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*

Purpose: This study aimed to identify the altered pathways and genes associated with freezing damage in human sperm during cryopreservation by multiomics analysis. Materials and Methods: Fifteen fresh human semen samples were collected for transcriptomic analysis, and another 5 fresh human semen samples were obtained for metabolomic analysis. For each semen sample, 1 mL was cryopreserved, and another 1 mL was left untreated for paired design. The results were then combined with previously published proteomic results to identify key genes/pathways. Results: Cryopreservation significantly reduced sperm motility and mitochondrial structure. Transcriptomic analysis revealed altered mitochondrial function, including changes in tRNA-methyltransferase activity and adenosine tri-phosphate/adenosine di-phosphate transmembrane transporter activity. Metabolomic analysis showed that the citrate cycle in mitochondria was significantly altered. Combining transcriptomic, proteomic, and metabolomic analyses revealed 346 genes that were altered in at least two omics analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that metabolic pathways were significantly altered and strongly associated with mitochondria. Five genes were altered in all three omics analyses: COL11A1, COL18A1, LPCAT3, NME1, and NNT. Conclusions Five genes were identified by multiomics analysis in human cryopreserved sperm. These genes might have specific functions in cryopreservation. Explorations of the functions of these genes will be helpful for sperm cryopreservation and sperm motility improvement or even for reproduction in the future.

Objective:To evaluate percutaneous mechanical thrombectomy (PMT) using Aspirex device for treating acute iliofemoral deep vein thrombosis (IFDVT).Methods:The clinical and follow-up data of 68 patients with IFDVT at our institution from Jan 2019 to Jun 2021 was retrospectively analyzed.Results:Twenty-six patients who had received PMT combined with auxiliary catheter directed thrombolysis (CDT) were included into group A, and 42 patients received CDT alone were into group B.The final thrombus clearance rates were more than 50%, and the clinical efficacy of thrombolysis was achieved. Group A associated a significant reduction in lysis duration and UK dosage and hospital days and degree of detumescence after 24 h compared with group B,and all aforementioned differences were statistically significant. Hospitalization costs in group A were more than group B. At one year follow-up, there were no significant differences between the two groups in the cumulative prevalence post-thrombotic syndrome (PTS) and the Villalta score and primary patency (92.0% vs. 90.0% , χ2=0.059, P=0.807). Conclusions:The application of PMT using the Aspirex device for acute IFDVT was safe and effective, which could accelerate the clearance of thrombus, and reduce UK dosage, lysis duration, hospital days. However, it increased the hospitalization costs.

Objective:To explore the team construction and treatment strategy of the Diabetic Foot-Multidisciplinary Team.Methods:The clinical data of 19 patients with severe ischemic diabetic foot treated by our Diabetic Foot-Multidisciplinary Team Center from Apr 2021 to Mar 2022 were collected, and the overall amputation rate, above-ankle major amputation rate, minor amputation rate and mortality, Diabetic Foot-Multidisciplinary Team consultation discipline participation rate and treatment participation degree were retrospectively analyzed.Results:Nineteen patients (15 males and 4 females) were enrolled, aged 26 to 94 (68.6±14.2). All were with severe ischemic diabetic foot ulcer:Rutherford grade 5 or up and dysfunction in 2 or more organs. Complications included arteriosclerosis obliterans of the lower extremities in 18 cases, heart diseases in 18, hypertension in 15, and renal insufficiencies in 10. The overall amputation rate was 36.8%, major amputation rate in 21.1%, minor amputation rate in 15.8%, and mortality rate was 15.8%. A total of 16 disciplines participated in Diabetic Foot-Multidisciplinary Team; the main participating disciplines were vascular surgery (19 times), endocrinology (12 times), and cardiology (11 times). The main treatment disciplines were vascular surgery (14 times), plastic surgery (3 times), and cardiology (2 times).Conclusion:For the diagnosis and treatment of diabetic foot, it is necessary to set up a multidisciplinary team as early as possible to control the causes of diabetic foot ulcer, prevent the recurrence of diabetic foot ulcer, reduce the mortality and amputation rate, and improve the quality of life of patients.

