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 establish a disk (CD) microfluidic chip detection platform for the rapid detection of CALR-1 and CALR-2 mutations in patients with cerebral infarction, and summarize its clinical application value.Methods:Based on microfluidic technology and loop mediated isothermal amplification technology, a CD microfluidic chip detection platform for simultaneous detection of CALR-1 and CALR-2 gene mutations were established, and the sensitivity, specificity, repeatability and accuracy of the platform were verified. A total of 124 patients with cerebral infarction treated in Huashan Hospital, Shanghai Medical College, Fudan University from November 2019 to March 2021 were prospectively selected into the experimental group; and 80 healthy subjects were included in the control group. The CALR-1 and CALR-2 gene mutations in anticoagulant peripheral blood samples were detected by the CD microfluidic chip. Each chip could detect 4 samples at the same time and synchronously detect 3 indexes of each sample. The detection results could be obtained after isothermal amplification for 40 min. At the same time, sequencing method was used to verify the test results, and the consistency of the results of the two detection methods was compared.Results:Using this CD microfluidic chip platform, the synchronous amplification of 3 indexes in the sample could be completed within 40 min without the need of thermal circulation, and the whole detection process of the sample could be completed within 60 min. For samples with a high concentration of target nucleic acid, typical positive signals could be visualized after amplification for 10 min, and the test results would be available within 30 minutes after receiving the samples. The detection sensitivity of CD microfluidic chip method for CALR-1 and CALR-2 mutation load concentration was 1.0% and 0.5% respectively. Nonspecific amplification was not observed for the non-target nucleic acid samples, indicating the high specificity of this method. The coincidence rates of intra and inter batch repeatability were 100% (20/20) respectively. Two samples with CALR gene mutation were found in the cerebral infarction group, both of which were CALR-1 mutations (L367fs*46). There was no CALR-1 or CALR-2 mutation in the control group. The detection results of CD microfluidic chip method were completely consistent with the sequencing verification results (100% [204/204]).Conclusions:The CD microfluidic chip method could be used for the detection of CALR-1 and CALR-2 gene mutations in clinical samples of patients with cerebral infarction. This method has the advantages of high detection sensitivity, good detection specificity, fast detection speed and high detection flux, which is helpful to clarify the etiology of patients with cerebral infarction.

Objective:To analyze the molecular epidemiological characteristics of the Corona virus disease 2019 (COVID-19) cases in Shijiazhuang, which can reveal the origin of the outbreak and provide a scientific basis for COVID-19 prevention and control.Methods:From January 2 to January 8, 2021, a total of 404 samples from 170 COVID-19 cases were collected from the Shijiazhuang Fifth Hospital. The consensus sequence of 2019 novel Coronavirus(2019-nCoV) was obtained through multiplex polymerase chain reaction-based sequencing. The sequences of 170 COVID-19 cases were analyzed by the PANGOLIN, and the data were statistically analyzed by T-test.Results:Among the 404 COVID-19 samples, a total of 356 samples obtained high quality genome sequences (>95%,100×sequencing depth). The whole genome sequences of 170 COVID-19 cases were obtained by eliminating repeated samples. All 170 sequences were recognized as lineage B1.1 using PANGOLIN. The number of single nucleotide polymorphism arrange from 18-22 and most of the single nucleotide polymorphism were synonymous variants. All of 170 genomes could be classified into 48 sub-groups and most of the genomes were classified into 2 sub-groups (66 and 31, respectively).Conclusions:All cases in this study are likely originated from one imported case. The viruses have spread in the community for a long time and have mutated during the community transmission.

Objective:To investigate the risk factors of duodenal papilla hemorrhage after endoscopic papillary balloon dilatation (EPBD) for choledocholithiasis.Methods:Clinical data of 411 cases of choledocholithiasis treated by EPBD in Hangzhou First People′s Hospital from January 2016 to December 2019 were analyzed retrospectively. Based on the development of hemorrhage after EPBD, patients were divided into the hemorrhage group and the non-hemorrhage group. The risk factors of hemorrhage after EPBD were analyzed by single and Logistic regression.Results:Among 411 patients who received EPBD, 29 patients had EPBD-related duodenal papilla hemorrhage and the overall incidence was 7.1%.Univariate analysis showed that there were significant differences between the hemorrhage group and the non-hemorrhage group in diameter≥1.2 cm of balloon dilation ( P=0.001), endoscopic sphincterotomy (EST) ( P=0.002)and the incision length of EST ( P<0.001). Logistic regression analysis showed that the incision length of EST ( OR=69.771, 95% CI: 7.544-645.296, P<0.001) was the independent risk factor for duodenal papilla hemorrhage after EPBD. Diameter≥1.2 cm of balloon dilation( OR=0.192, 95% CI: 0.071-0.524, P=0.001) was a protective factor. Conclusion:The incision length of EST is an independent risk factor of duodenal papilla hemorrhage after EPBD. Endoscopic papillary large balloon dilation is a protective factor for postoperative hemorrhage, which can reduce the incidence of bleeding.

