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Yuan Wang, Xiaoyu Song, Ru Cui, Xinlin Luo, Juntian Dong, Hanqi Wang, Chengcheng Song, Yifa Zhou, Sisi Cui Engineering Research Center of Glycoconjugates, Ministry of Education, Jilin Provincial Key Laboratory of Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun, 130024, People’s Republic of ChinaCorrespondence: Sisi Cui, Engineering Research Center of Glycoconjugates of Ministry of Education, Jilin Provincial Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun, 130024, People’s Republic of China, Email cuiss100@nenu.edu.cnPurpose: Conventional oral formulations for inflammatory bowel disease (IBD) treatment are less than satisfactory, due to the poor controllability of drug release and lack of specificity to the inflammation sites in the gastrointestinal (GI) tract. To overcome these limitations, we developed a multiple carbohydrate-based nanosystem with pH/ROS dual responsibility and charge-mediated targeting ability for IBD-specific drug delivery.Methods: In view of the overproduction of ROS and overexpression of cationic proteins in the inflammatory colon, the designed nanosystem was composed of oxidation-sensitive cyclodextrin (OX-CD), chitosan (CS) and pectin (AHP). OX-CD was utilized to load dexamethasone (DM) by the solvent evaporation method. CS and AHP with opposite charges were sequentially coated onto OX-CD to generate the nanosystems by the electrostatic self-assembly method. The physicochemical properties, stability, dual-sensitive drug release behavior, cytotoxicity, cellular uptake and anti-inflammatory activity were investigated in vitro. In vivo bio-distribution and therapeutic efficacy of the nanosystem were further evaluated in the ulcerative colitis (UC) mice.Results: The obtained AHP/CS/OX-CD-DM nanosystem (ACOC-DM) could maintain stability under the GI pH environments, and release drug in the inflammatory colon with pH/ROS sensitivity. Dual polysaccharide-coated ACOC-DM exhibited higher cellular uptake and anti-inflammatory efficacy in macrophages than single polysaccharide-coated CS/OX-CD-DM nanosystem (COC-DM). Orally administrated ACOC-DM could enhance inflammation targeting ability and therapeutic efficacy of DM in the UC mice.Conclusion: This carbohydrate-based nanosystem with pH/ROS dual sensitivity and inflammation targeting capacity may serve as a safe and versatile nanoplatform for IBD therapy. Keywords: PH/ROS sensitivity, pectin, chitosan, oxidation-sensitive cyclodextrin, inflammation targeting, oral delivery

Wenyi Du,1,* Yixuan Wang,2,* Chen Song,1 Zhiqiang Tian,1 Yuan Liu,1 Wei Shen1 1Department of General Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China; 2Medical Integration and Practice Center, Reproductive Hospital Affiliated to Shandong University, Jinan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuan Liu; Wei Shen, Department of General Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China, Tel +86 17852061572 ; +86 13385110723, Email liuyuanjs@outlook.com; shenweijs@outlook.comPurpose: Previous studies have shown a correlation between diabetes mellitus and gallstone formation. The atherogenic index of plasma (AIP) is associated with many metabolic diseases. However, insufficient evidence still exists to elucidate the association between AIP and gallstones. The primary objective of this study was to investigate the correlation between AIP and gallstones in US adults, and the secondary objective was to analyze whether diabetes plays a mediating role in the association.Patients and Methods: Using data from the National Health and Nutrition Survey (NHANES) conducted between 2017 and March 2020, this study investigated the association between AIP and gallstone incidence in US adults. A variety of statistical methods were used to analyze the data in this study, including multivariate logistic regression, subgroup analyses, restricted cubic spline curves (RCS), and mediation effects analysis. In addition, two-stage linear regression was used to detect possible threshold and saturation effects.Results: A total of 6952 subjects were enrolled in the trial, of which 748 patients were diagnosed with gallstones. A significant positive association between AIP and gallstones was observed by fully adjusted multivariate logistic regression analysis, with an odds ratio (OR) of 1.45 and a 95% confidence interval (CI) of (1.09, 1.93). In addition, a non-linear positive association and saturation effect between AIP and gallstones were found, with an inflection point of 0.2246. Mediation analysis showed that diabetes had a mediating effect of 16.9% in the association between AIP and gallstones.Conclusion: This study suggests that elevated levels of AIP are linked to an augmented vulnerability to gallstone development, with diabetes serving as a mediating factor. These findings present a novel perspective on clinical approaches to prevent and manage gallstones.Keywords: gallstones, atherogenic index of plasma, diabetes mellitus, National Health and Nutrition Examination Survey, cross-sectional studies

Zhongxin Hou,1,&ast; Yang Chen,1,2,&ast; Yunrui Sun,1 Chongwei Song,1 Haoyuan Deng,3 Nan Cheng,4 Xiaoyu Han,2 Jianghui Zhang,5 Qian Wang,1 Yi Li,1 Jianzhong Yin,1,6 Qiong Meng1 1School of Public Health, Kunming Medical University, Kunming, Yunnan, People’s Republic of China; 2Institute for Acute Communicable Disease Prevention and Control, Yunnan Provincial Center for Disease Control and Prevention, Kunming, Yunnan, People’s Republic of China; 3Medical Department, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, People’s Republic of China; 4Department of Hospital Infection, Wuhan Mental Health Center, Wuhan, Hubei, People’s Republic of China; 5AIDS Care Center, Yunnan Provincial Hospital of Infectious Disease, Anning, Yunnan, People’s Republic of China; 6Baoshan College of Traditional Chinese Medicine, Baoshan, Yunnan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qiong Meng, School of Public Health, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, Yunnan, 650500, People’s Republic of China, Tel +8615559789771, Email mengqiong2006@163.com Jianzhong Yin, Baoshan College of Traditional Chinese Medicine, Baoshan Longyang District Qingyang District Vocational Education Park, Baoshan, Yunnan, 678000, People’s Republic of China, Tel +8613987645337, Email yinjianzhong2005@sina.comPurpose: Depression and anxiety are two highly prevalent mental disorders that commonly coexist. However, little is known about the association between sleep and comorbid depressive and anxiety symptoms (CDAS). Therefore, this study aims to explore the relationship between sleep duration, insomnia and CDAS.Methods: This is a cross-sectional study of 22,004 community adults who participated in the China Multi-Ethnic Cohort (CMEC) study. Chinese version of Patient Health Questionnaire-2 and Generalized Anxiety Disorder-2 were used to screen CDAS in community adults. A positive score on each scale was considered indicative of CDAS. All participants received face-to-face interviews, medical examinations, and biochemical examinations to assess sleep duration and insomnia and collect covariates. The self-reported sleep duration was divided into three groups: < 7 hr, 7– 9 hr and > 9 hr groups. Logistic regression was used to analyze the association between sleep duration, insomnia and CDAS. A restricted cubic spline (RCS) was used to explore the dose–response relationship between sleep duration and CDAS. Stratified analysis based on gender and age was conducted.Results: Overall, 2.8% (95% CI 2.6%~3.0%) of participants reported having CDAS. After adjusting the potential covariates, sleep duration < 7 hr (OR=1.635, 95% CI=1.335~2.004) was significantly associated with CDAS, compared with sleep 7– 9 hr. After RCS analysis, there was a nonlinear relationship between sleep duration and CDAS. The increase in the number of types of insomnia, the greater the likelihood of CDAS (p for trend< 0.05). In sensitivity analysis, it was found that the results were consistent with those of the main analysis. After stratification by gender and age, the association between sleep duration and CDAS was only observed in female and young and middle-aged adults, not in men and older adults.Conclusion: Both insufficient sleep duration and insomnia are associated with a higher prevalence of CDAS in Chinese adults.Keywords: sleep duration1, insomnia2, comorbid depressive and anxiety symptoms3, adults4, cross-sectional study5

