OBJECTIVE To establish a proteomic pre-processing protocol for low input samples (1×104 cells) in order to map the proteomic aging atlas of individual mouse antral follicles and elucidate the aging patterns of mouse antral follicles.METHODS Using cell lines and primary mouse cells,the protocol was developed by optimizing lysis buffers,enzymatic digestion,and ultrasonication for 1 × 104 cells (about 1 μg protein).The sample pre-processing protocol established in this study was evaluated by comparing it with two commercial sample preparation kits:iST kit,EasyPept-Ex (Ex kit),in terms of protein identification overlap,overall grand average of hydropathy (GRAVY),theoretical isoelectric points,and protein molecular weight.The sample suitability of the current sample pretreatment process was assessed using primary spleen and liver cell samples of mice at three different scales:100,1000,and 10000.Using this sample pre-processing approach in conjunction with the highly sensitive timsTOF Pro 2 mass spectrometer,proteome maps of individual antral follicles from young-aged (2 months),middle-aged (12 months),and old-aged (22 months) mice were generated.Differential and time-series analyses identified age-related proteins,and elucidated the aging patterns of mouse antral follicles.RESULTS A proteomic pre-processing protocol for 1 × 104 cell samples was established,requiring only one single-step operation for lysis,reduction,and alkylation,with enzymatic digestion not necessitating ultrasonication.We found that for cell samples on the order of 1 × 104,increasing the concentration of sodium deoxycholate in the lysis buffer from 1％ to 10％ enhanced the number of identified proteins from 4089 to 4389,demonstrating improved lysis efficiency (P＜0.05).The addition of ammonium bicar-bonate buffer (50 mmol·L-1,90 μL) to the lysis solution significantly increased the number of identified proteins from 2579 to 4389 (P＜0.01).Both single trypsin and mixed enzyme (trypsin/Lys-C) treatments yielded similar proteolytic outcomes,identifying approximately 3950 proteins each.Reducing the diges-tion time from overnight to 0.5 hours increased the number of identified proteins from 4299 to 4632 (P＜0.05),thus saving time while achieving higher protein identification yields.Compared to commercial kits,approximately 92.3％of the proteins identified by our protocol could also be identified by the iST kit or Ex kit.Our protocol demonstrated no significant bias in terms of hydrophobicity,theoretical isoelectric points or molecular weight,indicating robust performance.As the number of cells in the sample increased,the variety of identified proteins increased significantly.For instance,in samples containing 100,1000,and 10000 cells,525,1650,and 3210 proteins were identified in primary mouse spleen cells,respectively,compared with 366,1160,and 3590 proteins in primary mouse liver cells (P＜0.05).Finally,using this protocol,proteomic profiling of individual antral follicles from young,middle-aged,and old mice (three follicles per age group) was performed,with each follicle identifying over 7500 proteins on average.Based on this data,principal component analysis was conducted in this study,revealing significant differences in protein expression profiles of antral follicles at different age stages,confirming that age made a big difference to the physiological state of follicles.Additionally,we observed a signifi-cant downregulation of chromosome separation-associated proteins in aging mouse follicles (P＜0.01),suggesting potential disruptions in chromosome segregation and meiotic dysregulation.Further analysis revealed 739 proteins significantly correlated with age,among which 378 exhibited positive correlations and 361 negative correlations.CONCLUSION This study provides a low-cost,easy-to-operate,high-throughput,and highly sensitive proteomic sample pre-processing protocol for low input samples.It has constructed dynamic proteome maps of individual antral follicles at different ages in mice,offering high-quality data resources for basic research on follicular aging and providing new insights for exploring potential therapeutic targets for ovarian aging and the development of drugs to enhance fertility.

