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*

Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016 pM.The mechanism was related to binding with Y453 of RBD deter-mined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quan-tum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)path-ways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.

Purpose::Mannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure. However, long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes. Given that neural stem cell (NSC) is a subpopulation of main regenerative cells in the central nervous system after injury, the effect of mannitol on NSC is still elusive. The present study aims to elucidate the role of mannitol in NSC proliferation.Methods::C57 mice were derived from the animal house of Zunyi Medical University. A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation. Initially, mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining. In order to investigate the impact of mannitol on NSC proliferation, both cell counting kit-8 assays and neurospheres formation assays were conducted. The in vitro effects of mannitol were examined at various doses and time points. In order to elucidate the role of Aquaporin 4 (AQP4) in the suppressive effect of mannitol on NSC proliferation, various assays including reverse transcription polymerase chain reaction, western blotting, and immunocytochemistry were conducted on control and mannitol-treated groups. Additionally, the phosphorylated p38 (p-p38) was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation. Finally, to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent (MAPK) signaling pathway in the observed inhibition of NSC proliferation by mannitol, SB203580 was employed. All data were analyzed using SPSS 20.0 software (SPSS, Inc., Chicago, IL). The statistical analysis among multiple comparisons was performed using one-way analysis of variance (ANOVA), followed by Turkey's post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test. Comparisons between 2 groups were determined using Student's t-test, if the data exhibited a normal distribution using a Shapiro-Wilk normality test. Meanwhile, data were shown as median and interquartile range and analyzed using the Mann-Whitney U test, if the data failed the normality test. A p < 0.05 was considered as significant difference. Results::Primary NSC were isolated from the mice, and the characteristics were identified using immunostaining analysis. Thereafter, the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8, neurospheres formation, and immunostaining of Nestin and Ki67 assays. During the process of mannitol suppressing NSC proliferation, the expression of AQP4 mRNA and protein was downregulated, while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction, immunostaining, and western blotting assays. Subsequently, the administration of SB203580, one of the p38 MAPK signaling pathway inhibitors, partially abrogated this inhibitory effect resulting from mannitol, supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol.Conclusions::Mannitol inhibits NSC proliferation through downregulating AQP4, while upregulating the expression of p-p38 MAPK.

We wished to establish an expert consensus on late stage of critical care (CC) management. The panel comprised 13 experts in CC medicine. Each statement was assessed based on the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) principle. Then, the Delphi method was adopted by 17 experts to reassess the following 28 statements. (1) ESCAPE has evolved from a strategy of delirium management to a strategy of late stage of CC management. (2) The new version of ESCAPE is a strategy for optimizing treatment and comprehensive care of critically ill patients (CIPs) after the rescue period, including early mobilization, early rehabilitation, nutritional support, sleep management, mental assessment, cognitive-function training, emotional support, and optimizing sedation and analgesia. (3) Disease assessment to determine the starting point of early mobilization, early rehabilitation, and early enteral nutrition. (4) Early mobilization has synergistic effects upon the recovery of organ function. (5) Early functional exercise and rehabilitation are important means to promote CIP recovery, and gives them a sense of future prospects. (6) Timely start of enteral nutrition is conducive to early mobilization and early rehabilitation. (7) The spontaneous breathing test should be started as soon as possible, and a weaning plan should be selected step-by-step. (8) The waking process of CIPs should be realized in a planned and purposeful way. (9) Establishment of a sleep-wake rhythm is the key to sleep management in post-CC management. (10) The spontaneous awakening trial, spontaneous breathing trial, and sleep management should be carried out together. (11) The depth of sedation should be adjusted dynamically in the late stage of CC period. (12) Standardized sedation assessment is the premise of rational sedation. (13) Appropriate sedative drugs should be selected according to the objectives of sedation and drug characteristics. (14) A goal-directed minimization strategy for sedation should be implemented. (15) The principle of analgesia must be mastered first. (16) Subjective assessment is preferred for analgesia assessment. (17) Opioid-based analgesic strategies should be selected step-by-step according to the characteristics of different drugs. (18) There must be rational use of non-opioid analgesics and non-drug-based analgesic measures. (19) Pay attention to evaluation of the psychological status of CIPs. (20) Cognitive function in CIPs cannot be ignored. (21) Delirium management should be based on non-drug-based measures and rational use of drugs. (22) Reset treatment can be considered for severe delirium. (23) Psychological assessment should be conducted as early as possible to screen-out high-risk groups with post-traumatic stress disorder. (24) Emotional support, flexible visiting, and environment management are important components of humanistic management in the intensive care unit (ICU). (25) Emotional support from medical teams and families should be promoted through"ICU diaries"and other forms. (26) Environmental management should be carried out by enriching environmental content, limiting environmental interference, and optimizing the environmental atmosphere. (27) Reasonable promotion of flexible visitation should be done on the basis of prevention of nosocomial infection. (28) ESCAPE is an excellent project for late stage of CC management.
Humans ; Consensus ; Critical Care/methods* ; Intensive Care Units ; Pain/drug therapy* ; Analgesics/therapeutic use* ; Delirium/therapy* ; Critical Illness

