Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

ObjectiveTo explore the effect of modified Lianpoyin formula （LPYJWF） in the treatment of Helicobacter pylori （Hp）-associated gastric mucosal damage based on mitochondrial autophagy and NLRP3 inflammasome signaling pathway. MethodsA total of 60 eight-week-old Balb/c male mice were assigned via the random number table method into control， model， high-dose LPYJWF （LPYJWF-H， 27.3 g·kg^-1·d^-1）， medium-dose LPYJWF （LPYJWF-M， 13.65 g·kg^-1·d^-1）， low-dose LPYJWF （LPYJWF-L， 6.83 g·kg^-1·d^-1）， and quadruple therapy groups. Except the control group， other groups were modeled for Hp infection. Mice were administrated with LPYJWF at corresponding doses by gavage. Quadruple therapy group was given omeprazole （6.06 mg·kg^-1·d^-1） + amoxicillin （303 mg·kg^-1·d^-1） + clarithromycin （151.67 mg·kg^-1·d^-1） + colloidal pectin capsules （30.3 mg·kg^-1·d^-1） by gavage. The control group was given an equal volume of 0.9% NaCl for 14 days. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of gastric mucosa， and Warthin-Starry （W-S） silver staining was used to detect Hp colonization. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of the gastric tissue， and immunofluorescence co-localization assay was adopted to detect the expression of mitochondrial transcription factor A （TFAM） and translocase of the outer mitochondrial membrane member 20 （TOMM20）. The water-soluble tetrazolium salt method and thiobarbituric acid method were used to determine the levels of superoxide dismutase （SOD） and malondialdehyde （MDA）， respectively， in the gastric tissue. Western blot was employed to measure the protein levels of PTEN-induced kinase 1 （PINK1）， Parkin， p62， microtubule-associated protein 1 light chain 3 （LC3）， NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18）. Real-time quantitative PCR was employed to assess the mRNA levels of PINK1， Parkin， p62， and LC3. ResultsCompared with the control group， the model group presented obvious gastric mucosal damage， colonization of a large number of Hp， severe mitochondrial damage， vacuolated structures due to excessive autophagy， reduced TOMM20 and TFAM co-expression in the gastric mucosal tissue， and reduced SOD and increased MDA （P<0.01）. In addition， the gastric tissue in the model group showed up-regulated protein and mRNA levels of PINK1， Parkin， and LC3 and down-regulated protein and mRNA levels of p62 （P<0.01， as well as increased expression of inflammasome-associated proteins NLRP3， ASC， IL-1β， and IL-18 （P<0.01）. Compared with the model group， the LPYJWF and quadruple therapy groups showed alleviated pathological damage of gastric mucosa， reduced Hp colonization， mitigated mitochondrial damage， and increased co-expression of TOMM20 and TFAM. The SOD level was elevated in the LPYJWF-L group （P<0.01）， and the MDA levels became lowered in the LPYJWF and quadruple therapy groups （P<0.05， P<0.01）. Furthermore， the LPYJWF and quadruple therapy groups showed down-regulated mRNA levels of PINK1， Parkin， and LC3 and protein levels of PINK1 and Parkin， and up-regulated mRNA level of p62 （P<0.01）. The LPYJWF-M， LPYJWF-H， and quadruple therapy groups showcased down-regulated LC3 Ⅱ/LC3 Ⅰ level （P<0.05， P<0.01） and up-regulated protein level of p62 （P<0.01）. The expression of inflammasome-associated proteins NLRP3， ASC， IL-1β， and IL-18 were reduced in the LPYJWF and quadruple therapy groups （P<0.05， P<0.01）. ConclusionLPYJWF ameliorates gastric mucosal damage and exerts mucosa-protective effects in Hp-infected mice， which may be related to the inhibition of excessive mitochondrial autophagy， thereby inhibiting the activation of the NLRP3 inflammasome pathway.

