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 analyze the system of first contact in community health institutions for migrant workers in Shenzhen city with the stakeholder theory. Methods:The report forms of the Social Insurance Fund Administration Bureau and the health bureau of Shenzhen city were used to analyze. All stakeholders of this policy were interviewed. Results:The system of first contact in community health institutions was good for all stakeholders. The number of in-sured migrant workers grew steadily, the business volume of community health service centers increased significantly, and the fund ran smoothly. Conclusions:The effects of the system of first contact in community health institutions for migrant workers in Shenzhen city were obvious. All the stakeholders felt satisfied with the policy.

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 investigate the clinical effect of modified Graeb criteria score and Glasgow coma score (GCS) in individualized treatment of intraventricular hemorrhage. Methods: 113 patients with intraventricular hemorrhage admitted to the department of neurosurgery of Second Affiliated Hospital of Guangxi Medical University from June 2014 to February 2018 were enrolled, and they were divided into 13-15, 9-12, and 3-8 groups according to GCS score at admission, and modified Graeb criteria score was classified as grade Ⅰ, Ⅱ and Ⅲ at the same time. In GCS 9-12 and 3-8 groups, patients with modified Graeb criteria score grade Ⅲ were treated with bilateral extra ventricular drainage, patients with modified Graeb criteria score grade Ⅱ were treated with bilateral extra ventricular drainage or lumbar cistern drainage (GCS 9-12 group was more prior to lumbar cistern drainage, 3-8 group was given priority to extra ventricular drainage), and patients with modified Graeb criteria score grade Ⅰ were treated conservatively. In GCS 13-15 group, bilateral extra ventricular cerebral drainage or lumbar cistern drainage was performed if the modified Graeb criteria score grade was Ⅲ, lumbar cistern drainage or conservative treatment was performed if the modified Graeb criteria score grade was Ⅱ, and conservative treatment was performed if the modified Graeb criteria score grade was Ⅰ. The changes in GCS score at 1 month after individualized treatment and the favourable prognosis rate at 6 months after treatment were observed [favourable prognosis was defined as Glasgow outcome score (GOS) Ⅳ-Ⅴ] as well as the basic clearance time of intraventricular hematomas, and the occurrence of complications such as intracranial infection, pulmonary infection and hydrocephalus were recorded. Results: 113 patients with intraventricular hemorrhage were enrolled in the final analysis, including 39 patients in GCS 13-15 group, 27 in 9-12 group, and 47 in 3-8 group; 21 patients with the first grade of modified Graeb criteria score, 42 with the second grade and 50 with the third grade. At 1 month after individualized treatment, the GCS scores in GCS 13-15 and 9-12 groups were significantly higher than those at admission (14.8±0.2 vs. 13.7±0.8, 13.1±1.7 vs. 10.7±1.1, both P < 0.05). When comparing the GCS score of the same patient at admission with that of 1 month after treatment, the GCS scores of the three groups were significantly improved, indicating that the consciousness of patients with different coma levels at admission had been significantly improved after individualized treatment. The basic clearance time of intracerebroventricular hematomas in patients with the second grade of modified Graeb criteria score was (7.0±2.8) days, in patients with the third grade was (6.1±2.0) days. At 6 months after individualized treatment, among 113 patients, GOS score was grade Ⅰ in 7 patients (6.2%), grade Ⅱ in 13 patients (11.5%), grade Ⅲ in 28 patients (24.8%), grade Ⅳ in 27 patients (23.9%), and grade Ⅴ in 38 patients (33.6%), with the favourable prognosis rate of 57.5% (65/113). Among 113 patients, intracranial infection occurred in 5 patients (4.4%), pulmonary infection in 22 patients (19.5%), hydrocephalus in 2 patients (1.8%) and rebleeding in 4 patients (3.5%). In 83 patients with lumbar cistern drainage, 1 patient had post-drainage infection (1.2%), 3 patients had plugging (3.6%), 6 patients had accidental drop of drainage tube (7.2%), and none of them had occipital macroforamen hernia after drainage. Seven of the 113 patients died including 2 patients died of cerebral hernia caused by rebleeding, 5 patients died of severe pneumonia or automatic discharge from hospital. Conclusion The combination of modified Graeb criteria score and GCS score can individualize treatment for patients with intraventricular hemorrhage and effectively improve the prognosis of patients with intraventricular hemorrhage.