Objective:In this study, the role of IL-17 in the pathogenesis of idiopathic myositis (IIM) was preliminarily investigated by detecting the expression of IL-17 in the muscle tissues of patients with idiopathic inflammatory myositis (IIM) and normal controls.Methods:Twenty-eight patients (20 in DM group with dermatomyositis and 8 in ASS group with anti-synthase syndrome) who were diagnosed with IIM after muscle biopsy and autoantibody detection in our hospital for the first time from October 2019 to August 2021 were included. Twelve cases with normal muscle tissue matched for age and sex were included as the control group. Western blot and immunohistochemical techniques were used to detect the expression level of IL-17 in muscle tissue, and enzyme-linked immunosorbent assay (ELISA) was used to detect serum IL-6. Mann-Whitney U rank sum test was used to compare the difference of IL-17 expression in muscle tissue between the two groups, and non-parametric test was used for comparison between multiple groups. Chi-square test and Spearman rank correlation analysis were used, and P<0.05 was considered statistically significant. Results:① The expression level of IL-17 in IIM muscle tissue[1.63(1.30, 2.05)pg/ml was higher than that in control group[1.00(0.96, 1.00)pg/ml, and the difference was statistically significant ( Z=-3.52, P<0.001). The difference be-tween DM[1.94(1.58, 2.14)pg/ml] and ASS[1.22(1.04,1.55)pg/ml was statistically significant ( Z=-3.20, P=0.001). ② Compared with healthy control group [4.08(3.01, 5.67)pg/ml, the expression of IL-6 in ⅡM serum[8.88(4.93, 13.64) was high ( Z=-3.01, P=0.003), which was positively correlated with the expression of IL-17 ( r=0.42, P=0.027). ③ The ex-pression of IL-17 in muscle tissue was higher in IIM associated with muscle weakness[1.91(1.56, 2.14) pg/ml vs 1.50(1.04, 2.00)pg/ml] ( Z=-1.38, P=0.020), dysphagia [2.06(1.99, 2.14)pg/ml vs 1.62(1.52, 2.04)pg/ml] ( Z=-2.74, P=0.010) and skin involvement[1.98(1.57, 2.14)pg/ml vs 1.04(0.86, 1.61)pg/ml] ( Z=-3.20, P<0.010), and the differences were statistically significant ( P<0.05). ④IL-17 was positively correlated with Myoact-total activity ( r=0.51, P=0.006), Myoact-muscle symptom ( r=0.45, P=0.016), erythrocyte sedimen tation ( r=0.48, P=0.020), and myoenzyme increase ( r=0.56, P=0.002). Conclusion:IL-17 and IL-6 are synergistically involved in the pathogenesis of IIM, suggesting that IL-17 is the therapeutic target of IIM.

