HCCs showed the hypodense shadow or slightly hypodense shadow in liver and were round-like,and some of the larger lesions were irregularly conglomerate,with a relatively clear tumor-liver boundary;tumor necrosis area showed patchy and irregular lower density,with small lesions around the port of tumors.Of 51 patients,34 were positive on PET and 17 were negative on PET,and some necrotic areas showed no uptake and located in the center of tumors.There was no abnormal 18 F-FDG uptake in other parts of the whole body.The maximum diameter of tumor was (6± 3)cm.The maximum standardized uptake value (SUVmax),and ratio of SUVmax of tumor to SUVmax of liver (SUVmax T/L) in all the lesions were 6.38±4.91 and 2.42±1.93,respectively.The mean standardized uptake value (SUVmean),metabolism of volume (MTV),total lesion of glycolysis (TLG) of 40 patients were 4.30± 2.46,43.82 cm3 (8.97 cm3,219.13 cm3) and 165.73 (28.26,794.50),respectively,and software could not automatically delineate lesions in other 11 patients due to low metabolism.Delayed imaging was found in 21 patients,and the delayed SUVmax and retention index (RI) were 7.22±6.26,19.66％ (-7.10％,50.84％),respectively.The cases with positive and negative on PET were 18,3 in the positive MVI group and 16,14 in the negative MVI group,respectively,with a statistically significant difference between groups (x2=5.829,P＜0.05).The maximum diameter of tumor in the positive MVI group and negative MVI group was respectively (7.7±2.9)cm and (5.2±3.1)cm,with a statistically significant difference between groups (t=-2.930,P＜0.05).(2) Multivariate analysis and diagnostic value affecting MVI of HCC:the results of multivariate analysis showed that maximum diameter of tumor was an independent factor affecting MVI of HCC (OR=1.276,95％ confidence interval:1.028-1.585,P＜0.05).The area under the ROC curve of the maximum diameter of tumor was 0.723 using MVI as a diagnostic standard.The sensitivity,specificity and Youden index were respectively 90.5％,50.0％ and 0.405,with 4.55 cm as the critical value.(3) Treatment and followup:all 51 patients underwent tumor resection.Twenty-two patients were followed up for 25 months (range,12-46 months).The 1-and 2-year overall survival rates were 81.8％ (18/22) and 63.6％ (14/22),respectively.The 1-and 2-year tumor-free survival rates were 59.1％ (13/22) and 45.5％ (10/22),respectively.Conclusion The positive rate on PET of 18F-FDG PET/CT examination in HCC patients with positive MVI is higher than that in HCC patients with negative MVI,and the maximum diameter of tumor is an independent factor predicting MVI of HCC,with a certainly reference value.

Objective To evaluate preoperative apparent diffusion coefficient (ADC) and exponential apparent diffusion coefficient (eADC) in diffusion weighted imaging (DWI) on microvascular invasion (MVI) of hepatocellular carcinoma (HCC).Methods We retrospectively reviewed 43 HCC patients with DWI data confirmed by surgery and pathology.ADC and eADC values were measured both in neoplasm and hepatic tissue,the ratio of tumor to liver (T/L) on ADC and eADC was calculated.The correlation between MVI and ADC value,T/L of ADC value,eADC value and T/L of eADC value was analysed,the best cut-off value of variables was analysed by ROC curve,multi factor analysis was carried out by Logistic regression model.Results The ADC value,T/L of ADC,eADC value and T/L of eADC were (1.458 ± 0.444) × 10-3 mm2/s,0.787 ± 0.186,0.289 ± 0.144 and 1.383 (1.179,1.655),respectively.Among them,the positive MVI group were (1.232 ± 0.480) × 10-3 mm2/s,0.683 ± 0.229,0.323 ±0.123 and 1.630(1.387,2.066),respectively.The negative MVI group were(1.545 ±0.404) × 10-3mm2/s,0.844 ±0.149,0.277 ±0.152 and 1.303(1.176,1.545),respectively.There was significant difference in ADC value,T/L of ADC value and T/L of eADC value (t =2.164,2.654,z =-2.058,all P ＜0.05),the area under the ROC curve were (1.085 × 10-3) mm2/s,0.685 and 1.475 using MVI as a diagnostic standard.Multivariate analysis showed that T/L of ADC value was an independent factor affecting MVI of HCC (OR=0.002,95％CI:1.380E-5-0.311,P＜0.05).Conclusions The ADC value in HCC patients with positive MVI is lower than in HCC patients with negative MVI,T/L of eADC value is higher than in HCC patients with negative MVI,and T/L of ADC value is an independent factor predicticing MVI of HCC.

