ObjectiveTo construct a stable monoclonal human embryonic kidney 293 （HEK293） cell line expressing recombinant human coagulation factor Ⅶ （rhFⅦ） and evaluate the expression level and procoagulant bioactivity of rhFⅦ. MethodsThe plasmid pCDNA3.1-EGFP-FⅦ was transfected into HEK293 cells to verify the effectiveness of the transfection system. The plasmid pCDNA3.1-FⅦ was transfected into HEK293 cells， and monoclonal stable transfected cell lines were selected using geneticin （G418）. The transcription of the FⅦ gene was identified by reverse transcription polymerase chain reaction （RT-PCR）. The expression level of rhFⅦ in the supernatant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay （ELISA）， and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay. ResultsHEK293 cells transfected with pcDNA3.1-EGFP-FⅦ showed green fluorescence， indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3.1-FⅦ. The monoclonal stable transfected cell line was obtained by G418 screening. RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line. The cell viability was good as detected by Cell Counting Kit-8， and a single band of rhFⅦ was obtained by purification of the cell supernatant. The highest rhFⅦ expression was （1.27±0.09） mg/L， and the highest procoagulant activity was （380.29±13.80）%. ConclusionThe monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent procoagulant bioactivity is successfully screened.

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 perform transient transfection of recombinant human Factor X(rhFX) into HEK293 cells,to optimize transfection parameters,to develop a high-yield in vitro expression system for rhFX production,and to assess the biological activity of expressed rhFX. Methods: The eukaryotic expression vector pcDNA3. 1-EGFP-FX was constructed by inserting the F10 gene into pcDNA3. 1-EGFP and subsequently transfected into HEK293cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3. 1-FX was generated through ligation of the F10 gene fragment with pcDNA3. 1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Transfection conditions were systematically optimized,and rhFX concentration was quantified by enzyme-linked immunosorbent assay(ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time(PT) assay and chromogenic substrate method. Results: rhFX was successfully expressed in the supernatant of HEK293 cells. rhFX was successfully expressed in the supernatant of HEK293 cells. The highest expression level of rhFX was achieved on the third day after transfection when the cell density was 80% and the ratio of plasmid DNA to polyethyleneimine(PEI) transfection reagent was 1 ∶ 2.Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5. 20 ng/mL in the supernatant.The prothrombin time(PT) of rhFX-containing supernatant was significantly shorter(P 50) of etoxaban was determined to be 1. 449 nmol/L using the chromogenic substrate method based on rhFX,which was in the same order of magnitude as the reported 0. 78 nmol/L in the literature. Conclusion HEK293cells successfully express biologically active recombinant human Factor X(rhFX) protein,laying a foundation for advancing the development of rhFX-based therapeutics.

BACKGROUND:Anterior subtotal corpectomy,decompression and fusion is a conventional method to treat cervical degenerative diseases.A titanium cage is an important implant to maintain the stability of the cervical spine after subtotal corpectomy.In recent years,many patients have complications such as titanium cage sinking,which are highly controversial. OBJECTIVE:To investigate the internal biomechanical relationship between the tilt angle of the titanium cage and postoperative titanium cage subsidence after anterior subtotal cervical corpectomy,decompression and fusion. METHODS:A three-dimensional finite element model of the C4-C6 segment was established by CT images of a normal human cervical spine,in which the anterior subtotal resection,decompression and fusion of the C5 vertebral body were simulated,and titanium cages with different tilt angles(-6° to-1° negative angle,that is,the front edge of titanium cage is shorter than the rear edge of titanium cage;1° to 6° positive angle,that is,the front edge of titanium cage is longer than the rear edge of titanium cage)were placed.After setting the boundary conditions,preloads of 50,100 and 150 N were applied respectively on the C4 vertebral body.The stress value of each contact point between the titanium cage and C4 lower-end plate and C6 lower-end plate(seven stress contact points on the contact surface of titanium mesh)was recorded and statistical analysis was conducted. RESULTS AND CONCLUSION:(1)The tilt angles of the titanium cage of the positive angle group and negative angle group under 50,100 and 150 N stress respectively were found by Mann Whitn test,with P<0.05,which was statistically significant.The dispersion coefficients of the positive angle group were smaller than those of the negative angle group under 50,100 and 150 N stress conditions.(2)Under 50,100 and 150 N stress conditions,the Wilcoxon sign rank test in the positive angle group of titanium cage tilt angle found that when the angle was set to 1° to 5°,the difference was not statistically significant(P>0.05).However,when the tilt angle of the titanium cage was set to 6°,the difference was statistically significant(P<0.05).(3)Under 50,100 and 150 N stress conditions,the Wilcoxon sign rank test in the negative angle group of titanium cage tilt angle found that when the tilt angle was set to-1° to-6°,the difference was not statistically significant(P>0.05).(4)It is concluded that in the sagittal position,the titanium cage with a positive tilt angle is more stable than with a negative tilt angle,which is more suitable for clinical use.The tilt angle of the titanium cage is relatively stable in the range of 1° to 5°.When the tilt angle is 6°,the stability starts to decline,which is easy to cause complications of titanium cage sinking after surgery.It is more suitable to select the titanium cage with a tilt angle of 1° to 5° according to the clinical situation during surgery to improve the efficacy.