Jining He,1– 3 Chenxi Song,1– 3 Rui Zhang,1– 4 Sheng Yuan,1– 3 Jianjun Li,1,3 Kefei Dou1– 4 1Cardiometabolic Medicine Center, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; 2Department of Cardiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; 3State Key Laboratory of Cardiovascular Disease, Beijing, People’s Republic of China; 4National Clinical Research Center for Cardiovascular Diseases, Beijing, People’s Republic of ChinaCorrespondence: Kefei Dou, Cardiometabolic Medicine Center, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 167A Beilishi Road, Xi Cheng District, Beijing, 100037, People’s Republic of China, Tel +86-13801032912, Fax +86-10-6831-3012, Email drdoukefei@126.com Jianjun Li, Department of Cardiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 167A Beilishi Road, Xi Cheng District, Beijing, 100037, People’s Republic of China, Email lijianjun938@126.comPurpose: Neutrophil to lymphocyte ratio (NLR), a novel inflammatory biomarker, has been shown to positively predict prognosis independent of high-sensitivity C-reactive protein (hsCRP) in patients with coronary artery disease (CAD). This study aimed to use discordance analysis to evaluate the effectiveness of NLR and hsCRP to predict adverse events in patients with stable CAD.Patients and Methods: This observational cohort study included 7827 consecutive CAD patients at Fuwai Hospital from March 2011 to April 2017. Discordant NLR with hsCRP was defined by the highest quartiles and medians. The primary endpoint was major adverse cardiovascular and cerebrovascular events (MACCEs), including cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, and unplanned revascularization.Results: During a median 36-month follow-up, 624 (8.0%) MACCEs occurred. Compared with the lowest NLR quartile, a significantly higher risk of MACCEs was observed in the highest NLR quartile after adjusting for confounding factors (hazard ratio [HR], 1.36; 95% confidence interval [CI], 1.09– 1.71). High NLR and low hsCRP discordance were also associated with an increased risk of MACCEs in the fully adjusted model (HR, 1.39; 95% CI, 1.05– 1.84).Conclusion: This study demonstrated that discordantly elevated NLR levels were associated with a greater risk of adverse clinical events in patients with stable CAD, suggesting the potential clinical significance of NLR as a goal of inflammatory risk management.Keywords: coronary artery disease, high-sensitivity C-reactive protein, inflammation, neutrophil to lymphocyte ratio, prognosis

Jonathan B Lin,1 Kristen M Pitts,1 Hani El Helwe,1 Cameron Neeson,1 Nathan E Hall,1 Henisk Falah,1 Stephanie A Schultz,2 Silas L Wang,1 Kristine Lo,1 Christian Song,1 Milica A Margeta,1,&ast; David Solá-Del Valle1,&ast; 1Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear, Boston, MA, USA; 2Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA&ast;These authors contributed equally to this workCorrespondence: David Solá-Del Valle; Milica A Margeta, Assistant Professor of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA, Email david_sola-delvalle@meei.harvard.edu; milica_margeta@meei.harvard.eduPurpose: Neurofilament light chain (NfL) is a neuronal cytoskeletal protein that has been identified as a marker of neurodegeneration in diseases of the central nervous system. In this study, we investigated whether NfL in the aqueous humor (AH) can serve as a marker of neurodegeneration in glaucoma in a racially diverse North American population.Design: Single-center, case–control study.Participants: We enrolled patients with various types and stages of glaucoma undergoing planned ophthalmic surgery as part of their routine care and compared them with patients without glaucoma undergoing phacoemulsification for age-related cataract.Methods: We collected AH from 39 glaucoma patients and 10 patients without glaucoma. AH NfL was quantified using the Single-Molecule Array (Simoa)® NF-light assay (Quanterix). Demographic information, such as age, body mass index, sex, and self-reported race, as well as clinical information, such as pre-operative intraocular pressure (IOP), maximum IOP, and number of pre-operative glaucoma medications, was obtained by reviewing the medical record.Main Outcome Measures: Levels of AH NfL.Results: In a model controlling for age and body mass index (BMI), NfL was significantly elevated in AH from glaucoma patients (mean: 429 pg/mL; standard deviation [SD]: 1136 pg/mL) compared to AH from patients without glaucoma (mean: 3.1 pg/mL; SD: 1.9 pg/mg): P = 0.002. Higher AH NfL was associated with higher maximum IOP (R = 0.44, P = 0.005), higher pre-operative IOP (R = 0.46, P = 0.003), and more pre-operative glaucoma medications (Rs = 0.61, P < 0.001). There was no association between AH NfL and Humphrey visual field mean deviation (R = − 0.20, P = 0.220), retinal nerve fiber layer thickness as measured with optical coherence tomography (R = 0.07, P = 0.694), or glaucoma stage (Rs = 0.015, P = 0.935).Conclusion: Our findings suggest that AH NfL may have clinical utility as a marker of glaucomatous neurodegeneration.Keywords: neurofilament light chain, glaucoma, neurodegeneration, biomarker

