Objective; To investigate the immunolocalization and significance of Notch-2 expression in the process of dental pulp repair after injury. Methods: An experimental animal model of injury-induced pulpitis was established to observe the time-sequenced alteration of the expression of Notch-2. Results: Three days post-operation, weak positive staining of Notch-2 was observed in pulp mesenchyme cells and pulp fibroblasts but not in vascular endothelial cells or odontoblasts. Five days post-operation, strong Notch-2 reactivity was found in subodontoblasts as well as newly bom capillary endothelial cells. Seven days after cavity preparation, Notch-2 staining became weaker in pulp mesenchyme cells and capillary endothelial cells, but stronger positive staining was found in odontoblasts. Two weeks post-operation, weak Notch-2 staining was seen in pulp mesenchyme cells and subodontoblastic layer cells and was absent from odontoblasts. Notch-2 immunoreactivity was completely absent in intact rat dental pulp. Conclusion: Notch-2 is strikingly up-regulated in dental pulp mesenchyme cells in the early days after dental injury and then shows progressively up-regulation in odontoblasts. Properly regulated activation of Notch signaling pathway is important for controlling cell fate and maintaining the correct balance among cell proliferation, differentiation and apoptosis during dental pulp repair process.

Objective Digital PCR ( dPCR ) was established to detect plasma epidermal growth factor receptor (EGFR) T790M mutation of non-small cell lung cancer (NSCLC) patients and was evaluated in terms of analytical performance and clinical application significance.Methods The specific primers and probes for EGFR T790M mutation and wildtype were designed to establish dPCR platform.Limit of blank, sensitivity and linearity of dPCR were evaluated by the detection of plasmids with different concentrations to set up optimal reporting system and reanalyzing process.The mutation of EGFR T790M in plasma and tissue samples from 10 patients with advanced NSCLC resistant to EGFR-TKI therapy who were enrolled in Zhongshan Hospital Fudan University from January 2014 to October 2015 were analyzed by dPCR and amplification refractory ( ARMS) , respectively.The consistency was evaluated between dPCR and ARMS by Chi-square test.The correlation of T790M abundance detected by dPCR between plasma and tissue samples was also analyzed by Peasrson correlation analysis.Results Limit of blank and sensitivity of dPCR was 10 copies and 0.01%, respectively.dPCR was evaluated as linear in the range of 0.01%-100%( Y=1.226X-3.984,R2 =0.999 ).The consistency between dPCR and ARMS of tissue samples was good ( kappa=0.80), while the positive rates of plasma T790M detected by dPCR was significantly higher than ARMS (50%vs 20%,P<0.05).It was found that T790M abundance detected by dPCR was highly correlated between lung cancer tissue and plasma ( R =0.923, P <0.05 ) using Pearson correlation analysis. Conclusions A new method of dPCR with high sensitivity and absolute quantification is established for the detection of EGFR T790M mutation in plasma from advanced NSCLC patients, which brings tumor liquid biopsy into real.It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in patients with advanced NSCLC resistant to EGFR-TKI.

Objective:To investigate the clinical effect of the transverse rectus abdominismuscle (TRAM) on reconstruction of the breast.Methods:The clinical data of 23 patients receiving TRAM breast reconstruction in our department from Jan. 2018 to Dec. 2019 were retrospectively analyzed.Results:The operation time of 23 patients ranged from 240 to 360 mins, andthe average time was about 300 mins. Intraoperative bleeding was about 120 to 200 ml, with an average of 170 ml. All the flaps survived successfully, but 2 cases were complicated with local fat necrosis. The postoperative period was between 6 and 12 months. No local tumor recurrence or metastasis was found inall patients during postoperative follow-up, and the breast shape was maintained in good condition.Conclusion:TRAM can make up for the regret of breast loss caused by breast cancer in female patients. It can bring confidence in life and work to female patients, and the technology is safe and reliable, which is worthy of promotion.

Objective To investigate the effect of interfering Hiwi gene on the apoptosis of MDA-MB-231 cells. Methods The mRNA and protein expression of Hiwi mRNA and its target protein were analyzed by qRT-PCR and Western Blot after transfection. MDA-MB-231 cells were divided into 6 groups according to the experimental design. Interference effects were screened as siRNA interference group (Hiwi10330 group), and then divided into 3 groups according to the experimental design: interference group, negative control group/NC, blank control group/Blank. The cell apoptosis rate was detected by flow cytometry after transfection. Results The expression of mRNA in the interference group was significantly lower than that in the siRNA group (P < 0.05), the expression of target protein of Hiwi gene was also significantly inhibited (P < 0.05). The apoptosis rate of MDA-MB-231 cells was significantly higher than that of NC and Blank groups (P<0.05). Conclusion The apoptosis rate of breast cancer cells MDA-MB-231 was significantly increased after siRNA targeting hiwi gene silencing.

