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 investigate the efficacy of Tanzhuo Decoction in the treatment of early diabetic nephropathy in patients with type 2 diabetes mellitus and its effect on cystatin C (Cys-C), C-reactive protein (CRP), urinary albumin excretion rate (UAER), and creatinine clearance rate (CCr). Methods:Eighty patients with type 2 diabetes mellitus complicated by early diabetic nephropathy who received treatment at Maanshan Hospital of Traditional Chinese Medicine from 2019 to 2021 were included in this randomized controlled study. They were divided into a control group ( n = 40) and a treatment group ( n = 40) using the random number table method. Patients in the control group received conventional therapy including blood glucose and blood pressure control, while those in the treatment group received Tangzhuo Decoction in addition to the same treatment as that given to the control group. Both groups of patients were treated for 30 days. The clinical efficacy as well as pre- and post-treatment Cys-C, CRP, UAER, and CCr were compared between the two groups. Results:The total response rate in the treatment group was 92.5% (37/40), which was significantly higher than 75.0% (30/40) in the control group ( χ2 = 4.50, P < 0.05). After treatment, Cys-C, CRP, and UAER in the treatment group were (2.04 ± 0.08) mg/L, (3.97 ± 1.71) mg/L, and (91.18 ± 18.68) μg/min, respectively, which were significantly decreased compared with those before treatment ( t = 12.14, 5.59, 4.73, all P < 0.05). After treatment, CCr in the treatment group was (56.3 ± 5.01) mL/min, which was significantly increased compared with that before treatment ( t = -8.56, P < 0.05). After treatment, Cys-C, CRP, and UAER in the control group were (2.17 ± 0.04) mg/L, (4.66 ± 1.47) mg/L, and (103.93 ± 22.62) μg/min, respectively, which were significantly decreased compared with those before treatment ( t = 4.05, 5.00, 2.24, all P < 0.05). After treatment, CCr in the control group was (45.9 ± 4.9) mL/min, which was significantly increased compared with that before treatment ( t = -3.98, P < 0.05). There were significant differences in Cys-C, UAER, and CCr between the treatment and control groups ( t = -7.42, -2.29, 7.82, all P < 0.05). Conclusion:Tanzhuo Decoction for the treatment of early diabetic nephropathy in patients with type 2 diabetes mellitus has a definite effect. It can effectively reduce levels of Cys-C and UAER, reduce inflammatory reactions, improve kidney function, and delay the progression of kidney injury.

Objective To investigate the computed tomography (CT) features of solitary nodular invasive mucinous lung adenocarcinoma (IMA) in stage IA and establish its prediction model. Methods We included 53 lesions of 53 patients with stage-IA IMA and 141 control lesions of 141 patients with invasive non-mucinous lung adenocarcinoma (NIMA) that were confirmed by surgical pathology in our hospital from January 2017 to December 2019. Univariable analysis was used to compare the demographics and CT signs of the two groups. Multivariable logistic regression analysis was performed to determine the main factors influencing solitary nodular IMA. A risk score prediction model was constructed based on the regression coefficients of the main influencing factors. A receiver operating characteristic (ROC) curve was used to assess the performance of the model. Results The univariable analysis showed significant differences between the two groups in age, largest nodule diameter, tumor-lung interface, lobulation, spiculation, air-bronchogram or vacuole sign, vessel abnormalities (P < 0.05). The spiculation sign was different between the two groups, which was longer and softer in the IMA group while shorter and harder in the NIMA group. There was no significant difference in sex, nodule shape, or pleural retraction (P > 0.05), but irregular shapes were slightly more frequent in the IMA group. The multivariable logistic regression analysis showed that obscure tumor-lung interface (odds ratio (OR = 20.930, P < 0.05), air-bronchogram or vacuole sign (OR = 7.126, P < 0.05), spiculation sign (OR = 4.207, P < 0.05), and vessel abnormalities (OR = 0.147, P < 0.05) were the main influencing factors. The prediction model based on those factors’ regression coefficients had an area under the ROC curve of 0.829 (P < 0.05). Conclusion Compared with those with NIMA, patients with solitary nodular IMA in stage IA were older and more likely to have the CT features of obscure tumor-lung interface, air-bronchogram or vacuole sign, and longer and softer spiculation. Based on the regression coefficients of tumor-lung interface, air-bronchogram or vacuole sign, spiculation, and vessel abnormalities, the risk score prediction model showed good predictive performance for solitary nodular IMA.