Changzheng Song,1 Qingwei Wang,2 Nianci Song3 1Erythrocrine Research Initiative of Translational Medicine, Shandong Academy of Medical Sciences, Jinan, People’s Republic of China; 2Cancer Therapy Center, Qilu Hospital of Shandong University, Jinan, People’s Republic of China; 3Biodesign Lab, Geriatricare Biorobotics Studio, Baltimore, MD, USACorrespondence: Changzheng Song, Erythrocrine Research Initiative of Translational Medicine, Shandong Academy of Medical Sciences, N-18877 Jingshi Road, Jinan, 250062, People’s Republic of China, Email songcz@sina.cnBackground and Objective: Cupping is a time-honoured traditional healing modality for pain management and remains favoured by professionals and lay people across several cultures today. However, the analgesic mechanism of cupping is still poorly understood. In addition, clinical guidelines for standardized applications of cupping are currently lacking. The awareness of cupping marks has provoked curiosity about the connection between skin color changes and their benefit for local pain relief. Computer simulation is a promising approach for numerical modeling the cupping-evoked erythrocyte emigration. Quantitative proteomic profiling of cupping-induced blister fluid exhibited a significant decrease in the abundance of haemoglobin β subunit. This finding provides a critical clue to paint a novel picture of the mechanism behind cupping. The hemorphins are a set of non-classical opioid peptides derived from the proteolysis of haemoglobin β subunit. In the present study, a probable mechanism of hemorphin-based cupping analgesia is proposed. The hemorphin could also act as a potential biomarker for objective and timely quantitative clinical assessment of cupping in the management of pain conditions. A seminal theory may open a new avenue for future translational research on promoting the efficacy and safety of cupping analgesia.Conclusion: The local analgesic effect of cupping is probable in the context of haemoglobin degradation that bestows the appearance of hemorphins along with engaging opioid receptor signalling. Exploring the potential novel mechanism of cupping analgesia facilitates seeking non-pharmacologic pain interventions.Keywords: cupping analgesia, erythrocyte, hemorphin, cupping mark, numerical simulation

Yuan Liu,&ast; Chen Song,&ast; Zhiqiang Tian, Wei Shen Department of General Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Wei Shen, Department of General Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China, Tel +86 13385110723, Email shenweijs@outlook.comPurpose: This study aims to construct a machine learning model that can recognize preoperative, intraoperative, and postoperative high-risk indicators and predict the onset of venous thromboembolism (VTE) in patients.Patients and Methods: A total of 1239 patients diagnosed with gastric cancer were enrolled in this retrospective study, among whom 107 patients developed VTE after surgery. We collected 42 characteristic variables of gastric cancer patients from the database of Wuxi People’s Hospital and Wuxi Second People’s Hospital between 2010 and 2020, including patients’ demographic characteristics, chronic medical history, laboratory test characteristics, surgical information, and patients’ postoperative conditions. Four machine learning algorithms, namely, extreme gradient boosting (XGBoost), random forest (RF), support vector machine (SVM), and k-nearest neighbor (KNN), were employed to develop predictive models. We also utilized Shapley additive explanation (SHAP) for model interpretation and evaluated the models using k-fold cross-validation, receiver operating characteristic (ROC) curves, calibration curves, decision curve analysis (DCA), and external validation metrics.Results: The XGBoost algorithm demonstrated superior performance compared to the other three prediction models. The area under the curve (AUC) value for XGBoost was 0.989 in the training set and 0.912 in the validation set, indicating high prediction accuracy. Furthermore, the AUC value of the external validation set was 0.85, signifying good extrapolation of the XGBoost prediction model. The results of SHAP analysis revealed that several factors, including higher body mass index (BMI), history of adjuvant radiotherapy and chemotherapy, T-stage of the tumor, lymph node metastasis, central venous catheter use, high intraoperative bleeding, and long operative time, were significantly associated with postoperative VTE.Conclusion: The machine learning algorithm XGBoost derived from this study enables the development of a predictive model for postoperative VTE in patients after radical gastrectomy, thereby assisting clinicians in making informed clinical decisions.Keywords: gastric neoplasms, gastrectomy, venous thromboembolism, risk factors, machine learning, prediction model

Yuan Liu,&ast; Chen Song,&ast; Zhiqiang Tian, Wei Shen Department of General Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Wei Shen, Department of General Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, Jiangsu Province, 214000, People’s Republic of China, Tel +86 13385110723, Email shenweijs@outlook.comPurpose: The occurrence of myocardial injury, a grave complication post complete mesocolic excision (CME), profoundly impacts the immediate and long-term prognosis of patients. The aim of this inquiry was to conceive a machine learning model that can recognize preoperative, intraoperative and postoperative high-risk factors and predict the onset of myocardial injury following CME.Patients and Methods: This study included 1198 colon cancer patients, 133 of whom experienced myocardial injury after surgery. Thirty-six distinct variables were gathered, encompassing patient demographics, medical history, preoperative examination characteristics, surgery type, and intraoperative details. Four machine learning algorithms, namely, extreme gradient boosting (XGBoost), random forest (RF), multilayer perceptron (MLP), and k-nearest neighbor algorithm (KNN), were employed to fabricate the model, and k-fold cross-validation, ROC curve, calibration curve, decision curve analysis (DCA), and external validation were employed to evaluate it.Results: Out of the four predictive models employed, the XGBoost algorithm demonstrated the best performance. The ROC curve findings indicated that the XGBoost model exhibited remarkable predictive accuracy, with an area under the curve (AUC) value of 0.997 in the training set and 0.956 in the validation set. For internal validation, the k-fold cross-validation method was utilized, and the XGBoost model was shown to be steady. Furthermore, the calibration curves demonstrated the XGBoost model’s high predictive capability. The DCA curve revealed higher benefit rates for patients who underwent interventional treatment under the XGBoost model. The AUC value for the external validation set was 0.74, which indicated that the XGBoost prediction model possessed good extrapolative capacity.Conclusion: The myocardial injury prediction model for patients undergoing CME that was developed using the XGBoost machine learning algorithm in this study demonstrates both high predictive accuracy and clinical utility.Keywords: colonic neoplasms, myocardial injury, surgery, prognosis, risk factor, machine learning