Accumulating epidemiological evidence shows that handgrip strength provides predictive potential in physical, mental, and reproductive health status. However, the associations between handgrip strength and semen characteristics have not been explored. We recruited 1382 eligible men at the Hubei Province Human Sperm Bank (Wuhan, China) who had their handgrip strength measured at recruitment and provided 6458 repeated semen specimens within a 6-month period. Semen characteristics, including semen volume, sperm motility parameters (immotility, nonprogressive motility, and progressive motility), and sperm concentration, were assessed. Mixed-effect models and restricted cubic spline functions were applied to investigate the relationship of handgrip strength with repeated measurements of semen characteristics. After adjusting for confounding factors, the mixed-effect models revealed that handgrip strength was positively associated with semen volume, sperm concentration, progressive motility, total motility, and total count (all P for trend < 0.05). Compared to men in the lowest quartile, those in the highest quartile of handgrip strength had higher semen volume, sperm concentration, progressive motility, total motility, and total count, with measurements of 14.2% (95% confidence interval [CI]: 5.9%-23.2%), 19.5% (95% CI: 7.3%‒33.1%), 9.5% (95% CI: 3.4%‒15.9%), 8.8% (95% CI: 3.2%‒14.6%), and 36.4% (95% CI: 18.9%‒56.5%), respectively. These positive dose-response relationships were further confirmed in restricted cubic splines, where handgrip strength was modeled as a continuous variable. Handgrip strength, as an indicator of muscular function and strength, was positively associated with semen characteristics in a dose-dependent manner.
Male ; Humans ; Semen ; Sperm Motility ; Hand Strength ; Sperm Count ; Semen Analysis ; Spermatozoa ; China/epidemiology*

OBJECTIVE: To explore the molecular biology of D variant blood group with RHD*95A genotype and the genetic mechanism of its generation. METHODS: A total of 6 samples from 3 generations of a family were analyzed. RHD blood group was identified by saline test tube and microcolumn gel card method. 10 exons of RHD gene were amplified by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) and analyzed by direct sequencing. Homology modeling was used to compare the structural differences between mutant RHD protein and wild-type RHD protein. RESULTS: The proband was identified as D variant by serological identification, RHD gene sequencing directly detected a c. 95 c > A mutation in exon 1 that leads to encoding the 32-bit amino acids by threonine Thr (T) into aspartic acid Asn (N), the rest of the exon sequences were normal compared with the normal RHD*01 gene. In the family, the proband's father, grandmather and uncle were all carried the same RHD*95A allele. Protein modeling results suggested that the hydrogen chain connected to the 32nd amino acid residue was changed after p.T32N mutation, which affected the structural stability of RHD protein. CONCLUSION The first genetic lineage of the RHD*95A gene was identified in a Chinese population. The c.95C>A mutation in RHD gene was found in the family, which resulted in reduced expression of RHD antigen and showed D variant, the mutation could be stably inheritable. Gene identification and protein structure analysis of D variant population is helpful to explore the molecular mechanism of its formation and ensure the safety of blood transfusion.
Humans ; Blood Group Antigens

This manuscript aims to investigate the effects of resibufogenin on the proliferation, migration and invasion of human hepatocellular carcinoma cells and its related mechanisms. MTT assay was used to determine the inhibitory effect of resibufogenin on the growth of four hepatocellular carcinoma cells in vitro. Wound-healing assay and Transwell assay were used to evaluate the migration and invasion ability of resibufogenin on MHCC-97H cells. Western blot assay was used to detect the expression of migration and invasion related proteins in MHCC-97H cells treated with different concentrations of resibufogenin. The results showed that resibufogenin significantly inhibited the proliferation of hepatocellular carcinoma cells in vitro. The half maximal inhibitory concentration (IC₅₀) values on MHCC-97H, HepG2, SK-Hep-1 and Huh-7 cells were 0.55 ± 0.06, 2.83 ± 0.24, 5.25 ± 0.49, 14.89 ± 2.28 μmol·L^-1, respectively. Resibufogenin also suppressed the migration and invasion of MHCC-97H cells in a concentration-dependent manner. The protein expression of integrin α2, integrin α6, integrin β1, N-cadherin, matrix metalloproteinase 2 (MMP2) and transcription factor Twist in MHCC-97H cells were decreased significantly with the increase of the concentration of resibufogenin, while the protein expression of E-cadherin increased. In addition, we found that p-PI3K/PI3K and p-AKT/AKT ratios were significantly reduced after treatment with resibufogenin. In conclusion, resibufogenin can inhibit the proliferation, migration and invasion of hepatocellular carcinoma MHCC-97H cells in vitro, which is related to the regulation of intracellular migration and invasion protein expression and PI3K/AKT signaling pathway.