To explore the effect of IL-33 on various tumor types,exogenous injection of recombinant mouse IL-33 protein (mIL-33) was used to study the efficacy of different subcutaneous tumor-bearing mouse models. In this study,mIL-33 has been found to significantly inhibit the growth of liver,lung,gastric,prostate,and colon cancers. However,the inhibition of tumor growth by mIL-33 was not completely consistent in different types of tumor. A lower dose of mIL-33 (10 μg/kg) significantly inhibited tumor growth in subcutaneous tumor-bearing mice of liver,lung,and gastric cancers,while a higher dose of mIL-33 (90 μg/kg) was required to exert the corresponding antitumor effect in subcutaneous tumor-bearing mouse models with prostate and colon cancers. In addition,the growth inhibitory effect of mIL-33 on subcutaneous tumors of colon cancer was also correlated with the duration of administration and the stage of tumor progression. The results of this study indicate that mIL-33 significantly inhibits the growth of a variety of tumors,suggesting that IL-33 might be an effective target for tumor treatment.

OBJECTIVE： To establish a method for simultaneous determination of nine components in Huoxiang zhengqi oral liquid， and to improve and perfect the quality standard of Huoxiang zhengqi oral liquid. METHODS： The contents of nine components in 10 batches of Huoxiang zhengqi oral liquid were determined by HPLC， such as licorice coumarin， isorlicin， liquiritinapioside， narirutin， liquiritin， saponins， hesperidin， magnolol and honokiol. The determination was performed on Kromasil Eternity XT-5-C18 column with mobile phase consisted of acetonitrile-0.05％ phosphoric acid solution （gradient elution） at the flow rate of 1 mL/min. The detection wavelength was set at 220 nm， and column temperature was 25 ℃. The sample size was 10 μL. RESULTS： The linear range of licorice coumarin， isorlicin， liquiritinapioside， narirutin， liquiritin， saponins， hesperidin， magnolol and honokiolin were 0.000 5-0.007 5， 0.000 8-0.025 0， 0.006 1-0.976 0， 0.001 6-0.250 0， 0.007 8-0.025 0， 0.000 4- 0.062 7， 0.008 6-0.276 0， 0.010 0-0.500 0， 0.010 0-0.500 0 mg/mL （r＝0.999 2-1.000 0）. The detection limits were 0.001 3， 0.000 1， 0.004 7， 0.005 0， 0.012 0， 0.001 3， 0.007 8， 0.007 7 0， 0.005 8 μg/mL， and the quantitative limits were 0.013 0， 0.000 8， 0.047 0， 0.050 0， 0.120 0， 0.013 0， 0.078 0， 0.070 0， 0.058 0 μg/mL， respectively； RSD of precision， stability and repeatability tests were less than 3.0％ （n＝6）. Average recovery rates were 98.67％， 101.85％， 98.97％， 103.05％， 100.00％， 97.78％， 97.91％， 100.13％， 101.95％； RSDs were 1.14％， 2.18％， 0.40％， 0.17％， 1.38％， 0.85％， 1.38％， 0.10％， 1.35％ （n＝6）. CONCLUSIONS： The established method is accurate and reliable， which can provide reference for the establishment of the overall quality control evaluation system and the improvement of quality standard for Huoxiang zhengqi oral liquid.

Objective:To determine drug dose and usage of timolol maleate eye drops in the treatment of superficial infantile hemangioma.Methods:A total of 250 superficial hemangioma infants were recruited and assigned into 5 groups (n=50 for each group):an external application group and 4 exterior coating groups (2,4,6,8 times per day).We evaluated the therapeutic effect of different methods for drug application (external application or exterior coating) and the frequency for drug administration on superficial infantile hemangioma.Results:The external application group (twice a day and 0.5 hour per time) showed better effect than that in the exterior coating group with twice a day (P＜0.001).The difference in therapeutic effect between the exterior coating group with 6 times a day and exterior coating group with twice a day or with 3 times a day was significant (P＜0.001).The differences in drug efficacy were not found among the exterior coating group with 6 times a day,the exterior coating group with 8 times a day,or the external application group with twice a day (All P＞0.05).Conclusion:Drug dose may affect the therapeutic effect oftimolol maleate eye drops in superficial hemangioma infants,and exterior coating with 6 times a day may achieve the best curative effect.

Researches show that exosome can take park in the development and progression of breast cancer by means of mediating the intercellular communication,which can promote cancer metastasis and drug resistance,thus influencing the treatment effect of patients with cancers.Exosome is closely related with clinical stage and prognosis of breast cancer,which has a potential value in the early diagnosis and biological therapy of breast cancer and provides a new hope for the treatment of breast cancer.

