The production of high-quality oocytes requires precisely orchestrated intercellular communication. Extracellular vesicles (EVs) are cell-derived nanoparticles that play a vital role in the transfer of bioactive molecules, which has gained much attention in the field of diagnosis and treatment. Over the past ten years, the participation of EVs in the reproductive processes of oocytes has been broadly studied and has shown great potential for elucidating the intricacies of female reproductive health. This review provides an extensive discussion of the influence of EVs on oocytes, emphasizing their involvement in normal physiology and altered cargo under pathological conditions. In addition, the positive impact of therapeutic EVs on oocyte quality and their role in alleviating ovarian pathological conditions are summarized.
Humans ; Extracellular Vesicles/physiology* ; Oocytes/cytology* ; Female ; Animals ; Cell Communication/physiology*

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*

Neoadjuvant therapy for hepatocellular carcinoma is the frontier and hot topic in the current field of liver cancer research.The fundamental purpose is to reduce the risk of postoperative recurrence through standardized preoperative treatment methods.From the attempts of Transcatheter Arterial Chemoembolization monotherapy for neoadjuvant therapy for hepatocellular carcinoma to systematic treatment represented by"targeted combined with immunotherapy",the latter has become the most promising neoadjuvant strategy due to its high objective response rate and potential to induce pathological complete remission.However,the field still faces challenges such as lack of evidence of overall survival benefit in Phase Ⅲ randomized controlled trials,treatment-related adverse reactions that may lead to delay in surgery,optimal population screening,and timing of surgery.This article aims to briefly discuss the current research status of the application of neoadjuvant therapy in resectable hepatocellular carcinoma,explore relevant diagnosis and treatment concepts,and further understand neoadjuvant therapy.

pAPN is a zinc-dependent metalloprotease,mediating the fusion between virus and host cell,and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV rep-lication,the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamH Ⅰ and Not Ⅰ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method,and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level,Western blot for N protein level,and TCID50.The results showed that the packaged lentivirus could infect Vero cells,and the monoclonal cell line Vero-pAPN(2C5)could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48 h(P＜0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(P＜0.05).In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.

Neoadjuvant therapy for hepatocellular carcinoma is the frontier and hot topic in the current field of liver cancer research.The fundamental purpose is to reduce the risk of postoperative recurrence through standardized preoperative treatment methods.From the attempts of Transcatheter Arterial Chemoembolization monotherapy for neoadjuvant therapy for hepatocellular carcinoma to systematic treatment represented by"targeted combined with immunotherapy",the latter has become the most promising neoadjuvant strategy due to its high objective response rate and potential to induce pathological complete remission.However,the field still faces challenges such as lack of evidence of overall survival benefit in Phase Ⅲ randomized controlled trials,treatment-related adverse reactions that may lead to delay in surgery,optimal population screening,and timing of surgery.This article aims to briefly discuss the current research status of the application of neoadjuvant therapy in resectable hepatocellular carcinoma,explore relevant diagnosis and treatment concepts,and further understand neoadjuvant therapy.

pAPN is a zinc-dependent metalloprotease,mediating the fusion between virus and host cell,and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV rep-lication,the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamH Ⅰ and Not Ⅰ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method,and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level,Western blot for N protein level,and TCID50.The results showed that the packaged lentivirus could infect Vero cells,and the monoclonal cell line Vero-pAPN(2C5)could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48 h(P＜0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(P＜0.05).In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.

Objective:To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS).Methods:HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting. Results:Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2 -ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2 -ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2 -ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2 -ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2 -ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2 -ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2 -ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2 -ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05]. Conclusion:High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.