BACKGROUND:Previous studies have found that qi deficiency and blood stasis syndrome is the main syndrome among various TCM syndromes of cervical spondylotic myelopathy.However,there is no report on proteomic markers as early diagnosis indicators for the transformation of developmental cervical spinal stenosis with qi deficiency and blood stasis syndrome to cervical spondylotic myelopathy. OBJECTIVE:To explore serum proteomics difference between developmental cervical spinal stenosis and cervical spondylotic myelopathy and to find and identify the potential serum biomarkers between them. METHODS:Serum samples of nine patients with cervical spondylotic myelopathy of qi deficiency and blood stasis syndrome(experimental group)and nine patients with developmental cervical spinal stenosis of qi deficiency and blood stasis syndrome(control group)were collected.The proteomic analysis was carried out by Tandem Mass Tag combined with liquid chromatography tandem mass spectrometry,so as to find and identify differentially expressed proteins. RESULTS AND CONCLUSION:A total of 1027 significantly differential proteins were initially screened by TMT technology and 89 significantly differential proteins were finally identified(P<0.05).Compared with the control group,there were 45 up-regulated proteins in the experimental group,such as α-actinin-4,α-actinin-1,cell division control protein 42 homolog,integrin-linked protein kinase and B-actin.Conversely,there were 44 down-regulated proteins in the experimental group compared with the control group,such as fibronectin,fibrinogen γ chain,fibrinogen α chain,fibrinogen β chain.Gene ontology enrichment analysis indicated that these differential proteins were involved in signal receptor binding,kinase binding,protein kinase activity,integrin binding,actin filament binding and other molecular functions.Based on the Kyoto Encyclopedia of Genes and Genomes pathway analysis,20 common differential signal/metabolic pathways were identified,including Rap1 signaling pathway,adherens junction,tight junction,platelet activation,and regulation of actin cytoskeleton.Protein-protein interaction analysis showed that ILK,FGA,FGB,FGG,FN1,Cdc42,ACTN1,ACTN4 and ACTB were located at the nodes of protein-protein interaction network and were closely related to bone formation and destruction system,nervous system,coagulation system,cellular inflammation and other systems.To conclude,the serum differentially expressed proteins between developmental cervical spinal stenosis and cervical spondylotic myelopathy can be successfully screened by Tandem Mass Tag combined with liquid chromatography tandem mass spectrometry.ILK,FN1,CDC42 and ACTN 4 are identified as specific markers for the transformation of developmental cervical spinal stenosis with qi deficiency and blood stasis syndrome into cervical spondylotic myelopathy.These findings provide a basis for further clarifying the transformation mechanism.

Objective:To investigate the detection status and genotypes distribution characteristics of norovirus(NoV)in environmental sewage from three monitoring points in Fujian province, and to explore the significance of its application to NoV monitoring.Methods:Sewage samples were collected monthly at 5 sampling sites in representative monitoring cities, enriched and concentrated. Partial gene fragments of norovirus VP1 were amplified by reverse transcription-semi nested polymerase chain reaction (RT-snPCR), TA cloned and sequenced. Genotypes were identified based on the sequencing.Results:A total of 56 sewage samples were collected from July 2022 to June 2023. The detection rates of GⅠ and GⅡ were 89.29% (50/56) and 94.64% (53/56), respectively. A total of 7 NoV GⅠ genotypes and 13 GⅡgenotypes were identified. GⅠ.1, GⅠ.4, GⅡ.4 and GⅡ.17 were the dominant genotypes. NoV genotypes detected in different sampling sites were not exactly the same. The detection rate of NoV was low from August to November 2022, and the prevalence of the dominant genotypes was different in different seasons. GⅠ.1 and GⅡ.4 were highly prevalent from August to November 2022, but were replaced by GⅠ.4 and GⅡ.17 from December 2022 to June 2023, respectively. More NoV genotypes were detected in January-June 2023, comparing to the July-December 2022. The dominant genotype GII.17, has multiple clades and new variants have been discovered that are different from the 2014/2015 circulating strains.Conclusions:The detection rates of NoV in environmental sewage were very high, and genotypes were diverse. Environmental sewage surveillance could be an important complementary method for NoV cases surveillance.