Objective:To observe the effects of static 70° head-up tilted standing and of repeated body repositioning on hemodynamics in healthy young and middle-aged persons.Methods:The hemodynamics of 24 middle-aged and 23 younger persons were studied. Both groups were requested to perform static 70° head-up tilted standing and to repeatedly change their body position from 0° to 70° of tilt at a velocity of 1°/second for ten minutes in a random order. Before, between and after each test the subjects rested supine for ten minutes. Hemodynamic variables and blood pressure were recorded non-invasively.Results:The average heart rate (HR) increased significantly in both groups when rising from supine to the testing positions. In 70° tilted standing the average HR of the youth group, 84.0±9.5bpm, was significantly higher than that in the other position and that of the middle-aged group in the same position. The average HR of the middle-aged group in 70° tilted standing was also significantly higher than in the other position. Among the middle-aged group, the average stroke volume (SV) in the testing positions was significantly lower than when resting. Significant differences were observed in the average diastolic blood pressure (DBP) between the testing and rest positions for both groups, with the average DBP of the middle-aged group significantly higher than that of the youth group in all three positions. Among the youth group, the average SV, CO and systolic blood pressure (SBP) of the males were significantly higher than among the females in all of the different body positions.Conclusions:Young persons mainly rely on an increased heart rate to maintain cardiac output while middle-aged participants appear to achieve this through increased peripheral resistance. Repeated position changes have less impact on hemodynamics than 70° inclined standing, making it a safer and more stable training method. However, the long-term effects of such intervention need to be confirmed in further studies.

Objective To study the different drying technology.on Wudang Lonicerae Flos (Ⅰ) in chlorogenic acid content.Methods Using HPLC method to research the difference in the contents of chlorogenic acid of Wudang Lonicerae Flos (Ⅰ) employing oven-drying method,air-dried method and steamed drying technologies.DIONEX C18 (4.6 mm×250 mm,5 μm) was used.The mobile phases consisted of acetonitrile-0.4％ phosphoric acid aqueous solution (17 ∶ 83).The detection wavelengths of chlorogenic acid was 327nm.The flow rate was 0.4 ml/min.The column temperature was 30 ℃.Results The Separation of Chlorogenic acid content was satisfactory.The concentration and peak area showed a good linearity relationship with the range of 0.285-2.850 μg.Precision,repeatability and stability met the requirements.The average recovery was 100.26％ with RSD 1.95％.Chlorogenic acid content in the steamed drying Wudang Honeysuckle (Ⅰ) was higher.Conclusions The method of steam drying is better than that of oven-drying method and drying.

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 assess the clinical and radiologic outcomes between laminoplasty with forominotomy(LF) and anterior cervical discectomy and fusion(ACDF) in treating cervical radiculomyelopathy(CRM). Methods Datas of 68 patients (ACDF=33, LF=35) from January 2008 to January 2010 was collected retrospectively, the follow-up is at least 2 years. The Japa-nese Orthopedic Association (JOA) score and associated recovery rate were evaluated. For radiographic evaluation, the lordotic an-gle and range of motion (ROM) at C2-C7 were investigated. The Neck Disabilitv Index Scale(NDI) was used to evaluate the degree of patient’pain at the last follow-up. Results Patients’demographics were similar between the two groups. The differences be-tween ACDF and LF in operative time (187min VS 154min),the blood loss (127 ml VS 235 ml) and the sensation of lower extremity (64.0%VS 66.0%) are significant(t=4.170, P=0.000;Z=-6.888, P=0.000;Z=-7.512, P=0.000). 1 case with failed fusion of bone graft 3 months post-operation. 3 cases of adjacent segment degenerative changes occurred at the 2nd year follow-up in ACDF group. But no such complications occurred in the EOLF group. In addition, ACDF group showed lower NDI score than LF group in extracting and amusing (Z=-3.947, P=0.000;t=-7. 523, P=0.000). Cervical lordosis of ACDF increased from 13.7° to 16.2°, while that of LF group decreased from 14.6° to 13.3°(Z=-3.374,P=0.001);Both of the two groups (ACDF/LF) exhibited decreased cervi-cal ROM (14.8° VS 16.5°, t=-2.167, P=0.034). Conclusion The two surgical procedures have similar clinical effects in treating multi-segmental CRM. However, the LF group demonstrated shorter operative time, fewer short-term complications, so it proved to be effective and safe surgical procedure.

OBJECTIVE: To explore the laws of traditional Chinese medicine (TCM) syndromes in unstable angina, and to establish the preliminary diagnostic criteria for TCM syndromes. METHODS: Multi-center prospective research on TCM syndromes in 815 cases of unstable angina was done with the nonlinear dimension reduction by factor analysis. RESULTS: There were five extracted factors in factor analysis: F1, F2, F3, F4 and F5. F1 was yin deficiency of heart and kidney, F2 was deficiency of both heart and spleen, F3 was intermingled phlegm and blood stasis, F4 was qi deficiency and blood stasis, and F5 was yang deficiency and coagulated cold. Qi deficiency and blood stasis (F4) syndrome accounted for the maximum proportion. The diagnostic criteria for TCM syndromes were preliminarily and respectively established. CONCLUSION: Qi deficiency and blood stasis is the key factor of pathogenesis. The factor analysis can help us classify traditional Chinese medicine syndromes and establish the preliminary diagnostic criteria.