OBJECTIVE To establish a proteomic pre-processing protocol for low input samples (1×104 cells) in order to map the proteomic aging atlas of individual mouse antral follicles and elucidate the aging patterns of mouse antral follicles.METHODS Using cell lines and primary mouse cells,the protocol was developed by optimizing lysis buffers,enzymatic digestion,and ultrasonication for 1 × 104 cells (about 1 μg protein).The sample pre-processing protocol established in this study was evaluated by comparing it with two commercial sample preparation kits:iST kit,EasyPept-Ex (Ex kit),in terms of protein identification overlap,overall grand average of hydropathy (GRAVY),theoretical isoelectric points,and protein molecular weight.The sample suitability of the current sample pretreatment process was assessed using primary spleen and liver cell samples of mice at three different scales:100,1000,and 10000.Using this sample pre-processing approach in conjunction with the highly sensitive timsTOF Pro 2 mass spectrometer,proteome maps of individual antral follicles from young-aged (2 months),middle-aged (12 months),and old-aged (22 months) mice were generated.Differential and time-series analyses identified age-related proteins,and elucidated the aging patterns of mouse antral follicles.RESULTS A proteomic pre-processing protocol for 1 × 104 cell samples was established,requiring only one single-step operation for lysis,reduction,and alkylation,with enzymatic digestion not necessitating ultrasonication.We found that for cell samples on the order of 1 × 104,increasing the concentration of sodium deoxycholate in the lysis buffer from 1％ to 10％ enhanced the number of identified proteins from 4089 to 4389,demonstrating improved lysis efficiency (P＜0.05).The addition of ammonium bicar-bonate buffer (50 mmol·L-1,90 μL) to the lysis solution significantly increased the number of identified proteins from 2579 to 4389 (P＜0.01).Both single trypsin and mixed enzyme (trypsin/Lys-C) treatments yielded similar proteolytic outcomes,identifying approximately 3950 proteins each.Reducing the diges-tion time from overnight to 0.5 hours increased the number of identified proteins from 4299 to 4632 (P＜0.05),thus saving time while achieving higher protein identification yields.Compared to commercial kits,approximately 92.3％of the proteins identified by our protocol could also be identified by the iST kit or Ex kit.Our protocol demonstrated no significant bias in terms of hydrophobicity,theoretical isoelectric points or molecular weight,indicating robust performance.As the number of cells in the sample increased,the variety of identified proteins increased significantly.For instance,in samples containing 100,1000,and 10000 cells,525,1650,and 3210 proteins were identified in primary mouse spleen cells,respectively,compared with 366,1160,and 3590 proteins in primary mouse liver cells (P＜0.05).Finally,using this protocol,proteomic profiling of individual antral follicles from young,middle-aged,and old mice (three follicles per age group) was performed,with each follicle identifying over 7500 proteins on average.Based on this data,principal component analysis was conducted in this study,revealing significant differences in protein expression profiles of antral follicles at different age stages,confirming that age made a big difference to the physiological state of follicles.Additionally,we observed a signifi-cant downregulation of chromosome separation-associated proteins in aging mouse follicles (P＜0.01),suggesting potential disruptions in chromosome segregation and meiotic dysregulation.Further analysis revealed 739 proteins significantly correlated with age,among which 378 exhibited positive correlations and 361 negative correlations.CONCLUSION This study provides a low-cost,easy-to-operate,high-throughput,and highly sensitive proteomic sample pre-processing protocol for low input samples.It has constructed dynamic proteome maps of individual antral follicles at different ages in mice,offering high-quality data resources for basic research on follicular aging and providing new insights for exploring potential therapeutic targets for ovarian aging and the development of drugs to enhance fertility.

Objective:To explore the value of pre-treatment contrast-enhanced computed tomography (CT)-based texture analysis in predicting response to non-small cell lung cancer (NSCLC) immunotherapy.Methods:From January to July 2018, a total of 51 lesions from 42 patients with advanced non-small cell lung cancer receiving immunotherapy at Shanghai Chest Hospital were selected in this retrospective study. Pre-treatment contrast-enhanced CT-based texture features were extracted by MaZda software. Ten optimal texture features were chosen based on three different methods: Fisher coefficient, mutual information measure (MI) and minimization of classification error probability combined average correlation coefficients(POE+ ACC), respectively. According to the efficacy of the first immunotherapy, 51 lesions were divided into non-progressive disease (non-PD, n=26) and progressive disease (PD, n=25). The differences were tested in each texture feature set between the two groups. The immunotherapy effects of target lesions were analyzed by principal component analysis(PCA), linear discriminant analysis (LDA) and nonlinear discriminant analysis (NDA). The sensitivity, specificity, accuracy, positive-predictive value (PPV) and negative-predictive value (NPV) were calculated. The area under the curve (AUC) was used to quantify the predictive accuracy of the three analysis models for each texture feature set and compare them with the actual classification results. Results:In all of three texture feature sets, the texture parameter differences of Perc.50%, Perc.90%, "S(5, 5)SumEntrp" and "S(4, 4)SumEntrp" were higher in PD group than those in non-PD group (all P<0.05). The classification result of texture feature set chosen by POE+ ACC and analyzed by NDA was identified as the best model (AUC=0.802, 95% CI: 0.674-0.930), and its sensitivity, specificity, accuracy, PPV and NPV were 72%, 88.5%, 80.4%, 85.7%, 76.7%, respectively. Conclusion:Pre-treatment contrast-enhanced CT-based texture characteristics of NSCLC may function as non-invasive biomarkers for the evaluation of response to immunotherapy.