ObjectiveTo explore the effect of modified Lianpoyin formula （LPYJWF） in the treatment of Helicobacter pylori （Hp）-associated gastric mucosal damage based on mitochondrial autophagy and NLRP3 inflammasome signaling pathway. MethodsA total of 60 eight-week-old Balb/c male mice were assigned via the random number table method into control， model， high-dose LPYJWF （LPYJWF-H， 27.3 g·kg^-1·d^-1）， medium-dose LPYJWF （LPYJWF-M， 13.65 g·kg^-1·d^-1）， low-dose LPYJWF （LPYJWF-L， 6.83 g·kg^-1·d^-1）， and quadruple therapy groups. Except the control group， other groups were modeled for Hp infection. Mice were administrated with LPYJWF at corresponding doses by gavage. Quadruple therapy group was given omeprazole （6.06 mg·kg^-1·d^-1） + amoxicillin （303 mg·kg^-1·d^-1） + clarithromycin （151.67 mg·kg^-1·d^-1） + colloidal pectin capsules （30.3 mg·kg^-1·d^-1） by gavage. The control group was given an equal volume of 0.9% NaCl for 14 days. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of gastric mucosa， and Warthin-Starry （W-S） silver staining was used to detect Hp colonization. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of the gastric tissue， and immunofluorescence co-localization assay was adopted to detect the expression of mitochondrial transcription factor A （TFAM） and translocase of the outer mitochondrial membrane member 20 （TOMM20）. The water-soluble tetrazolium salt method and thiobarbituric acid method were used to determine the levels of superoxide dismutase （SOD） and malondialdehyde （MDA）， respectively， in the gastric tissue. Western blot was employed to measure the protein levels of PTEN-induced kinase 1 （PINK1）， Parkin， p62， microtubule-associated protein 1 light chain 3 （LC3）， NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18）. Real-time quantitative PCR was employed to assess the mRNA levels of PINK1， Parkin， p62， and LC3. ResultsCompared with the control group， the model group presented obvious gastric mucosal damage， colonization of a large number of Hp， severe mitochondrial damage， vacuolated structures due to excessive autophagy， reduced TOMM20 and TFAM co-expression in the gastric mucosal tissue， and reduced SOD and increased MDA （P<0.01）. In addition， the gastric tissue in the model group showed up-regulated protein and mRNA levels of PINK1， Parkin， and LC3 and down-regulated protein and mRNA levels of p62 （P<0.01， as well as increased expression of inflammasome-associated proteins NLRP3， ASC， IL-1β， and IL-18 （P<0.01）. Compared with the model group， the LPYJWF and quadruple therapy groups showed alleviated pathological damage of gastric mucosa， reduced Hp colonization， mitigated mitochondrial damage， and increased co-expression of TOMM20 and TFAM. The SOD level was elevated in the LPYJWF-L group （P<0.01）， and the MDA levels became lowered in the LPYJWF and quadruple therapy groups （P<0.05， P<0.01）. Furthermore， the LPYJWF and quadruple therapy groups showed down-regulated mRNA levels of PINK1， Parkin， and LC3 and protein levels of PINK1 and Parkin， and up-regulated mRNA level of p62 （P<0.01）. The LPYJWF-M， LPYJWF-H， and quadruple therapy groups showcased down-regulated LC3 Ⅱ/LC3 Ⅰ level （P<0.05， P<0.01） and up-regulated protein level of p62 （P<0.01）. The expression of inflammasome-associated proteins NLRP3， ASC， IL-1β， and IL-18 were reduced in the LPYJWF and quadruple therapy groups （P<0.05， P<0.01）. ConclusionLPYJWF ameliorates gastric mucosal damage and exerts mucosa-protective effects in Hp-infected mice， which may be related to the inhibition of excessive mitochondrial autophagy， thereby inhibiting the activation of the NLRP3 inflammasome pathway.

Objective:To preliminarily examine the feasibility and outcome of single-port laparoscopic radical prostatectomy with full-length urethral preservation (FLUP-SPRP).Method:This study was a prospective case series study. A total of 25 patients with prostate cancer who met the enrollment criteria and agreed to this surgical procedure from March 2022 to December 2022 were collected at the Department of Urology, the Second Affiliated Hospital of Nanjing Medical University. The age of the patients was (67.2±7.6) years (range: 61 to 76 years). This novel procedure was performed by an experienced surgeon who performed single hole radical prostatectomy skillfully. Patient urinary control, tumor control, and related surgical complications after surgery were regularly monitored. Postoperative urinary control was evaluated using the daily amount of urine pad, 0 to 1 piece of urine pad was to restore urinary control, and 0 to 1 piece of pad within 24 hours after catheter removal was immediate urinary control.Result:All prodecures were successfully completed without transit to open surgery. The surgical time was (128.4±22.4) minutes (range: 100 to 145 minutes), the intraoperative blood loss was (68.2±13.7) ml (range: 50 to 120 ml). The urethral injury occurred in 4 cases during surgery and was repaired by sutures. The urinary control recovery rates within 24 hours, 1 week, 4 weeks, and 7 weeks after surgery were 80.0%, 84.0%, 92.0% and 100%, respectively. Postoperative large section pathology revealed 1 case with a positive basal margin of the prostate and negative margins of all prostate glands around the urethra. Postoperative complications included urinary tract infection in 3 cases, urodynia in 2 cases, and acute urinary retention in 1 case. MRI follow-up 3 months after surgery showed normal anatomy of the bladder and urethra. The follow-up values of prostate specific antigen at 3 and 6 months after surgery were less than 0.1 μg/L.Conclusions:The preliminary results of this study indicate that the FLUP-SPRP procedure is safe and feasible. The early results of postoperative urinary control and oncology are as expected.