Ning Li,1,&ast; Chao Song,2 Xian Huang,3,&ast; Hongjiang Zhang,1 Juan Su,4 Lichun Yang,5 Juhua He,6 Guihua Cui1 1Department of Ultrasound, Anning First People’s Hospital, Kunming City, People’s Republic of China; 2Department of Radiology, Anning First People’s Hospital, Kunming City, People’s Republic of China; 3Department of Ultrasound, Kunming City Maternal and Child Health Hospital, Kunming City, People’s Republic of China; 4Department of Ultrasound, Yulong People’s Hospital, Lijiang City, People’s Republic of China; 5Department of Ultrasound, Yunnan Cancer Hospital, Kunming City, People’s Republic of China; 6Department of Function Examination, Yunnan Provincial Hospital of Traditional Chinese Medicine, Kunming City, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Chao Song, Department of Radiology, Anning First People’s Hospital, Ganghe South Road, Anning City, Kunming City, Yunnan Province, 650302, People’s Republic of China, Tel + 86-13908848395, Email songchaodr@aliyun.com Hongjiang Zhang, Department of Ultrasound, Anning First People’s Hospital, Ganghe South Road, Anning City, Kunming City, Yunnan Province, 650302, People’s Republic of China, Tel +86- 13308809792, Email zhanghj246@163.comBackground: Axillary lymph node dissection (ALND) can be safely avoided in women with T1 or T2 primary invasive breast cancer (BC) and one to two metastatic sentinel lymph nodes (SLNs). However, cancellation of ALND based solely on SLN biopsy (SLNB) may lead to adverse outcomes. Therefore, preoperative assessment of LN tumor burden becomes a new focus for ALN status.Objective: This study aimed to develop and validate a nomogram incorporating the radiomics score (rad-score) based on automated breast ultrasound system (ABUS) and other clinicopathological features for evaluating the ALN status in patients with early-stage BC preoperatively.Methods: Totally 354 and 163 patients constituted the training and validation cohorts. They were divided into ALN low burden (< 3 metastatic LNs) and high burden (≥ 3 metastatic LNs) based on the histopathological diagnosis. The radiomics features of the segmented breast tumor in ABUS images were extracted and selected to generate the rad-score of each patient. These rad-scores, along with the ALN burden predictors identified from the clinicopathologic characteristics, were included in the multivariate analysis to establish a nomogram. It was further evaluated in the training and validation cohorts.Results: High ALN burdens accounted for 11.2% and 10.8% in the training and validation cohorts. The rad-score for each patient was developed based on 7 radiomics features extracted from the ABUS images. The radiomics nomogram was built with the rad-score, tumor size, US-reported LN status, and ABUS retraction phenomenon. It achieved better predictive efficacy than the nomogram without the rad-score and exhibited favorable discrimination, calibration and clinical utility in both cohorts.Conclusion: We developed an ABUS-based radiomics nomogram for the preoperative prediction of ALN burden in BC patients. It would be utilized for the identification of patients with low ALN burden if further validated, which contributed to appropriate axillary treatment and might avoid unnecessary ALND.Keywords: axillary lymph node, sentinel lymph node biopsy, invasive breast cancer, radiomics, nomogram, automated breast ultrasound system, tumor burden

Hongyan Xu, Wenjing Wang, Junlei Yin, Chengcun Song, Lin Li, Zhi Sun Department of Chemotherapy, Weihai Municipal Hospital, Cheeloo College of Medicine, Shandong University, Weihai, People’s Republic of ChinaCorrespondence: Hongyan Xu, Department of Chemotherapy, Weihai Municipal Hospital, No. 248, Tongyi Road, Huancui District, Weihai, Shangdong, 264200, People’s Republic of China, Tel +860631-5287510, Email xuhongyan19701212@163.comAim: To investigate the efficacy and safety of preoperative neoadjuvant therapy (PD-1 inhibitor plus nab-PTX and nedaplatin) for resectable stage III lung squamous cell carcinoma (SCC) patients.Methods: Patients with locally advanced lung SCC (stage IIIA, IIIB) who received PD-1 inhibitor combined with nab-PTX and NED between February 2019 and June 2021 in Weihai Municipal Hospital were included and underwent surgical treatment 4 weeks after 2– 4 cycles neoadjuvant therapy. The rate of resection R0, the effective rate, the complete pathological remission rate (pCR) and the rate of major pathological remission (MPR) were observed.Results: A total of 14 initially unresectable male patients with lung SCC were included and received neoadjuvant treatment after evaluation. Nine out of 14 patients (64.3%) experienced treatment-related adverse events (TRAE), among which 8 (57.1%) experienced grade (G) I–II TRAEs including nausea, vomiting, fatigue, constipation, elevated ALT and AST, hyperthyroidism, hypothyroidism, rash, granulocytopenia, and thrombocytopenia, and 1 (7.1%) experienced grade III–V TRAEs (G), including granulocytopenia and atelectasis. Thirteen patients (92.86%) achieved RECIST-assessed partial remission (PR), while 1 patient (7.14%) achieved stable disease (SD) on imaging assessment after neoadjuvant treatment and continued to be progression-free for 26 months. Of the 11 patients who underwent resection, all were alive and recurrence/progression-free. MPR and pCR were observed in 2 (18.18%) and 9 (81.82%), respectively. IHC results exhibited that all NSCLC patients exhibited positive PD-L1 expression (9/14, TPS ≥ 50% or greater; 5/14, 1% < TPS < 50%). Two were negative for ALK, EGFR, and ros-1, and the rest were not examined for driver oncogene mutation.Conclusion: The neoadjuvant therapy of the PD-1 inhibitor combined with nab-PTX and NED demonstrated remarkable therapeutic efficacy and good safety on stage III lung SCC without increasing the risk of TRAE, mortality and surgery-related complications, or impede surgery feasibility.Keywords: lung squamous cell lung cancer, neoadjuvant therapy, immunotherapy, complete pathological remission, pCR, major pathological remission, MPR

Qun Li,1,2 Fengting Yu,1,2 Chuan Song,1– 3 Hongxin Zhao,1,2 Qing Xiao,1,2 Xiaojie Lao,1,2 Siyuan Yang,1,2 Yunxia Tang,1,2 Fujie Zhang1,2 1Beijing Ditan Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Clinical Center for HIV/AIDS, Capital Medical University, Beijing, People’s Republic of China; 3Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, People’s Republic of ChinaCorrespondence: Fujie Zhang, Clinical and Research Center of Infectious Disease, Beijing Ditan Hospital, Capital Medical University, Beijing, 100015, People’s Republic of China, Tel +86 10 84322581, Email treatment@chinaaids.cnObjective: In this study, we aimed to determine drug-resistance mutations (DRMs) in HIV-1 patients with low-level viremia (LLV) and explored the performance of next-generation sequencing (NGS) in detecting HIV DRMs by using LLV samples.Methods: Overall, 80 samples with LLV were amplified and sequenced using a commercial Sanger sequencing (SS) genotyping kit. Furthermore, 51 samples successfully sequenced using SS were simultaneously subjected to NGS. Genotyping success rates of various viremia categories by two sequencing methods were calculated. Stanford HIV-1 drug-resistance database (HIVdb version 8.9) was used to analyze the DRMs. In the NGS assay, a threshold of 5% was considered for reporting low-frequency variants, and the DRMs detected using SS and NGS were compared.Results: The overall success rate of PR/RT regions was 88.1% (67/80) using SS and 86.3% (44/51) using NGS. Furthermore, a significant linear trend was noted between viral load and the genotyping success rate. A total of 38.8% (26/67) participants harbored at least one mutation, as revealed through SS. Moreover, the prevalence of DRMs in persistent LLV was significantly higher than that in intermittent LLV (62.1% vs. 21.1%; P < 0.05). A total of 69 DRMs were detected using the two sequencing methods at the threshold of 5%. Moreover, 10 DRMs missed by SS were detected using NGS, whereas 8 DRMs missed by NGS were detected by SS.Conclusion: Our data suggested that the genotyping resistance testing is necessary to guide antiretroviral therapy optimization in LLV patients.Keywords: HIV-1, low-level viremia, drug-resistance mutation, next-generation sequencing