Objective:To propose a model that could improve image quality of cone-beam computed tomography(CBCT),which based on region-discriminative generative adversarial networks(GAN),in radiotherapy for cervical cancer,so as to meet the requirements of self-adaptive radiotherapy for image quality.Methods:We employed a region-discriminative strategy and a generative adversarial networks idea to construct a model of improving CBCT image quality that could focus on local details of the images of radiotherapy for cervical cancer,which discriminator could improve the quality of generating local details of images.This model of image quality was applied to CBCT images of radiotherapy for cervical cancer.And then,the effects of processing image were evaluated through quantitative indicators and visualization.Results:Both texture clarity and contrast were significantly enhanced after CBCT image quality was improved.The signal to noise ratio of peak value of images was increased by 47.2%,and the indicator of similarity of structure was enhanced to>0.838.Compared with other model,both visualization and indicators can appear better efficiency of model.Compared with Unet network and CycleGAN network,the similarities of structure were respectively increased by 11.88% and 19.54%,and the signal to noise ratios were respectively increased by 19.75% and 25.99%.Conclusion:The GAN bases on region-discrimination can significantly improve the quality of generating integral and detailed CBCT image of radiotherapy for cervical cancer,which can provide new technical pathway for image quality of CBCT with low dose,and can play an important role for improving safety and effectiveness of radiotherapy.It has importantly clinical value for formulating and executing radiotherapy plan.

Objective:To investigate the incidence and prothrombotic risk factors of postoperative venous thromboembolism(VTE) in Cushing′s disease and to further develop an assessment model to identify those at high risk of postoperative VTE events.Methods:A retrospective study was performed in 82 patients who were admitted to Huashan Hospital, Fudan University during January 2019 and January 2020 and diagnosed with Cushing′s disease. These patients underwent the evaluation about their clinical, hormonal, and coagulation parameters, as well as ultrasonography and pulmonary angio-CT when necessary. The least absolute shrinkage and selection operator(LASSO) regression analysis was used to screen independent risk factors, and a nomogram model for postsurgical VTE risk assessment in Cushing′s disease was initially established, and Bootstrap method was used for internal verification. Finally, the predictive model was evaluated for calibration and clinical applicability in the study cohort.Results:Nineteen patients(23.17%) developed VTE events, with 14 cases occurring after endoscopic transsphenoidal surgery. Compared to patients without VTE, those in the VTE group were older( P<0.001), had longer postoperative bed rest, higher rates of current infection, higher HbA 1C levels, and more severe glucose tolerance impairment(all P<0.05). Through LASSO regression analysis, two independent risk factors for postoperative VTE were identified: Age and current infection. Then a VTE risk assessment nomogram model was established to predict the patients at high risk of VTE. In the nomogram model for VTE risk assessment, the area under the receiver operating characteristic curve was 0.868(95% CI 0.787-0.949), with the calibration curve closely aligning with the ideal diagonal line and the clinical decision curve exceeding the two extreme curves. Conclusions:Advanced perioperative assessment needs to be taken to screen those with high VTE risks in patients diagnosed with Cushing′s disease. Additionally, during the perioperative period, patients with Cushing′s disease should undergo mandatory physical activity or prophylactic anticoagulant therapy.

Objective To achieve personalized assessment of cumulated activity distribution in patients undergoing 18F-FDG PET scans using the biokinetic model based on the single-time-point PET images.Methods A biokinetic compartmental model for the organs of interest was established,and the variation of activities in each compartment was described as a set of differential equations.The PET image data at a specific time point of each patient was used to obtain the kinetic model parameters which were then optimized by a simulated annealing algorithm to calculate the cumulated activity of each organ.Additionally,the effects of different voiding patterns on the cumulated activity of the bladder were analyzed.Considering the heterogeneity of radioactive nuclide distribution in organs,a whole-body cumulated activity distribution map was computed,and the accuracy of the proposed method was evaluated by comparison with the existing methods.Results For the 11 cases analyzed in the study,the calculation results of the biokinetic model generally showed a good agreement with experimental data,with an average deviation of less than 15%.Meanwhile,under different urinary excretion conditions,the average cumulated activity in the bladder differed by up to 2.4 times,indicating the positive significance of enforced urination in reducing radiation exposure.The comparison with the biokinetic data obtained from ICRP Publication 128 revealed relatively large deviations for certain organs such as the brain and heart,with standard deviations of 61.0%and 46.3%,respectively.Conclusion The constructed biokinetic model can effectively estimate the individualized cumulated activity distribution in patients undergoing PET scans,providing valuable insights for the internal dosimetry assessment.