Objective: To study the relationship between morphological characteristics, grading, diagnosis and prognosis in phyllodes tumors (PT) of the breast. Methods: A retrospective study was carried out on 83 PTs diagnosed between 1999 and 2003 that were classified semi-quantitatively according to the WHO recommendation. Follow-up data was available for some cases, and Cox regression analysis was used to evaluate factors affecting metastasis and recurrence. Results: All cases were classified into the benign (57.8%), borderline (28.9%) and malignant (13.3%). The overall recurrence rate for the 72 cases with follow-up data was 20.8% (15/72), and was 17.5% (7/40) in benign, 22.7% (5/22) in borderline and 3/10 in malignant PT, respectively, with no significant difference (P>0.05). The median interval between the initial diagnosis and the first recurrence was 24 months. Lung or bone metastases occurred in 1/22 borderline and 3/10 malignant PT patients 5 years post-surgery. The mitotic count and the degree of stromal cell atypia were significantly correlated with recurrence (P=0.001 and P=0.006). Multivariate analysis showed that severe stromal cell atypia was an independent predictor of recurrence-free survival in PT [HR=6.40 (95% CI=1.378 to 29.732), P=0.018]. Conclusions Each parameter in the histological grading of PT may have different prognostic value, and markedly increased mitotic count and were predictive of relapse.

Objective To investigate the effect of erythropoietin(EPO)on the expression of aquaporin - 2 (AQP2)in the kidney of young SD rats after release of bilateral ureter obstruction(BUO - R). Methods Thirty - two young SD rats were equally divided into 4 groups randomly(BUO group,BUO - R group,BUO - R ﹢ EPO group and Sham group,8 rats in each group). The BUO model was built through bilateral ureteral ligation. EPO(500 U/ kg)was given to BUO - R ﹢ EPO rats at 2 h after release of BUO,and then repeated 6 h,12 h,24 h and 36 h thereafter and the same volume of 9 g/ L saline was simultaneously given to BUO - R rats. The Sham group was prepared in parallel by laparotomy and free dissection of bilateral ureters but not ligated. Both side kidneys were harvested 48 h(72 h for Sham group)after release of BUO to examine the effect of EPO on the expression of AQP2 in inner medulla by immunohisto-chemistry,Real - time PCR and Western blot. The urine samples were collected by using metabolic cage before death. Results The osmotic pressure of BUO - R ﹢ EPO group was higher than that of BUO - R group,but lower than that of Sham group(P ﹤ 0. 05). Immunohistochemistry showed that the collecting duct wall thinned and lumen enlarged. After the pictures were analysized by using Image - Pro Plus software,it showed that the expression of AQP2 in collecting duct in BUO group was significantly down - regulated compared with that in Sham group,whereas,it was slightly weaker in BUO - R group and BUO - R ﹢ EPO group than Sham group(P ﹤ 0. 05). These results were further confirmed by a-dopting Western blot,and the relative quantity of AQP2 in BUO group was also the lowest of the four groups(P ﹤0. 05). Real - time PCR showed that the level of AQP2 mRNA in Sham group was(24. 30 ± 1. 03)folds of BUO group,(10. 60 ± 1. 05)folds of BUO - R group and(5. 70 ± 1. 01)folds of BUO - R ﹢ EPO group,respectively. Conclusion EPO could promote not only the recovery of AQP2 mRNA and protein expression but also the recovery of AQP2 function in young BUO - R rats.