Objective:To explore the value of pre-treatment contrast-enhanced computed tomography (CT)-based texture analysis in predicting response to non-small cell lung cancer (NSCLC) immunotherapy.Methods:From January to July 2018, a total of 51 lesions from 42 patients with advanced non-small cell lung cancer receiving immunotherapy at Shanghai Chest Hospital were selected in this retrospective study. Pre-treatment contrast-enhanced CT-based texture features were extracted by MaZda software. Ten optimal texture features were chosen based on three different methods: Fisher coefficient, mutual information measure (MI) and minimization of classification error probability combined average correlation coefficients(POE+ ACC), respectively. According to the efficacy of the first immunotherapy, 51 lesions were divided into non-progressive disease (non-PD, n=26) and progressive disease (PD, n=25). The differences were tested in each texture feature set between the two groups. The immunotherapy effects of target lesions were analyzed by principal component analysis(PCA), linear discriminant analysis (LDA) and nonlinear discriminant analysis (NDA). The sensitivity, specificity, accuracy, positive-predictive value (PPV) and negative-predictive value (NPV) were calculated. The area under the curve (AUC) was used to quantify the predictive accuracy of the three analysis models for each texture feature set and compare them with the actual classification results. Results:In all of three texture feature sets, the texture parameter differences of Perc.50%, Perc.90%, "S(5, 5)SumEntrp" and "S(4, 4)SumEntrp" were higher in PD group than those in non-PD group (all P<0.05). The classification result of texture feature set chosen by POE+ ACC and analyzed by NDA was identified as the best model (AUC=0.802, 95% CI: 0.674-0.930), and its sensitivity, specificity, accuracy, PPV and NPV were 72%, 88.5%, 80.4%, 85.7%, 76.7%, respectively. Conclusion:Pre-treatment contrast-enhanced CT-based texture characteristics of NSCLC may function as non-invasive biomarkers for the evaluation of response to immunotherapy.

OBJECTIVE：To systematically e valuate the efficacy and safety of duloxetine in the improvement of pain symptoms of knee osteoarthritis （KOA），and to provide evidence-based reference for clinical treatment of KOA. METHODS ：Retrieved from PubMed，Embase，Medline，Cochrane Library ，CNKI，VIP and Wanfang database ，during the establishment of the database to Sept. 2019，RCTs about duloxetine （trial group ）vs. placebo （control group ）in the improvement of pain symptoms of KOA were collected. After data extraction and quality evaluation using Cochrane system evaluator ’s manual 5.1.0，Meta-analysis was performed by using Stata 14.0 software for WOMAC total score ，WOMAC pain score ，WOMAC stiffness score ，WOMAC function score and BPI-S score ，as well as the incidence of adverse reaction such as dry mouth ，somnolence and nausea. RESULTS：A total of 6 RCTs were included ，involving 2 059 patients. Meta-analysis results showed that WOMAC total score [MD＝－0.34，95％CI（－0.48，－0.20），P＜0.05]，WOMAC pain score [MD ＝－0.41，95％CI（－0.54，－0.29），P＜0.05]， WOMAC stiffness score [MD ＝－0.24，95％ CI（－0.37，－0.12），P＜0.05]，WOMAC function score [MD ＝－0.43，95％CI （－0.55，－0.31），P＜0.05]，BPI-S score [MD ＝－0.38，95％CI（－0.48，－0.28），P＜0.05] of trial group were significantly lower than those of control group ；the incidence of dry mouth [RR ＝3.55，95％CI（2.00，6.29），P＜0.05]，somnolence [RR ＝3.23， 95％CI（1.88，5.54），P＜0.05] and nausea [RR ＝6.95，95％CI（2.99，16.15），P＜0.05] in trial group were significantly higher than control group ，with significant difference. CONCLUSIONS ：Duloxetine can relieve the pain and improve knee function in patients with KOA ，but it is necessary to pay attention to its adverse reactions.