OBJECTIVE: To investigate the clinical effect of modified Graeb criteria score and Glasgow coma score (GCS) in individualized treatment of intraventricular hemorrhage. METHODS: 113 patients with intraventricular hemorrhage admitted to the department of neurosurgery of Second Affiliated Hospital of Guangxi Medical University from June 2014 to February 2018 were enrolled, and they were divided into 13-15, 9-12, and 3-8 groups according to GCS score at admission, and modified Graeb criteria score was classified as grade I, II and III at the same time. In GCS 9-12 and 3-8 groups, patients with modified Graeb criteria score grade III were treated with bilateral extra ventricular drainage, patients with modified Graeb criteria score grade II were treated with bilateral extra ventricular drainage or lumbar cistern drainage (GCS 9-12 group was more prior to lumbar cistern drainage, 3-8 group was given priority to extra ventricular drainage), and patients with modified Graeb criteria score grade I were treated conservatively. In GCS 13-15 group, bilateral extra ventricular cerebral drainage or lumbar cistern drainage was performed if the modified Graeb criteria score grade was III, lumbar cistern drainage or conservative treatment was performed if the modified Graeb criteria score grade was II, and conservative treatment was performed if the modified Graeb criteria score grade was I. The changes in GCS score at 1 month after individualized treatment and the favourable prognosis rate at 6 months after treatment were observed [favourable prognosis was defined as Glasgow outcome score (GOS) IV-V] as well as the basic clearance time of intraventricular hematomas, and the occurrence of complications such as intracranial infection, pulmonary infection and hydrocephalus were recorded. RESULTS: 113 patients with intraventricular hemorrhage were enrolled in the final analysis, including 39 patients in GCS 13-15 group, 27 in 9-12 group, and 47 in 3-8 group; 21 patients with the first grade of modified Graeb criteria score, 42 with the second grade and 50 with the third grade. At 1 month after individualized treatment, the GCS scores in GCS 13-15 and 9-12 groups were significantly higher than those at admission (14.8±0.2 vs. 13.7±0.8, 13.1±1.7 vs. 10.7±1.1, both P < 0.05). When comparing the GCS score of the same patient at admission with that of 1 month after treatment, the GCS scores of the three groups were significantly improved, indicating that the consciousness of patients with different coma levels at admission had been significantly improved after individualized treatment. The basic clearance time of intracerebroventricular hematomas in patients with the second grade of modified Graeb criteria score was (7.0±2.8) days, in patients with the third grade was (6.1±2.0) days. At 6 months after individualized treatment, among 113 patients, GOS score was grade I in 7 patients (6.2%), grade II in 13 patients (11.5%), grade III in 28 patients (24.8%), grade IV in 27 patients (23.9%), and grade V in 38 patients (33.6%), with the favourable prognosis rate of 57.5% (65/113). Among 113 patients, intracranial infection occurred in 5 patients (4.4%), pulmonary infection in 22 patients (19.5%), hydrocephalus in 2 patients (1.8%) and rebleeding in 4 patients (3.5%). In 83 patients with lumbar cistern drainage, 1 patient had post-drainage infection (1.2%), 3 patients had plugging (3.6%), 6 patients had accidental drop of drainage tube (7.2%), and none of them had occipital macroforamen hernia after drainage. Seven of the 113 patients died including 2 patients died of cerebral hernia caused by rebleeding, 5 patients died of severe pneumonia or automatic discharge from hospital. CONCLUSIONS The combination of modified Graeb criteria score and GCS score can individualize treatment for patients with intraventricular hemorrhage and effectively improve the prognosis of patients with intraventricular hemorrhage.
Cerebral Hemorrhage ; China ; Glasgow Coma Scale ; Humans ; Hydrocephalus ; Prognosis ; Retrospective Studies ; Treatment Outcome

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.

Objective:To investigate the correlation of the expression of Ephrin A receptor 2(EphA2)and its promoter region DNA hypomethylation with the occurrence of pyroptosis in invasive breast cancer. Methods:The expression level of pyroptosis-related protein EphA2 in normal breast tissue,paracancerous tissues and cancer tissues from 42 breast cancer patients was examined by Immunofluorescence staining and Western blotting.The expression level of pyroptosis related protein nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)was analyzed by immunofluorescence staining and Western blotting.The expression levels of apoptosis related proteins Caspase 1 and inflammatory cytokine interleukin-1β(IL-1 β)were studied by Western blotting.The DNA methylation level in the promoter region of EphA2 was investigated by nested methylation-specific PCR(nMS-PCR).The expression levels of DNA methyltransferase 1(DNMT1)and DNA methyltransferase 3a(DNMT3a)were examined by Western blotting.The correlation of the protein expression and methylation level of EphA2 in cancer tissues with the expression NLRP3,Caspase 1,IL-1 β,DNMT1 and DNMT3a was analyzed by Pearson correlation coefficient. Results:Compared with normal breast tissues and paracancerous tissues,the expression level of EphA2 protein was significantly increased(P＜0.01),while that of NLRP3,Caspasel and IL-1 βwas significantly decreased(P＜0.05)in breast cancer tissues.Meanwhile,compared with normal breast tissues and paracancerous tissues,the DNA methylation level of EphA2 promoter in breast cancer tissues was significantly decreased(P＜0.05),the expression level DNMT3a protein was significantly decreased(P＜0.01,P＜0.05),and the difference in the expression level of DNMT1 protein was not statistically significant.Pearson correlation analysis showed that the expression level of EphA2 protein is negatively correlated with that of NLRP3(r=-0.651 2,P＜0.05),Caspasel(r=-0.571 2,P＜0.05),IL-1β(r=-0.654 6,P＜0.05)or DNMT3a(r=-0.537 4,P＜0.05),while the methylation level of EphA2 was positively correlated with the protein expression level of NLRP3(r=0.634 1,P=0.026 8),Caspase1(r=0.672 8,P=0.01 6 5),IL-1 β(r=0.694 0,P=0.01 2 3)and DNMT3a(r=0.687 1,P=0.01 3 6). Conclusion:The expression of EphA2 protein is upregulated in breast cancer tissues is negatively correlated with pyroptosis.DNMT3a may be involved in the process of DNA hypomethylation in the promoter region of EphA2.

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.