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is one of the most important targeted drugs for lung cancer patients carrying EGFR sensitive mutations.However,almost all patients that are effective to this treatment will eventually develop secondary resistance to EGFR-TKI.The most accepted mechanisms of resistance mainly include T790M mutation,MET amplification,PIK3CA mutation,down-regulation of PTEN expression and activation of Fas-transcription factor-κB.Recent years,many new drugs are developed to overcome this resistance.Although most of drugs are in the stages of cell or animal experiment,some new drugs get positive clinical results.

Objective: Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR)-activating mutations have higher response rate and more prolonged survival following treatment with single-agent EGFR tyrosine kinase inhibitor (EGFR-TKI) compared with patients with wild-type EGFR. However, all patients treated with reversible inhibitors develop acquired re-sistance over time. The mechanisms of resistance are complicated. The lack of established therapeutic options for patients after a failed EGFR-TKI treatment poses a great challenge to physicians in managing this group of lung cancer patients. This study evaluates the in-fluence of EGFR-TKI retreatment following chemotherapy after failure of initial EGFR-TKI within at least six months on NSCLC pa-tients. Methods:The data of 27 patients who experienced treatment failure from their initial use of EGFR-TKI within at least 6 months were analyzed. After chemotherapy, the patients were retreated with EGFR-TKI (gefitinib 250 mg qd or erlotinib 150 mg qd), and the tumor progression was observed. The patients were assessed for adverse events and response to therapy. Targeted tumor lesions were as-sessed with CT scan. Results:Of the 27 patients who received EGFR–TKI retreatment, 1 (3.7%) patient was observed in complete re-sponse (CR), 8 (29.6%) patients in partial response (PR), 14 (51.9%) patients in stable disease (SD), and 4 (14.8%) patients in progres-sive disease (PD). The disease control rate (DCR) was 85.2%(95%CI=62%-94%). The median progression-free survival (mPFS) was 6 months (95%CI=1-29). Of the 13 patients who received the same EGFR-TKI, 1 patient in CR, 3 patients in PR, 8 patients in SD, and 2 patients in PD were observed. The DCR was 84.6%, and the mPFS was 5 months. Of the 14 patients who received another EG-FR-TKI, 0 patient in CR, 6 patients in PR, 6 patients in SD, and 2 patients in PD were observed. The DCR was 85.7%, and the mPFS was 9.5 months. Significant difference was found between the 2 groups in progression-free survival but not in response rate or disease control rate. Conclusion:Retreatment of EGFR-TKIs can be considered an option after failure of chemotherapy for patients who were
previously controlled by EGFR-TKI treatment.

Objective To evaluate the significance of COLD-PCR in detecting KRAS mutation of pancreatic cancer and colorectal cancer patients. Methods First, set up COLD-PCR and compared the sensitivities of COLD-PCR/Sanger sequencing with PCR/Sanger sequencing using mixed cell lines ( KRAS wild-type cell line SW116 and KRAS mutant cell line SW480).Then, detected KRAS mutation of 20 formalin-fixed paraffin-embedded samples of pancreatic cancer and 39 formalin-fixed paraffin-embedded samples of colorectal cancer using PCR/Sanger sequencing and COLD-PCR/Sanger sequencing, respectively and compared the coincidence rate and consistency. Results The low detection limits of PCR/Sanger respectively. KRAS frequency detected by COLD-PCR/Sanger sequencing [75% (15/20)] in 20 cases of pancreatic cancer was higher than that detected by regular PCR/Sanger sequencing [40% ( 8/20 ) ,x2 =5.013, P < 0.05]. KRAS frequency detected by COLD-PCR/Sanger sequencing [44% (17/39)] in 39 cases of colorectal cancer was higher than that detected by regular PCR/Sanger sequencing [31% (12/39) ,x2 =1. 372, P = 0. 174]. The coincidence rate of these two methods was 0. 730 and the difference had no statistical significance. The coincidence rate of detecting KRAS mutation was 65% in pancreatic cancer and the results showed a good correlation between two methods and the two methods had bad agreement in diagnosis (Kappa = 0. 364, P < 0. 05 ). COLD-PCR/Sanger sequencing could detect more cases of KRAS mutations from pancreatic caner than regular PCR/Sanger sequencing. This was because there were many non-tumor cells in pancreatic tumor tissue and COLD-PCR/Sanger sequencing was more sensitive than regular PCR/Sanger sequencing. The coincidence rate of detecting KRAS mutations was 87% in colorectal cancer and the results were showed a good correlation between two methods and the two methods had substantical agreement in diagonsis ( Kappa = 0. 730, P < 0. 05 ) . Conclusion COLD-PCR/Sanger sequencing is highly sensitive to screen KRAS mutation in pancreatic cancer and colorectal cancer patients.