Objective: To discuss the effect and safety of continuous pumping for home enteral nutrition after esophagectomy. Methods: The current study retrospectively analyzed the esophageal cancer patients who underwent transthoracic esophagectomy between January 2017 and November 2017 at First Department of Thoracic Surgery, Peking University Cancer Hospital and Institute. There were totally 108 cases, including 88 males and 20 females, with an average age of 62 years. The patients were divided into pump feeding group (n=56) and traditional tube feeding group (n=52). The postoperative short-term safety, weight maintenance, enteral nutrition tolerance and nutritional support complete rate of the 2 groups were compared by χ² test, Fisher exact test and t test, respectively. Results: Compared with traditional tube feeding group, the patient safety in pumping feeding group was significantly better, with complications within 2 months after discharge were 11/52 and 4/56 respectively (χ²=2.393, P=0.035); the weight maintenance was significantly better, the weight loss within 4 weeks after discharge were 3.90 kg and 0.13 kg, respectively (t=7.720, P=0.000); the general enteral complications were significantly lower (26/52 vs. 5/56, χ²=22.225, P=0.000), the nutritional support complete rate was significantly higher (23/52 vs. 55/56, χ²=39.167, P=0.000). Conclusions Continuous pump feeding enteral nutrition support after discharge postoperatively could help improve patient safety after discharge, which is better for weight maintenance of the patients. Pump feeding could also enhance tolerability of tube feeding and ensure the effective accomplishment of nutritional support.

BACKGROUND: Thoracoscopic surgery has gradually become the major procedure for lung cancer surgery in our department. Its characteristics are minimal trauma and quick recovery, which make approximately 90% of patients discharge from the hospital after surgery. However, the postoperative complications still happen now and then. We analyzed the patients who had been hospitalized for longer than 7 days after thoracoscopic lung cancer surgery, aiming to summarize the types and risk factors of complications, and improve postoperative safety of patients. METHODS: The data were come from the prospective database of Thoracic Surgery Unit One in Peking Cancer Hospital, and patients that underwent thoracoscopic pulmonary surgery between Jan. 2010 and Dec. 2014 with length of stay more than 7 days were included in the study. The classifications of the complications were investigated and graded as mild or severe complications according to modified Claviengrading, the relationship between clinical factors and degrees of complications was also analyzed. RESULTS: The hospitalization of 115 cases were longer than 7 days after surgery, accounting for 10.3% (115/1,112) of the whole patients that underwent surgery during the same period. Eighty-one cases had mild complications, accounting for 7.3% (81/1,112) of the whole cases that underwent surgery during the same period and 70.4% (81/115) of the cases with prolonged length of stay; the proportions of severe complications in both groups were 3.1% (34/1,112) and 29.6% (34/115), respectively; and the proportions of complications that caused perioperative deaths were 0.18% (2/1112) and 1.7% (2/115), respectively. Among all the postoperative complications, the most common was air leakage for more than 5 days after surgery, with a total of 20 cases (1.8% and 17.4%). The other common complications were: atelectasis (19 cases, 1.7% and 16.5%), pulmonary infection (18 cases, 1.6% and 15.7%), etc. The less common complications was bronchopleural fistula (4 cases, 0.36% and 3.5%) with very high risk, and 2 cases died perioperatively due to the combination of acute respiratory distresssyndrome (ARDS). In the clinical factors, only preoperative low pulmonary function (FEV1%<70%) was the potential risk factor for postoperative severe complications (45.8% vs 23.6%, P=0.038). There was no significant difference either regarding the 5 year disease free survival or the 5 year overall survival between mild complication group and severe complication group, with 5 year DFS being 52.2% and 51.9%, respectively (P=0.894) , and 5 year overall survival being 64.0% and 53.5%, respectively (P=0.673). CONCLUSIONS Continuous postoperative air leakage, atelectasis and pulmonary infections were the major causes for prolonged hospitalization after thoracoscopic surgery for lung cancer, and bronchopleural fistula was the most perilous complications. Patients with low preoperative pulmonary function were more likely to have severe postoperative complication, however, this would not influence the long term survival of the patients.
Adult ; Aged ; Aged, 80 and over ; Female ; Hospitalization ; Humans ; Length of Stay ; Lung Neoplasms ; complications ; surgery ; therapy ; Male ; Middle Aged ; Postoperative Complications ; epidemiology ; Postoperative Period ; Prospective Studies ; Thoracic Surgery, Video-Assisted