Objective:To preliminarily examine the feasibility and outcome of single-port laparoscopic radical prostatectomy with full-length urethral preservation (FLUP-SPRP).Method:This study was a prospective case series study. A total of 25 patients with prostate cancer who met the enrollment criteria and agreed to this surgical procedure from March 2022 to December 2022 were collected at the Department of Urology, the Second Affiliated Hospital of Nanjing Medical University. The age of the patients was (67.2±7.6) years (range: 61 to 76 years). This novel procedure was performed by an experienced surgeon who performed single hole radical prostatectomy skillfully. Patient urinary control, tumor control, and related surgical complications after surgery were regularly monitored. Postoperative urinary control was evaluated using the daily amount of urine pad, 0 to 1 piece of urine pad was to restore urinary control, and 0 to 1 piece of pad within 24 hours after catheter removal was immediate urinary control.Result:All prodecures were successfully completed without transit to open surgery. The surgical time was (128.4±22.4) minutes (range: 100 to 145 minutes), the intraoperative blood loss was (68.2±13.7) ml (range: 50 to 120 ml). The urethral injury occurred in 4 cases during surgery and was repaired by sutures. The urinary control recovery rates within 24 hours, 1 week, 4 weeks, and 7 weeks after surgery were 80.0%, 84.0%, 92.0% and 100%, respectively. Postoperative large section pathology revealed 1 case with a positive basal margin of the prostate and negative margins of all prostate glands around the urethra. Postoperative complications included urinary tract infection in 3 cases, urodynia in 2 cases, and acute urinary retention in 1 case. MRI follow-up 3 months after surgery showed normal anatomy of the bladder and urethra. The follow-up values of prostate specific antigen at 3 and 6 months after surgery were less than 0.1 μg/L.Conclusions:The preliminary results of this study indicate that the FLUP-SPRP procedure is safe and feasible. The early results of postoperative urinary control and oncology are as expected.

Objective To measure the anatomical parameters of the first maxillary molars in children of different age groups and evaluate the age-related changes in dental crowns.Methods A retrospective analysis was conduc-ted on cone beam computed tomography(CBCT)images of 4-8-year-old children.NNT software was used to ana-lyze multiple important indicators of maxillary first molar.Results A total of 308 first maxillary molars,including 154 pediatric patients,were evaluated in this study.The thickness of the pulp apex H1(left,P=0.01;right,P=0.02)and the thickness of the pulp chamber floor H3(left and right P＜0.01)were positively correlated with age,while the height of the pulp cavity H2(left and right P＜0.01)and the height of the palate tip D1(left P=0.003,right P=0.002)showed a negative correlation with age.There was no significant correlation between the height of the buccal tip and age(P＞0.05).There were significant differences in H1 and H3 between the 4-year-old and 5-year-old age groups between the 8-year-old age group(P＜0.05),as well as significant differences in H2 and D1 between the 4-year-old and 5-year-old between the 6-year-old,7-year-old and 8-year-old age groups(P＜0.05).Conclusion The age-related changes in the crowns of the first maxillary molars are important references for the clinical treatment,and can be accurately measured through CBCT data.

Objective To evaluate and compare the treatment efficacy between concentrated growth factor(CGF)and blood clots(BC)as scaffolds in regenerative endodontic procedures(REPs).Methods Twenty young permanent teeth from 18 healthy children with pulp necrosis or periapical periodontitis were randomly divided into CGF group and BC group.In the CGF group(n=10),after ap-ical bleeding,CGF was inserted into the root canal as a stent.In the BC group(n=10),by stimulating apical bleeding,blood entered the root canal and produced blood clots as scaffolds.Clinical examination and apical X-ray shooting were conducted for each follow-up visit.Cone beam computed tomographic(CBCT)images were acquired preoperatively and at the 24-month recall.The increase of root length,root wall thickness,and newly-formed calcified tissue were calculated.Results The root length increased by(1.68±0.90)mm in the CGF group and(2.36±1.34)mm in the BC group.Root wall thickness increased by(0.44±0.34)mm in the CGF group and(0.50±0.31)mm in the BC group.There was no statistically significant difference in root lengthening and root wall thickening between two groups(P＞0.05).The amount of newly formed calcified tissue in the CGF group((22.13±19.12)mm3)was significantly less than that in the BC group((42.97±22.69)mm3)(P＜0.05).According to the goals for success outlined by American Association of Endodontists(AAE),90%(9/10)of the CGF cases and100%(10/10)of the BC cases achieved the primary and secondary goals(P＞0.05).40%of the CGF cases(4/10)and 60%of the BC cases(6/10)achieved the tertiary goal(P＞0.05).Conclusion CGF is found to be use-ful as a scaffold for REPs,but the success rate is slightly lower than that of BC group and the difference is not statistically significant.