The repair and reconstruction of long ureteral defects is a technical challenge faced by urologists. The ileal ureter replacement is currently the main surgical method for treating long segment ureteral defects. After the ileal ureter replacement surgery, mucin 2 (MUC2) and mucin 3 (MUC3), which are the main functional components of ileal mucus, continue to be strongly expressed. In addition, the expression of MUC2 gene after surgery is not limited to goblet cells, but can even be expressed in absorption cells of the ileum, ultimately leading to a large amount of mucin synthesis and mucus secretion. Mucus can cause many complications such as obstruction and infection, seriously affecting the treatment effect. At present, the industry has tried various methods to reduce the secretion of mucus and related complications after ileal ureteral replacement surgery, such as cutting the ileum to reduce mucosal area and mucosal exfoliation to reduce mucus production, and has achieved certain results. This article provides a review of research progress on the types, molecular mechanisms, and solutions of mucus related complications after ileal ureteral replacement surgery.

Objective:To explore the clinicopathological and genetic characteristics of sporadic medullary thyroid carcinoma (MTC) and new therapeutic targets for sporadic MTC.Methods:Based on family and personal disease history, we identified 32 sporadic MTC who underwent surgical resection from Jan 2010 to Dec 2022. Clinicopathological and immunohistochemical features were analyzed in all patients, while 6 of them were subject to the whole exome sequencing (WES).Results:Compared with those of low-grade sporadic MTC, patients with high-grade tumors were more likely to have lymph node metastasis at presentation ( χ2=4.428, P=0.040); less likely to be cured by biochemical treatment ( χ2=4.072, P=0.044). Pathological grading scheme, biochemical cure, and TNM stage were independent risk factors of disease free survival. WES was performed on 6 pairs of normal tissues. We screened RET and RAS as driver mutations, and the mutation ratio was 3/6 respectively. Patients with RET or RAS mutations had no recurrence. In addition, we detected PDGFRA somatic mutation, with a mutation ratio of 1/6. Conclusions:For sporadic MTC cases, the pathological grading system has important prognostic value, and RET and RAS somatic mutations are the main driver mutations. PDGFRA are potential therapeutic targets for sporadic MTC.

Periodontitis is a critical risk factor for the occurrence and development of diabetes.Porphyromonas gingivalis may participate in insulin resistance(IR)caused by periodontal inflammation,but the functional role and specific mechanisms of P.gingivalis in IR remain unclear.In the present study,clinical samples were analysed to determine the statistical correlation between P.gingivalis and IR occurrence.Through culturing of hepatocytes,myocytes,and adipocytes,and feeding mice P.gingivalis orally,the functional correlation between P.gingivalis and IR occurrence was further studied both in vitro and in vivo.Clinical data suggested that the amount of P.gingivalis isolated was correlated with the Homeostatic Model Assessment for IR score.In vitro studies suggested that coculture with P.gingivalis decreased glucose uptake and insulin receptor(INSR)protein expression in hepatocytes,myocytes,and adipocytes.Mice fed P.gingivalis tended to undergo IR.P.gingivalis was detectable in the liver,skeletal muscle,and adipose tissue of experimental mice.The distribution sites of gingipain coincided with the downregulation of INSR.Gingipain proteolysed the functional insulin-binding region of INSR.Coculture with P.gingivalis significantly decreased the INSR-insulin binding ability.Knocking out gingipain from P.gingivalis alleviated the negative effects of P.gingivalis on IR in vivo.Taken together,these findings indicate that distantly migrated P.gingivalis may directly proteolytically degrade INSR through gingipain,thereby leading to IR.The results provide a new strategy for preventing diabetes by targeting periodontal pathogens and provide new ideas for exploring novel mechanisms by which periodontal inflammation affects the systemic metabolic state.

Objective:To investigate whether murine peroxidase reductase-5(mPRDX5)has anti-tumor activity in mice,so as to further confirm the anti-tumor activity and mechanism of recombinant peroxidase reductase-5.Methods:High purity mPRDX5 was obtained by heterologous expression and purification in vitro.Pancreatic cancer Pan02 cells were inoculated subcutaneously on the left axillary back of mice to establish a tumor bearing mouse model.Mice were randomly divided into PBS(solvent control)group,GEM(gemcitabine)50.0 mg/kg group and mPRDX5 10.0 mg/kg group,with 10 mice in each group,and the tumor related indexes were detected in mice.Results:Compared with PBS group,weight of tumor-bearing mice in GEM group was decreased obviously,while weight of mPRDX5 group was increased to a certain extent.Tumor growth was good in PBS group,according to tumor volume,com-pared with PBS group,tumor growth inhibition rates in D7 were 87.07%in GEM group and 52.82%in mPRDX5 10.0 mg/kg group,re-spectively;according to tumor weight,compared with PBS group,GEM group and mPRDX5 10.0 mg/kg group had tumor growth inhibi-tion rates of 95.39%and 48.33%in D7,respectively.Polarization state of macrophages in tumor tissues of mice in PBS group and mPRDX5 group was analyzed,and it was found that compared with PBS group,M1 macrophages expressing CD86 in tumor tissues of mice in mPRDX5 group were significantly increased,while M2 macrophages expressing CD206 were significantly decreased.Conclu-sion:mPRDX5 has significant anti-pancreatic cancer activity in mice,and the activity is exerted by promoting M1-type polarization of macrophages in the tumor microenvironment.