Objective To investigate the voiding patterns of term and preterm newborns and whether voiding in term and preterm neonates was accompanied by any cortical arousal. Methods Between May 2013 and September 2013,64 hospitalized newborns at Neonatal Intensive Cave Unit in the Frist Affiliated Hospital of Zhengzhou University were recruited in this study. In these patients,31 cases were term newborns(20 male,11 female)and 33 cases were preterm newborns(19 male,14 female). The term and preterm newborns gestational ages at birth were(38. 2 ± 1. 2) weeks and(32. 1 ± 1. 6)weeks,weighted(3. 3 ± 0. 4)kg and(1. 7 ± 0. 3)kg,respectively and postnatal ages at study were[4 - 16(10. 5 ± 3. 6)]days and[4 - 16(11. 2 ± 3. 1)]days. The voiding volume(VV),post - void residual volumes(PRV),body movement rate and voiding frequency(VF)in 4 hours as well as the volume of milk and liquid fed at the same time frame were recorded and analyzed,retrospectively. At the same time electrocardiogram(ECG)and electroencephalogram(EEG)were recorded. The changes of heart rate(HR),EEG frequency,respiratory frequency (RF)during the 5 s period and 30 s before and after voiding onset were compared respectively. For cortical arousal definition the recommendations of the International Pediatric Work Group on Arousals(2005)were used. Results A total of 184 times of voiding were recorded. In preterm newborns,the VV and body movements rate were significantly lower compared with the term newborns[(21. 8 ± 7. 9)mL and(41 ± 21)% vs(26. 4 ± 8. 7)mL and(62 ± 19)% , t = 3. 75,4. 20,all P ﹤ 0. 05]. However,the VF and PRV were significantly higher in preterm newborns[(1. 7 ± 0. 9) mL and(3. 2 ± 1. 1)times vs(1. 2 ± 0. 7)mL and(2. 6 ± 0. 9)times,t = 2. 47,2. 38,all P ﹤ 0. 05]. Bladder voiding in these infants happened only during QS. In term newborns,HR frequency was higher during the 5 s interval before and after voiding onset when compared with the 30 s period before voiding onset[(152 ± 6)times/ min and(152 ± 5) times/ min vs(147 ± 6)times/ min,t = 5. 30,5. 76,all P ﹤ 0. 05]and the EEG frequency[(2. 6 ± 0. 1)Hz and (2. 6 ± 0. 1)Hz vs(1. 5 ± 0. 1)Hz,t = 70. 0,70. 0,all P ﹤ 0. 05]. While the HR and EEG frequency of preterm neo-nate was not changed before and after bladder voiding onset. The RF of both term and preterm neonates were not changed before and after bladder voiding onset. Conclusions The voiding patterns between term and preterm were sig-nificantly different and cortical arousal was found only in term neonates,which indicate the term newborns have better mature bladder function and development of nervous system.

OBJECTIVETo summarize our experience in surgical treatment of traumatic lower limb pseudoaneurysm.

METHODSTwenty patients with traumatic lower limb pseudoaneurysm were surgically treated in our department from January 2007 to January 2012. The treatment protocols included interventional covered-stent placement (10 cases), spring coil embolization (2 cases), and surgical operation (8 cases). Surgical operations included pseudoaneurysm repair (2 cases), autologous-vein transplantation (1 case), and artificial-vessel bypass graft (5 cases).

RESULTSAll the patients were successfully treated without aggravating lower limb ischemia. Pseudoaneurysm disappeared after treatment. A surgical operation is suitable to most pseudoaneurysms, but its damage is relatively obvious and usually leads to more bleeding. It also requires a longer operating time. Compared to a surgical operation, interventional therapy is less traumatic and patients usually have a quicker recovery (P<0.05). All patients were followed up once per month for 12-36 months by color Doppler ultrasound examination. There were no cases of pseudoaneurysm recurrence.

CONCLUSIONBoth surgical operation and interventional therapy are safe and effective in the treatment of pseudoaneurysm.

Adult ; Aged ; Aneurysm, False ; diagnostic imaging ; etiology ; surgery ; Angiography, Digital Subtraction ; Embolization, Therapeutic ; Female ; Humans ; Leg Injuries ; complications ; diagnostic imaging ; surgery ; Male ; Middle Aged ; Stents ; Treatment Outcome ; Ultrasonography, Doppler