Objective:To explore the safety and effectiveness of the application of sevoflurane inhalation sedation and analgesia during dressing changes in children with extensive burns.Methods:A prospective randomized controlled research was conducted. From March 2020 to January 2023, 216 children with extensive burns who met the inclusion criteria were admitted to the Department of Burns and Plastic Surgery of Kunming Children's Hospital. According to the random number table, the children were divided into sevoflurane group and ibuprofen group, with 103 cases left in sevoflurane group (67 males and 36 females, aged 1 (1, 2) years), and 98 cases left in ibuprofen group (67 males and 31 females, aged 1 (1, 2) years) after the exclusion of several dropped-out children. Children in sevoflurane group received sevoflurane inhalation for sedation and analgesia during dressing changes, while those in ibuprofen group took oral ibuprofen for analgesia before dressing changes. The heart rate, mean arterial pressure (MAP), and percutaneous arterial oxygen saturation (SpO 2) of the children were monitored and recorded at 30 minutes before the start of dressing changes, immediately after debridement, and at 30 minutes after the completion of dressing changes. The face, legs, activity, cry, and consolability scale and Ramsay sedation scale were used to evaluate the pain intensity and degree of sedation, respectively, at 30 minutes before the start of dressing changes, immediately after debridement, and at 30 minutes after the completion of dressing changes. The duration of dressing changes and the total number of dressing changes during hospitalization were recorded. The Houston Pain Outcome Instrument questionnaire was used to assess the satisfaction of the dressing-changing surgeons and a family member of the child with the analgesic effects during the process of dressing change when the children were discharged from the hospital. The occurrence of adverse reactions such as respiratory depression and hypoxemia that occurred during the process of dressing change were monitored and recorded. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, Wilcoxon rank-sum test, chi-square test, and Fisher's exact probability test. Results:At 30 minutes before the start of dressing changes and 30 minutes after the completion of dressing changes, there were no statistically significant differences in heart rate, MAP, and SpO 2 between children in the two groups ( P>0.05). Immediately after debridement, compared with those in ibuprofen group, the heart rate and MAP of children in sevoflurane group were significantly decreased (with t values of 8.10 and 4.37, respectively, P<0.05), while the SpO 2 was significantly increased ( t=21.77, P<0.05). At 30 minutes before the start of dressing changes and 30 minutes after the completion of dressing changes, there were no statistically significant differences in the score of pain intensity and score of sedation degree between children in the two groups ( P>0.05). Immediately after debridement, compared with that in ibuprofen group, the score of pain intensity of children in sevoflurane group was significantly decreased, while the score of sedation degree was significantly increased (with t values of 42.87 and 72.45, respectively, P<0.05). The duration of dressing changes and the total number of dressing changes during hospitalization of patients in sevoflurane group were (18±3) min and (4.1±1.0) times, respectively, which were both significantly shorter than (26±7) min and less than (6.6±1.4) times in ibuprofen group, respectively (with t values of -4.44 and 14.17, respectively, P<0.05). Upon discharge, the satisfaction scores of dressing-changing surgeons and the family members of children with the analgesic effects during the process of dressing change in sevoflurane group were significantly higher than those in ibuprofen group (with t values of 44.23 and 36.55, respectively, P<0.05). There were no statistically significant differences in the incidence of respiratory depression, hypoxemia, hypotension, coughing, nausea, and vomiting during the process of dressing change between children in the two groups ( P>0.05). Conclusions:Application of sevoflurane inhalation during dressing changes in children with extensive burns can safely and effectively control pain and sedation, shorten the time for dressing change, with fewer adverse reactions. This method can be used for routine dressing change in pediatric burn wards.

Objective To explore the association of nocturnal serum cortisol levels with diabetic microvascular complications in overweight or obese patients with type 2 diabetes mellitus. Methods Serum cortisol levels of 316 overweight or obese type 2 diabetic patients were tested at midnight by the method of chemiluminescence. Diabetic microvascular complications were compared among various groups according to nocturnal serum cortisol levels. All the patients with nocturnal serum cortisol level > 50 nmol/L were asked to undergo overnight low-dose dexamethasone suppression test to rule out the possibility of subclincal Cushing's syndrome. The incidences of diabetic nephropathy ( DN ) , diabetic retinopathy ( DR ) , and diabetic peripheral neuropathy ( DPN ) were examined in all the patients. Results (1)The incidence of DN was gradually increased from 13.3％to 27.7％and 44.2％in patients with low, medium, and high cortisol level groups, showing a statistical difference among 3 groups ( P<0.05) . The incidences of DR in medium and high cortisol level groups were higher than that in low cortisol level group (40.6％and 47.7％vs 22.7％, both P<0.01). The incidence of DPN in high cortisol level group was higher as compared with low cortisol level group (60.5％ vs 38.7％, P<0.01). (2) Nocturnal serum cortisol level in patients with diabetic microvascular complications was higher than that in patients without complications [ (136.87 ± 105.78 vs 97.55 ± 93.48) nmol/L, P<0.01]. Nocturnal serum cortisol level in patients with multiple diabetic microvascular complications was higher than that in patients with single diabetic microvascular complication [ (151.66±114.54vs117.69±90.26)nmol/L,P<0.05].(3)Singlefactorlogisticregressionanalysisshowedthat higher nocturnal serum cortisol level was a risk factor for diabetic microvascular complications in addition to female, age, longer diabetic duration, higher fasting plasma glucose ( FPG ) . Multivariate logistic regression analysis showed that higher nocturnal serum cortisol level was still a risk factor for diabetic microvascular complications after adjusted by diabetic duration, FPG, HbA1C, and the use of insulin (P=0.013). Conclusion Nocturnal serum cortisol level seems to be a risk factor for diabetic microvascular complications in overweight or obese patients with type 2 diabetes mellitus.