Objective: To understand the relationships of social exclusion, personality trait and emotion regulation with willingness to seek help after being bullied, and to provide reference for rationalized intervention of campus bullying among middle school students. Methods: A tatal of 2 040 middle school students from a middle school in Jiangxi Province were selected as the research objects, and surveyed by general situation questionnaire, Olweus Bullying Questionnaire, Willingness to Seek Help Scale, social exclusion scale, personality scale and Emotion Regulation Scale. Among them, a further survey of 381 bullies was conducted and SPSS 26.0 was used for statistical processing of data. Results: About 55.88% (133/238) and 58.74% (84/143) reported willingness to seek help after being bullied among middle and high school students, respectively( χ 2=0.30, P >0.05). There were no significant differences in gender and residency( P >0.05). In junior middle school students, compared with the non help willingness group( 3.83± 0.78，3.35±1.03，3.33±1.03，29.81±7.77), the rejected scores of the help willingness group were lower(3.57±0.75), scores of affinity and openness in personality traits were higher(3.69±0.88，3.72±0.79), the cognitive reappraisal scores were higher( 32.42 ±8.25). Among senior middle school students, the rejected and expression suppression scores of the help willingness group were lower(3.51±0.67，26.96±7.47), while extroversion personality traits were higher(3.61±0.95). Multivariate unconditional Log binomial regression analysis showed that high score of expression suppression was associated with less willingness to seek help( OR=0.94, P =0.02). Conclusion Social exclusion, personality trait and emotional regulation may have certain influences on willingness to seek help after being bullied among junior and senior middle school students, effects varies by grade level.

Breast cancer is one of the most common cancers in women,but there is currently a lack of accurate prognos-tic assessment systems.The Naples Prognostic Score(NPS)is a prognostic prediction system that incorporates inflammatory and nutritional indicators.It has been proven to have important clinical utility in predicting the prognosis of patients with malignancies such as colon cancer,gallbladder cancer,endometrial cancer,and lung cancer.In recent years,research has found that NPS may be superior to TNMstaging in predicting the prognosis of breast cancer patients.It is an independent predictor of overall sur-vival(OS)and progression-free survival(PFS)in breast cancer patients.This suggests that NPS has great potential for applica-tion in predicting the prognosis of breast cancer.

Objective:To investigate the feasibility of the surgical mode of immediate implant-based breast reconstruction (IIBR) with silicone implants under subcutaneous tissue directly after the total mastectomy in breast cancer.Methods:Data of 53 patients who underwent (skin-sparing mastectomy, SSM) or (nipple-areola-complex-sparing mastectomy, NSM) combined with IIBR in Department of Breast Surgery, Maoming People’s Hospital were retrospectively analyzed. Patients were divided into two groups, 31 cases with the silicone implant placed in different anatomical locations of the chest wall, including subcutaneous tissue, and 22 cases with subpectoral space implantation followed NSM or SSM. The two groups were compared in terms of the short-term and long-term complications, as well as the aesthetic outcome. Within 12 months the local recurrence rate was collected to evaluate the treatment safety of the two groups preserving the thickness of subcutaneous adipose after NSM or SSM.Results:There was no nipple-areola-complex (NAC) or skin flap for both groups, and the time of removing the drainage tube had no significant difference ( P>0.05) . There was no significant difference between the two groups in terms of the short-term complications within 6 months (repeated local infection and unknown effusion occurrence) , the long-term complications after 6 months (local skin with wrinkles sign, prosthesis displacement, and grade III-IV capsular contracture ( P>0.05) . However, the subcutaneous tissue implant group were superior ( P<0.05) in cosmetic outcome because of the breast had better symmetry. What’s more, no local tumor recurrence occurred in either group within 12 months. Conclusions:IIBR of subcutaneous tissue implantation (without patches) is an economical, novel, safe, and effective surgical mode for breast reconstruction, and the key to this operation mode depends on quality control of surgical procedures and the thickness of skin flap ≥ 1cm covering silicone implants. However, due to the cases enrolled in this study is not enough and short follow-up time, further clinical studies are still needed.