Yiqiu Zhang,1,* Chenlu Song,1,* Wei Ni,1,2 Qing Pei,1 Caixia Wang,1 Youguo Ying,3 Min Yao1 1Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 2Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 3Department of Intensive Care Unit, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Yao; Youguo Ying, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, People’s Republic of China, Email my058@vip.sina.com; yingyouguo@sina.comPurpose: Acute kidney injury (AKI) is one of the most severe complications of sepsis, the pathological features of which are excessive inflammation and programmed cell death of resident renal cells. Heat shock protein 70 (HSP70) is a critical stress protein for repressing inflammation, however, its role in AKI is not fully understood. The current study aimed to determine the protective effect of HSP70 on septic AKI and its underlying mechanisms.Methods: Hsp70.1 knockout and wildtype mice were used for creating sepsis model by cecal ligation and puncture (CLP). Renal function, histological changes, pro-inflammatory cytokines, and apoptosis were analyzed with H&E, PAS, ELISA, western-blot, and immunofluorescence. Moreover, the effects of HSP70 on renal proximal tubular epithelial (HK-2) cells with LPS were assessed by measuring the levels of nuclear factor kappa B (NF-κB) signaling and downstream cytokines, viability, and apoptosis using western-blot, qRT-PCR, flow-cytometry, and immunofluorescence. Immunoprecipitate and immunoblotting were used for determining the interaction of HSP70 with tumor necrosis factor receptor-associated factor 6 (TRAF6). Exogenous HSP70 was applied to further identify its biological significance at the cellular and animal level.Results: Hsp70.1 deficiency significantly aggravated renal dysfunction with increasing serum levels of BUN, SCr, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL), and shortened survival in CLP mice. Furthermore, hsp70.1 knockout caused renal-tissue structural damage, especially proximal tubular, and inflammatory cascade and increased apoptotic cells, along with elevated Bax, caspase 3 and cleaved caspase 3, as well as decreased Bcl2 in vivo and vitro. Significantly, HSP70 directly interacted with TRAF6 in HK-2 cells, leading to suppression of inflammatory response and apoptosis. Moreover, exogenous HSP70 alleviated renal damage, decreased apoptosis and elevated survival rate in septic AKI in vivo and vitro.Conclusion: Our findings demonstrated that HSP70 played a critical role in sepsis-induced AKI via interaction with TRAF6 and inhibiting inflammation and apoptosis.Keywords: heat shock protein 70, sepsis, acute kidney injury, tumor necrosis factor receptor-associated factor 6, nuclear factor kappa B, apoptosis

Wenchao Wang, Yue Yuan, Xiaomeng Zhang, Chao Song Beijing Key Laboratory of Applied Experimental Psychology, National Demonstration Center for Experimental Psychology Education (Beijing Normal University), Faculty of Psychology, Beijing Normal University, Beijing, People’s Republic of ChinaCorrespondence: Chao Song, Faculty of Psychology, Beijing Normal University, No. 19 Xinjiekouwai Street, Beijing, 100875, People’s Republic of China, Tel +86 18810161767, Email songchao0010@163.comPurpose: Although the risk effect of perceived discrimination on left-behind youth’s mental health (mainly emotional problems) has been demonstrated in prior studies, there is a lack of longitudinal studies examining the effect of perceived discrimination on behavioral problems, particularly in emerging adults with early left-behind experience. In addition, little is known about individual differences in terms of this association. In the present study, we draw on the social information processing model of aggression to examine the effect of perceived discrimination and mindfulness on cyber aggression.Methods: We used two-wave longitudinal panel data involving 535 emerging adults with early left-behind experience in rural China (M age = 19.89 years; SD = 1.20; 57.2% female). To test the moderating role of mindfulness, hierarchical multiple regression and simple slope test analyses were performed.Results: The results of linear regression analysis demonstrated that higher levels of perceived discrimination at T1 were significantly associated with higher cyber aggression at T2; the strength of this association was buffered by mindfulness over time.Conclusion: These findings suggest that the facilitation of mindfulness training may be an effective strategy for reducing the risk of perceived discrimination leading to cyber aggression in emerging adults with early left-behind experience.Keywords: perceived discrimination, cyber aggression, mindfulness, emerging adults with early left-behind experience

Mengyao Bian,&ast; Wenhao Wang,&ast; Chengjie Song, Lai Pan, Yanmin Wu, Lili Chen Department of Periodontology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Lili Chen, Department of Periodontology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China, Tel +86 571-87784576, Email chenlili_1030@zju.edu.cnPurpose: The goal of this study was to identify the crucial autophagy-related genes (ARGs) in periodontitis and construct mRNA-miRNA-lncRNA networks to further understand the pathogenesis of periodontitis.Methods: We used the Gene Expression Omnibus (GEO) database and Human Autophagy Database (HADb) to identify differentially expressed mRNAs, miRNAs, and ARGs. These ARGs were subjected to Gene Ontology (GO), KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, and PPI (protein–protein interaction) network analysis. Two databases (miRDB and StarBase v2.0) were used to reverse-predict miRNAs while the miRNA-lncRNA interaction was predicted using the StarBase v2.0 and LncBase Predicted v.2 databases. After excluding the lncRNAs only present in the nucleus, a competing endogenous RNA (ceRNA) network was built. Finally, we used quantitative real-time PCR (qRT-PCR) to confirm the levels of mRNA expression in the ceRNA network.Results: The differential expression analysis revealed 10 upregulated and 10 downregulated differentially expressed ARGs. After intersecting the reverse-predicted miRNAs with the differentially expressed miRNAs, a ceRNA network consisting of 4 mRNAs (LAMP2, NFE2L2, NCKAP1, and EGFR), 3 miRNAs (hsa-miR-140-3p, hsa-miR-142-5p, and hsa-miR-671-5p), and 30 lncRNAs was constructed. In addition, qRT-PCR results revealed that EGFR expression was downregulated in diseased gingival tissue of periodontitis patients.Conclusion: Four autophagy-related genes, especially EGFR, may play a key role in periodontitis progression. The novel ceRNA network may aid in elucidating the role and the mechanism of autophagy in periodontitis, which could be important in developing new therapeutic options.Keywords: periodontitis, autophagy, ceRNA, bioinformatics analysis, EGFR