Purpose To investigate the expression and significance of ArhGAP29 and E-cadherin in endometrial tissue of intrauterine adhesions (IUAs) and to explore their roles in the pathogenesis of IUAs.Methods The expression of ArhGAP29 and E-cadherin was detected by immunohistochemical PV 9000 two-step method.The correlation between ArhGAP29 and E-cadherin expression and clinical features was analyzed.Results (1) The immunoreactive score (IRS) of ArhGAP29 and E-cadherin in normal endometrial tissue were higher than those in IUAs endometrial tissue (P =0.017,P =0.004).(2) IRS of ArhGAP 29 and E-cadherin in moderate IUAs patients were higher than that in severe IUAs patients (P =0.020,P =0.026).In IUAs patients without amenorrhea,the IRS of ArhGAP29 and Ecadherin were higher (P =0.019,P =0.031) than that in IUAs patients with amenorrhea.(3) The decrease of ArhGAP29 expression had a significantly parallel relationship with the negative expression of E-cadherin (r =0.725,P ＜ 0.001).Conclusion The expression of ArhGAP29 and E-cadherin decreases in endometrial tissue of IUAs patients,which is related with degree of IUAs severity.ArhGAP29 and E-cadherin may participate in the IUAs formation.

Objective To assess the efficacy and safety of tacrolimus (TAC) combined with methotrexate (MTX) for the treatment of refractory rheumatoid arthritis (RA),and to compare it with cyclophosphamide (CTX) added to MTX for the treatment of refractory RA.Methods Thirty-six cases of refractory RA patients were divided into the observation group and the control group.TAC+MTX were used in the observation group,and CTX+MTX were used in the control group.We used repeated measures to analyze the variance and Fisher exact probability method to analyze the efficacy at 8 weeks and 24 weeks.Results The effective rate of the observation group in 8 weeks,24 weeks were 77.8％(14 cases) and 100％(18 cases) respectively,while those of the control group were 11.1％ (2 cases) and 44.4％(8 cases),it showed that both TAC+MTX and CTX+MTX in the treatment of refractory RA were effective,but the efficacy of TAC+MTX was better than CTX+MTX,the difference of C reactive protein (CRP) and disease activity score (DAS)28 was statistically significant (P＜0.05),and it could significantly improve the clinical symptoms and laboratory indexes.Conclusion TAC+MTX is effective and safe in treating refractory RA,and is worth of spreading.

Objective To explore the possible role of proline-rich tyrosine kinase (Pyk2) in the pathogenesis of systemic lupus erythematosus (SLE).Methods The expression of Pyk2 in the peripheral blood mononuclear cells (PBMCs) of 50 patients with SLE and 36 healthy controls were tested with RT-PCR assay.The activation of Pyk2 was inhibited using specific inhibitor Pyk2 (TyrA9).Semi-quantitative PCR methodwas used to detect the Blys expression of PBMCs.One-way ANOVA and Pearson's correlation analysis were used for statistical analysis.Results The Pyk2 expression level (28.31 ±0.91) of SLE patients was significantly higher than that in healthy controls (33.69±0.04),the difference was statistically significant (P＜0.05).The activation of Pyk2 was stimulated and the expression levels of Blys in the PBMCs of patients with SLE was elevated.By inhibiting the activation of Pyk2,the BLyS expression levels decreased significantly.Conclusion Pyk2 may be involved in the abnormal activation of lymphocytes which lead to the pathogenesis of SLE.Pyk2 expression is associated with SLE disease activity,disease aggravation,and the Pyk2 expression levels is also increased significantly.In addition,the expression level of Pyk2 is higher in patients with renal involvement than those patients with other organ involvement.