BACKGROUND: Currently, there is no consensus on the follow-up strategy (follow-up time interval and content) of non-small cell lung cancer (NSCLC) in the world, and the relevant clinical evidence is also very limited. In this study, we aimed to summarize the recurrence/metastasis sites and timings of stage I NSCLC patients based on their follow-up data, aiming to provide a basis of follow-up time interval and content for this group of patients. METHODS: We retrospectively analyzed the 416 stage I NSCLC patients that underwent continuous anatomic lobectomy between Jan. 2000 to Oct. 2013 in our prospective lung cancer database. According to the recurrence/metastasis sites and timings, the long term follow-up time interval and content were explored. RESULTS: The 5-yr disease free survival (DFS) and overall survival (OS) in the whole group were 82.4% and 85.4%, respectively. There were 76 cases (18.3%) had recurrence/metastasis during follow-up, among which the most frequent site was pulmonary metastasis (21 cases, 5.0%), followed by brain metastasis (20 cases, 4.8%), bone metastasis (12 cases, 2.9%), and mediastinal lymph node metastasis (12 cases, 2.9%). Among the factors that could influence recurrence/metastasis, patients with pT2a suffered from a higher recurrence/metastasis rate compared to patients with pT1 (P=0.006), with 5-yr DFS being 73.8% and 87.3%, respectively (P=0.002), and the 5-yr OS being 77.7% and 90.3%, respectively (P=0.011). CONCLUSIONS The commonest recurrence/metastasis sites of stage I NSCLC after anatomic lobectomy are lung, brain and mediastinal lymph nodes, the risk of recurrence/metastasis within 2 years were equal to that between 3 years and 5 years. The follow-up frequencies and content within 2 years could be adjusted according to T stages.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; mortality ; pathology ; surgery ; Female ; Follow-Up Studies ; Humans ; Lung Neoplasms ; mortality ; pathology ; surgery ; Lymph Nodes ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Pneumonectomy ; Prospective Studies ; Retrospective Studies ; Young Adult

BACKGROUND: It has been believed mesenchymal stem cells (MSCs) play a role in treatment through paracrine mechanism. Various side effects such as embolism, tumorigenesis and immunological reaction caused by direct injection of MSCs can be avoided by extracting MSC lysate. However, there is a larger difference in current collection methods and standards of MSC lysate. OBJECTIVE: To compare repeated freeze-thaw and ultrasonication for the collection of lysate of MSCs. METHODS: Adipose-derived mesenchymal stem cells (ADMSCs) were isolated from the abdominal subcutaneous fat of healthy individuals, and purified with adherence screening method, followed by in vitro amplification using fetal bovine serum medium. The common surface makers of these cells were tested by flow cytometry (1×109, 2×109, 4×109/L). Repeated freeze-thaw and ultrasonication were employed for cell cytoclasis at three different densities respectively in saline and double distilled water, and a comprehensive comparison was performed on cytoclasis rate and the content of protein in cell lysate between the two methods. RESULTS AND CONCLUSION: (1) ADMSCs obtained from in vitro isolated human adipose tissue grew in a swirl or radial pattern with a homogenous size and neat arrangement. CD44, CD90, CD105 and other commonly used surface markers were highly expressed. (2) The study for optimization of lysate collection revealed that the higher cell density implicated a longer time for cell wall disruption and cytoclasis, as well as significantly increased cytoclasis rate. (3) BCA protein assay showed that the highest content of protein was obtained in saline solvent using ultrasonication method. Comprehensive analysis on the results leads to a conclusion that ultrasonication method with saline as the solvent is the optimized method for extraction of ADMSCs lysate, and the cell concentration of less than 4×109/L is recommended.

Objective To study the effects of diet and exercise intervention on prognosis of nonalcoholic fatty liver disease (NAFLD).Methods Fifty two patients with NAFLD received diet and exercise intervention.The treatment effects before and after intervention were analyzed.Result The body mass index(BMI),triglyceride(TG),cholesterol(TC)were significantly lower than those in control group after three-month intervention(all P<0.05).Conclusions Diet and exercise intervention can effectively improve liver function and promote the prognosis of nonalcoholic fatty liver,thus promote their quality of life.