Changho Song,1 Yongho Lee,2 Shin Kim3– 5 1Department of Obstetrics and Gynecology, School of Medicine, Keimyung University, Daegu, 42601, Republic of Korea; 2Department of Anesthesiology and Pain Medicine, Keimyung University Dongsan Hospital, Daegu, 42601, Republic of Korea; 3Department of Immunology, School of Medicine, Keimyung University, Daegu, 42601, Republic of Korea; 4Institute of Medical Science, Keimyung University, Daegu, 42601, Republic of Korea; 5Institute for Cancer Research, Keimyung University Dongsan Medical Center, Daegu, 42601, Republic of KoreaCorrespondence: Shin Kim, 1095 Dalgubeol-Daero, Dalseo-gu, Daegu, 42601, Republic of Korea, Tel +82-53-258-7359, Fax +82-53-258-7355, Email god98005@dsmc.or.krBackground: Cervical cancer is one of the leading causes of death in women. Among the sodium ion channels associated with cancer development, voltage gated sodium channel plays an important role in pathophysiology of cervical cancer; however, the clinicopathological implication of epithelial sodium channel (ENaC) has not been explored.Purpose: This study focused on identifying dysregulation of ENaC encoding genes, including SCNN1A, SCNN1B, and SCNN1G, and their relationship with clinicopathologic features in cervical cancer patients.Materials and Methods: RNA sequencing data of ENaC-encoding genes, clinicopathologic data, and survival data of cervical cancer patients were obtained from The Cancer Genome Atlas cohort. Microarray data of ENaC-encoding genes were obtained from Gene Expression Omnibus datasets: GSE6791 and GSE63514.Results: The expression levels of SCNN1A, SCNN1B, and SCNN1G were positively correlated with each other. SCNN1A, SCNN1B, and SCNN1G are significantly overexpressed in normal tissues than in tumor tissues. Survival analysis showed that simultaneous overexpression of all three genes associated with better overall survival (OS). Each overexpression of SCNN1B and SCNN1G was significantly associated with better OS. Moreover, each expression level of SCNN1A, SCNN1B, and SCNN1G was negatively correlated with histologic grade of tumor.Conclusion: ENaC-encoding genes might be potential biological markers to better predict survival outcomes in cervical cancer patients.Keywords: sodium ion channel, cervical cancer, survival outcome, ENaC-encoding genes

Xin Hua,1,&ast; Fangfang Duan,1,&ast; Wenyu Zhai,2,&ast; Chenge Song,1 Chang Jiang,1 Li Wang,1 Jiajia Huang,1 Huanxin Lin,3 Zhongyu Yuan1 1Department of Medical Oncology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China; 2Department of Thoracic Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China; 3Department of Radiotherapy, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhongyu YuanDepartment of Medical Oncology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, 510060, People’s Republic of China, Email yuanzhy@sysucc.org.cnHuanxin LinDepartment of Radiotherapy, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, 510060, People’s Republic of China, Email linhx@sysucc.org.cnPurpose: We attempted to explore the prognostic value of baseline inflammatory and nutritional biomarkers at diagnosis in patients with early-stage breast cancer and develop a novel scoring system, the inflammatory-nutritional prognostic score (INPS).Patients and Methods: We collected clinicopathological and baseline laboratory data of 1259 patients with early-stage breast cancer between December 2010 and November 2012 from Sun Yat-sen University Cancer Center. Eligible patients were randomly divided into training and validation cohorts (n = 883 and 376, respectively) in a 7:3 ratio. We selected the most valuable biomarkers to develop INPS by the least absolute shrinkage and selection operator (LASSO) Cox regression model. A prognostic nomogram incorporating INPS and other independent clinicopathological factors was developed based on the stepwise multivariate Cox regression method. Then, we used the concordance index (C-index), calibration plot, and time-dependent receiver operating characteristic (ROC) analysis to evaluate the prognostic performance and predictive accuracy of the predictive nomogram.Results: Four inflammatory-nutritional biomarkers, including neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR), prognostic nutritional index (PNI), and albumin-alkaline phosphatase ratio (AAPR), were selected using the LASSO Cox analysis to construct INPS, which remained an independent prognostic indicator per the multivariate Cox regression analysis. Patients were stratified into low- and high-INPS groups based on the cutoff INPS determined by the maximally selected rank statistics. The prognostic model for overall survival consisting of INPS and other independent clinicopathological indicators showed excellent discrimination with C-indexes of 0.825 (95% confidence interval [CI]: 0.786– 0.864) and 0.740 (95% CI: 0.657– 0.822) in the training and validation cohorts, respectively. The time-dependent ROC curves showed a higher predictive accuracy of our prognostic nomogram than that of traditional tumor-node-metastasis staging.Conclusion: Baseline INPS is an independent indicator of OS in patients with early-stage breast cancer. The INPS-based prognostic nomogram could be used as a practical tool for individualized prognostic predictions.Keywords: early-stage breast cancer, LASSO Cox analysis, inflammatory-nutritional biomarker, prognostic nomograms, overall survival