Objective:To evaluate the safety and effectiveness of stent-assisted coil embolization in treatment of acute intracranial rupture wide-necked aneurysms.Methods:A total of 249 patients with acute intracranial wide-necked aneurysms (301 aneurysms) treated with stent-assisted coil embolization in our hospital from January 2014 to December 2018 were chosen in our study; these patients were divided into ruptured group ( n=114) and non-ruptured group ( n=135). The differences in the prognoses (modified Rankin scale [mRS] scores) and perioperative complications, mortality, and aneurysm recurrence rate were retrospectively analyzed. Univariate analysis and multivariate Logistic regression analysis were used to screen the risk factors for perioperative complications and recurrence of aneurysms in patients from the ruptured group. Results:The incidence of perioperative complications in the ruptured group (10.5%) was slightly higher than that in the un-ruptured group (6.6%), and the recurrence rate of aneurysms in the un-ruptured group (12.8%) was slightly higher than that in the ruptured group (9.6%), but the differences were not statistically significant ( P>0.05); except for 2 patients in the ruptured group who died of postoperative recurrent hemorrhage, the prognosis of the rest patients in the 2 groups was good (mRS scores≤3). Univariate analysis showed that there was significant difference in the age distribution between the complication group and the non-complication group in the ruptured patients ( P<0.05), and the sizes and degrees of embolization between the ruptured group and un-ruptured group showed significant differences ( P<0.05). Multivariate Logistic regression analysis showed that ages at 40-60 years was independent risk factor for perioperative complication ( OR=17.819, 95%CI: 2.356-137.719, P=0.005), and aneurysm diameter>10 mm and aneurysm Raymond grading 3 embolization were independent risk factors for aneurysm recurrence ( OR=36.823, 95%CI: 0.862-768.308, P=0.000; OR=52.813, 95%CI: 2.967-938.152, P=0.007). Conclusion:Stent-assisted coil embolization in the treatment of acute intracranial rupture wide-necked aneurysms is safe and effective, but patients aged 40-60 years should be wary of high incidence of perioperative complications, and patients with large aneurysms and incomplete embolization should be wary of their high risk of aneurysm recurrence.

Adult ; Capillary Permeability ; Extracorporeal Membrane Oxygenation ; Extravascular Lung Water ; Humans ; Lung/diagnostic imaging* ; Prognosis ; Respiratory Distress Syndrome/therapy*

To investigate the effects of thanatos-associated protein 11 (THAP11) on the proliferation and apoptosis of esophageal cancer cell and the underlying mechanism.
 Methods: Expression of THAP11 in human esophageal epithelial cells (Het-1A) and esophageal cancer cells (Eca109, TE-1, Ec 9706) were detected by Western blotting. Esophageal cancer TE-1 cells were divided into 3 groups: a normal control (NC) group, a negative control (LV-NC) group and a THAP11 (LV-THAP11) group. Then the cell proliferation were detected by MTT assay, cell apoptosis were detected by flow cytometry, caspase-3 and caspase-9 levels were detected by caspases kits. Ubiquitination of p53 was determined in esophageal cancer TE-1 cells.
 Results: Expression of THAP11 was reduced in esophageal cancer cells compared with human esophageal epithelial cells (P<0.05). After transfection with LV-THAP11 in TE-1 cells, cell viability was reduced (P<0.05), while apoptosis rate and caspase-3 and caspase-9 levels were increased (P<0.05), indicating that THAP11 inhibited growth of esophageal cancer cells. In addition, the THAP11 increased the levels of p53 (P<0.05) and inhibited the ubiquitination of p53 regulated by MDM2. 
 Conclusion: THAP11 may inhibit the proliferation of esophageal cancer cells by inhibiting ubiquitination of p53.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; Humans ; Repressor Proteins ; metabolism ; Tumor Suppressor Protein p53 ; Ubiquitination