AIM:To investigate the role of specific long noncoding RNA SLC25a6(lncSLC25a6)in homocys-teine(Hcy)-induced cuproptosis in cardiomyocytes.METHODS:Rat cardiomyocytes were cultured in vitro and divided into control group and Hcy group.After 48 h of intervention,the expression levels of cuproptosis-related proteins,ferre-doxin 1(FDX1)and heat shock protein 70(HSP70),were detected by Western blot and immunofluorescence staining.The oxidative stress state of cardiomyocytes was assessed using fluorescence staining,and the intracellular Cu2+levels were measured using a copper ion assay kit.Furthermore,the impact of Hcy on the expression of cuproptosis-related proteins in cardiomyocytes was analyzed following overexpression of lncSLC25a6.RESULTS:Compared with the control group,80 μmol/L Hcy significantly accelerated cardiomyocyte damage,with a notable underexpression of lncSLC25a6(P<0.05).Western blot results indicated that,compared with the control group,the expression level of FDX1 in the Hcy intervention group was significantly reduced(P<0.05),while the expression level of HSP70 was significantly elevated(P<0.05),and the expression level of copper ions in cardiomyocytes of the Hcy group was increased(P<0.05).Immunofluorescence staining showed a significant reduction in FDX1 fluorescence intensity and a significant increase in HSP70 fluorescence in-tensity in the Hcy group.Further overexpression of lncSLC25a6 significantly mitigated Hcy-induced cuproptosis in cardio-myocytes,resulting in elevated expression of FDX1 and reduced expression of HSP70(P<0.05).Pearson correlation analysis demonstrated that the expression level of lncSLC25a6 was negatively correlated with FDX1 protein expression(r=-0.676,P=0.046)and positively correlated with HSP70 expression(r=0.680,P=0.044).CONCLUSION:lnc-SLC25a6 significantly mitigates Hcy-induced cuproptosis in cardiomyocytes,positioning it as a potential therapeutic target for managing Hcy-induced cardiac injury.

BACKGROUND:Hyperhomocysteinemia is closely related to the function of islet β cells,but its specific molecular mechanism is not fully understood. OBJECTIVE:To investigate the role of N6 methyltransferase-like 3(METTL3)in homocysteine(Hcy)-induced autophagy of mouse islet β cells. METHODS:The 3rd and 4th generation mouse islet β cells were taken for the experiment.(1)Cell modeling and grouping:cells in control group were not treated with Hcy,while those in homocysteine group were treated with 100 μmol/L Hcy for 48 hours.(2)The mouse islet β-cells were transfected with the plasmids overexpressing Ad-METTL3 and si-METTL3 according to the instructions of LipofectamineTM 2000.Three different interfering fragments were designed,and the one with the best interfering efficiency was verified and screened by PCR.(3)After transfection,the cells were divided into control group,Hcy group,Ad-NC(negative control)+Hcy group,Ad-METTL3+Hcy group,si-NC(negative control)+Hcy group and si-METTL3+Hcy group.(4)qRT-PCR and western blot were used to detect the expression levels of METTL3 and autophagy-related proteins LC3Ⅱ/Ⅰ and p62 in cells.Insulin level was determined by ELISA to evaluate insulin secretion capacity of islet cells.Autophagy-related proteins and insulin level were detected after overexpression and interference with METTL3. RESULTS AND CONCLUSION:Compared with the control group,the expression level of LC3Ⅱ/Ⅰ was increased(P＜0.05),the expression of p62 was significantly reduced(P＜0.05),and the insulin secretion capacity was significantly decreased(P＜0.05)in the Hcy group.Compared with the control group,the protein and mRNA levels of METTL3 were reduced in the Hcy group(P＜0.05).After METTL3 silencing in islet β cells,Hcy further upregulated the expression of LC3Ⅱ/Ⅰ(P＜0.05),significantly dowregulated the expression of p62(P＜0.05),and increased the insulin level(P＜0.05).After overexpression of METTL3,Hcy significantly decreased the LC3Ⅱ/Ⅰ expression and increased the p62 expression in islet β cells(P＜0.05).To conclude,METTL3 is involved in the Hcy-induced autophagy regulation of islet β cells and plays a role in the regulation of insulin secretion.

Objective To study the role of ubiquitin-conjugating enzyme 9(UBC9)in the pyroptosis of homocysteine-induced macrophages mediated by small ubiquitin-like modifier(SUMO)modification.Methods First,the effects of homocysteine at different concentrations(0 μmol/L,50 μ.mol/L,100 μmol/L,150 μmol/L and 200 μmol/L)on the viability and pyrodeath of mouse macrophages(RAW264.7)were detected by CCK-8 and Western blot.Western blot was used to detect the expression levels of UBC9,SUMO-1,and the inflammatory cytokine IL-1β in different groups of cells.qRT-PCR was used to detect the mRNA expression of UBC9 before and after RNA interference and the expression of UBC9,pyrogen-related protein,and SUMO-1 after RNA interference.Results After stimulation with 100 μmol/L homocysteine,the effect of macrophage activity was minimal,and NLRP3 and Caspase-1 were the proteins with the most obvious increase in expression(P＜0.05).Compared with the Control group,the Hcy group's expression of IL-1β and SUMO-1 was increased(P＜0.01).Compared with the Control group,the Hcy group's UBC9 protein and mRNA levels were increased(P＜0.05).The expression of NLRP3,Caspase-1,IL-1β,UBC9,and SUMO-1 was decreased in the si-UBC9+Hcy group compared with the si-NC+Hcy group(P＜0.01).Conclusions Homocysteine induces pyroptosis in macrophages,and its mechanism of action is related to the up-regulation of UBC9 to induce SUMO modification.