Jing Yuan,1,&ast; Fangfang Duan,2,&ast; Wenyu Zhai,3,&ast; Chenge Song,2 Li Wang,2 Wen Xia,2 Xin Hua,2 Zhongyu Yuan,2 Xiwen Bi,2 Jiajia Huang2 1Departments of Experimental Research, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China; 2Departments of Medical Oncology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China; 3Departments of Thoracic Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jiajia Huang; Xiwen BiDepartment of Medical Oncology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, 510060, People’s Republic of ChinaEmail huangjiaj@sysucc.org.cn; bixw@sysucc.org.cnBackground: Aging, an inevitable process characterized by functional decline over time, is a significant risk factor for various tumors. However, little is known about aging-related genes (ARGs) in breast cancer (BC). We aimed to explore the potential prognostic role of ARGs and to develop an ARG-based prognosis signature for BC.Methods: RNA-sequencing expression profiles and corresponding clinicopathological data of female patients with BC were obtained from public databases in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). An ARG-based risk signature was constructed in the TCGA cohort based on results of least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis, and its prognostic value was further validated in the GSE20685 cohort.Results: A six ARG-based signature, including CLU, DGAT1, MXI1, NFKBI, PIK3CA and PLAU, was developed in the TCGA cohort and significantly stratified patients into low- and high-risk groups. Patients in the former group showed significantly better prognosis than those in the latter. Multivariate Cox regression analysis demonstrated that the ARG risk score was an independent prognostic factor for BC. A predictive nomogram integrating the ARG risk score and three identified factors (age, N- and M-classification) was established in the TCGA cohort and validated in the GSE20685 cohort. Calibration plots showed good consistency between predicted survival probabilities and actual observations.Conclusion: A novel ARG-based risk signature was developed for patients with BC, which can be used for individual prognosis prediction and promoting personalized treatment.Keywords: breast cancer, aging, prognostic signature, risk stratification

Zhiying Pang,&ast; Zengxin Jiang,&ast; Runwen Zhu, Chunfeng Song, Han Tang, Lu Cao, Changan Guo Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Changan Guo; Lu CaoDepartment of Orthopedic Surgery, Zhongshan Hospital, Fudan University, NO. 180 Feng Lin Road, Xuhui District, Shanghai, 200032, People’s Republic of ChinaTel +86 13681975321; +86 13482794964Email changanguo@hotmail.com; caolu922@126.comPurpose: Oxidative stress-induced chondrocyte apoptosis and extracellular matrix (ECM) degradation plays an important role in the progression of osteoarthritis (OA). Bardoxolone methyl (BM), a semisynthetic triterpenoid, exerts strong effect against oxidative stress. The purpose of the present study was to determine the effectiveness of bardoxolone-methyl (BM) in preventing oxidative stress-induced chondrocyte apoptosis and extracellular ECM degradation in vitro and the role of alleviating OA progression in vivo.Methods: Oxidative damage was induced by 25 mM tert-butyl hydroperoxide (TBHP) for 24 h in rat chondrocytes. 0.025 and 0.05 μM bardoxolone-methyl (BM) were used in vitro treatment. Ex-vivo cartilage explant model was established to evaluate the effect of BM on oxidative stress-induced ECM degradation. The mouse OA model was induced by surgical destabilization of the medial meniscus.Results: In vitro, 0.025 and 0.05 μM BM reduced TBHP-induced excessive ROS generation, improved cell viability, increased malondialdehyde level and decreased superoxide dismutase level. 0.025 and 0.05 μM BM prevented TBHP-induced mitochondrial damage and apoptosis in chondrocytes BM activated heme oxygenase-1 (HO-1)/NADPH quinone oxidoreductase 1 (NOQ1) signaling pathway through targeting nuclear factor erythroid derived-2-related factor 2 (Nrf2). Additionally, BM treatment enhanced the expression levels of aggrecan and collagen II and inhibited the expression levels of matrix metalloproteinase 9 (MMP 9), MMP 13, Bax and cleaved-caspase-3. BM increased proteoglycan staining area and IOD value in ex vivo cultured experiment cartilage explants and improved the OARSI score, stands, max contact mean intensity, print area and duty cycle in mouse OA model.Conclusion: BM prevented oxidative stress-induced chondrocyte apoptosis and ECM degradation in vitro and alleviated OA in vivo, suggesting that BM serves as an effective drug for treatment with OA.Keywords: osteoarthritis, chondrocyte apoptosis, extracellular matrix degradation, oxidative stress, bardoxolone-methyl

Haitao Li,1 Yi Li,2 Chao Song,2 Yongbin Hu,3 Minhui Dai,2 Ben Liu,4 Pinhua Pan2 1First Department of Thoracic Medicine, Hunan Cancer Hospital/The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha City, Hunan Province, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, National Key Clinical Specialty, Xiangya Hospital, Central South University, Changsha City, Hunan Province, People’s Republic of China; 3Department of Pathology, Xiangya Hospital, Central South University, Changsha City, Hunan Province, People’s Republic of China; 4Department of Emergency, Xiangya Hospital, Central South University, Changsha City, Hunan Province, People’s Republic of ChinaCorrespondence: Pinhua PanDepartment of Respiratory and Critical Care Medicine, National Key Clinical Specialty, Xiangya Hospital, Central South University, Changsha City, Hunan Province, People’s Republic of ChinaEmail pinhuapan668@csu.edu.cnBackground: Uncontrollable inflammation is a critical feature of gram-negative bacterial pneumonia-induced acute respiratory distress syndrome (ARDS). Both neutrophils and alveolar macrophages participate in inflammation, but how their interaction augments inflammation and triggers ARDS is unclear. The authors hypothesize that neutrophil extracellular traps (NETs), which are formed during neutrophil NETosis, partly cause alveolar macrophage pyroptosis and worsen the severity of ARDS.Methods: The authors first analysed whether NETs and caspase-1 are involved in clinical cases of ARDS. Then, the authors employed a lipopolysaccharide (LPS)-induced ARDS model to investigate whether targeting NETs or alveolar macrophages is protective. The AIM2 sensor can bind to DNA to promote AIM2 inflammasome activation, so the authors studied whether degradation of NET DNA or silencing of the AIM2 gene could protect alveolar macrophages from pyroptosis in vitro.Results: Analysis of aspirate supernatants from ARDS patients showed that NET and caspase-1 levels were correlated with the severity of ARDS and that the levels of NETs and caspase-1 were higher in nonsurvivors than in survivors. In vivo, the NET level and proportion of pyroptotic alveolar macrophages in bronchoalveolar lavage fluid (BALF) were obviously higher in LPS-challenged mice than in control mice 24 h after injury. Administration of DNase I (a NET DNA-degrading agent) and BB-Cl-amidine (a NET formation inhibitor) alleviated alveolar macrophage pyroptosis, and Ac-YVAD-cmk (a pyroptosis inhibitor) attenuated NET levels in BALF and neutrophil infiltration in alveoli. All treatments markedly attenuated the severity of ARDS. Notably, LPS causes NETs to induce alveolar macrophage pyroptosis, and degradation of NET DNA or silencing of the AIM2 gene protected against alveolar macrophage pyroptosis.Conclusion: These findings shed light on the proinflammatory role of NETs in mediating the neutrophil–alveolar macrophage interaction, which influences the progression of ARDS.Keywords: ALI/ARDS, NETs, macrophage pyroptosis, AIM2 inflammation