China prospective multicenter birth cohort (Prospective Omics Health Atlas birth cohort, POHA birth cohort) study was officially launched in 2022. This study, in collaboration with 12 participating units, aims to establish a high-quality, multidimensional cohort comprising 20 000 naturally conceived families and assisted reproductive families. The study involves long-term follow-up of parents and offspring, with corresponding biological samples collected at key time points. Through multi-omics testing and analysis, the study aims to conduct multi-omics big data research across the entire maternal and infant life cycle. The goal is to identify new biomarkers for maternal and infant diseases and provide scientific evidence for risk prediction related to maternal diseases and neonatal health.

Objective To investigate the effects of syncytial formation induced by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(SARS-2-S)on the proliferation and migration of human lung adenocarcinoma A549 cells and mouse melanoma B16 cells in vitro.Methods Plasmids expressing human angiotensin-converting enzyme 2(hACE2)and SARS-2-S were constructed and respectively co-transfected with lentiviral packaging plasmids into HEK-293FT cells before the lentiviral supernatant was collected and infected with A549 cells which were screened by puromycin to obtain the A549 cells respectively that were stably transfected with hACE2(A549-A)and SARS-2-S(A549-S).The protein expression of A549-A and A549-S cells was verified by Western blotting.A549-A and A549-S cells were co-cultured before their syncytia were observed by fluorescence microscopy.Conditioned media(syncytial supernatant and non-syncytial supernatant)was collected to culture A549 cells,ovalbumin(OVA)-gene-modified B16 cells(B16-OVA),and B16-F10 cells in vitro.The CCK-8 assay and colony formation assay were used to assess the proliferation capacity of tumor cells,while the wound healing assay was employed to evaluate the migration capacity of tumor cells.Results Stable A549 cell lines expressing hACE2 and SARS-2-S were constructed.The SARS-2-S-induced syncytial formation system was established after co-culture of A549-A and A549-S cells.Both syncytial and non-syncytial supernatants significantly promoted the proliferation and migration of A549,B16-OVA,and B16-F10 cells in vitro,especially the syncytial supernatant.Conclusion SARS-2-S-induced syncytial formation promotes the proliferation and migration properties of A549 and B16 cells in vitro.

【Objective】 To observe the penetration and biological activity of PTD4-Cu, Zn-SOD into human astrocytes and whether it can mitigate hypoxia damages. 【Methods】 ①Immunohistochemistry and fluorescence test: We labeled the Cu, Zn-SOD by a monoclonal antibody, combined it with the fluorescent secondary antibody labeled with fluorescein isothiocyanate (FITC) to observe the effect of transduced PTD4-Cu, Zn-SOD on the viability of human astrocytes. ② The experimental group: After hypoxic damage model, the cells were divided into three groups: blank control, group Cu, Zn-SOD, and group PTD4-Cu, Zn-SOD. Group blank was added with DMEM medium (excluding serum) as control; DMEM medium was added to the other two for one hour (excluding serum) with its fusion proteins (Cu, Zn-SOD and PTD4 -Cu, Zn-SOD) with the final concentration of 2 μmoL/L. After the intervention, we used SOD and MDA test kits to observe PTD4-Cu, Zn-SOD and Cu, Zn-SOD in astrocytes after fusion protein intervention. 【Results】 The PTD4-Cu, Zn-SOD fusion protein could have aggregation distribution in the nucleus by FITC fluorescently labeled. After the intervention, it could increase the SOD activity in astrocytes in group PTD4-Cu, Zn-SOD and group Cu, Zn-SOD compared with control group, but the SOD activity was more obvious in the fusion proteins PTD4-Cu, Zn-SOD group. And the dose of MDA was reduced in group PTD4-Cu, Zn-SOD compared with group Cu, Zn-SOD and control group. 【Conclusion】 PTD4-Cu, Zn-SOD fusion protein can transcellular membrane of human astrocytes. The fusion protein PTD4-Cu, Zn-SOD can increase the SOD activity and reduce the content of MDA by human astrocytes from hypoxia injury.