Fangfang Duan,&ast; Chenge Song,&ast; Yuyu Ma, Kuikui Jiang, Fei Xu, Xiwen Bi, Jiajia Huang, Ruoxi Hong, Zhangzan Huang, Qianyi Lu, Zhongyu Yuan, Shusen Wang, Wen Xia Departments of Medical Oncology, Sun Yat-sen University Cancer Center, The State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Wen XiaDepartment of Medical Oncology, Sun Yat-sen University Cancer Center, The State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng Road East, Guangzhou, Guangdong, 510060, People’s Republic of ChinaEmail xiawen@sysucc.org.cnBackground: There are no clinically available prognostic models for patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer treated with everolimus. We aimed to develop a tool to predict the progression-free survival (PFS) and overall survival (OS) of these patients and to identify optimal candidates who would benefit from everolimus-based treatment in this heterogeneous patient population.Methods: The clinical data of patients with HR+, HER2- metastatic breast cancer receiving everolimus between May 2012 and January 2018 at Sun Yat-sen University Cancer Center were retrospectively retrieved. Based on potential prognostic factors derived from multivariate Cox analysis, we established predictive nomogram models for PFS and OS and evaluated their predictive values by means of the concordance index (C-index). Calibration curves were used to estimate the consistency between the actual observations and the nomogram-predicted probabilities.Results: A total of 116 patients with HR+, HER2- metastatic breast cancer were enrolled in this study. Three independent prognostic factors, including the line of everolimus in the metastatic setting, everolimus clinical benefit rate and number of liver metastatic lesions, were identified from the multivariate Cox analysis. Prognostic models for individual survival prediction were established and graphically presented as nomograms. The C-index was 0.738 (95% confidence interval [CI]: 0.710– 0.767) for the PFS nomogram and 0.752 (95% CI: 0.717– 0.788) for the OS nomogram, which showed favourable discrimination. The calibration curves for the probabilities of 6-, 9-, and 12-month PFS and 1-, 2-, and 3-year OS suggested satisfactory consistency between the actual observations and the predicted probabilities.Conclusion: We constructed convenient nomogram models for patients with HR+, HER2- metastatic breast cancer to individually predict their potential benefits from everolimus in the metastatic setting. The models showed good performance in terms of accuracy, discrimination capacity and clinical application value.Keywords: prognostic nomogram, metastatic breast cancer, everolimus, progression-free survival, overall survival

Cheng Zhou,1 Chunlin Song,1 Xiang Huang,1 Shufen Chen,1 Yan Long,2 Shanshui Zeng,2 Hongling Yang,2 Min Jiang2 1Laboratory of Molecular Diagnostics, Southern Medical University Affiliated Maternal & Child Health Hospital of Foshan, Foshan, 528000, People’s Republic of China; 2Department of Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou, 510623, People’s Republic of ChinaCorrespondence: Hongling Yang; Min JiangDepartment of Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, No. 9, Jinsui Road, Guangzhou, 510623, People’s Republic of ChinaTel +86-20-38857723; +86-20-38076256Email hlyang62@163.com; jiangmin2011@126.comBackground: Gestational hypertension (GH), a hypertensive disorder of pregnancy (HDP), is a leading cause of maternal and fetal mortality due to the lack of clarity on its exact etiology and clinically feasible prediction models. This study was performed to discover novel biomarkers before 20 weeks gestation and thereby construct an early GH prediction model.Methods: This study was designed based on differentially expressed protein screening followed by clinical validation. In the screening phase, a nested case-controlled study was conducted by plasma proteomic analyses using label-free LC-MS/MS and plasma samples from seven pre-GH cases before 20-week gestation and seven age- and gestational week-matched controls. In the validation phase, 10 proteins with differential expression in the screening phase were validated by ELISA or electrochemiluminescence in an independent study consisting of 29 pre-GH cases before 20-week gestation and 29 matched controls.Results: In the screening phase, 149 proteins were found to be differentially expressed between the two groups and were predominantly involved in complement and coagulation cascades, platelet degranulation and positive regulation of cell motility. Further validation showed that serpin family C member 1 (SERPINC1), serpin family A member 5 (SERPINA5), complement factor H-related protein 5 (CFHR5), clusterin, cytokeratin 18 (CK18) and histidine-rich glycoprotein (HRG) levels were significantly higher in women who later developed GH compared to women with uncomplicated pregnancies (P< 0.05). Binary logistic regression analysis was used to determine the combination efficacy of models for early prediction of GH. The model with a combination of SERPINC1, CK18 and HRG had a significantly better discriminatory power (AUC = 0.91, 95% CI 0.83– 0.98) compared to the models with those proteins alone as independent predictors of GH.Conclusion: Plasma levels of SERPINC1, SERPINA5, CFHR5, clusterin, CK18 and HRG are potential novel predictive biomarkers of GH, and a prediction model using a combination of SERPINC1, CK18 and HRG has good discriminatory performance for GH before 20 weeks gestation.Keywords: label-free LC-MS/MS, gestational hypertension, biomarkers

Peng Chen,1 Dongsheng Lin,2 Chen Wang,2 Cuiwen Song,1 Wenjing Wang,1 Jinglian Qu,1 Zhibing Wu2 1Basic Medical School, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou, People’s Republic of China; 2First Clinical Medical School, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of ChinaCorrespondence: Zhibing WuDepartment of Neurology, First Clinical Medical College of Guangzhou University of Chinese Medicine, 12 Jichang Road, Guangzhou, Guangdong, People’s Republic of ChinaTel +86 020 36585577Email wuzhibing2021@126.comBackground: Neuropathic pain (NP) is a syndrome of pain mediated by distinct pathophysiological processes, and current treatments are not fully satisfactory. Emodin is an effective component of Chinese traditional medicine and has an alleviating effect on NP, but the pharmacological mechanism is not clear.Methods: We used isobaric tags for relative and absolute quantitation (iTRAQ) technique integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify potential targets of emodin in a rat peripheral nerve chronic constriction injury (CCI) model.Results: A total of 177 differentially expressed proteins were identified among the sham group, CCI group, and emodin group, with a threshold of 1.2-fold change and a P value ≤ 0.05. Among them, 100 differentially expressed proteins (51 up-regulated and 49 down-regulated) were identified in the CCI group compared with sham group. Moreover, 108 differentially expressed proteins (65 up-regulated and 43 down-regulated) were identified in the emodin group with the CCI group as reference. The enrichment analysis of Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed an important role of calcium signaling pathway, neurotransmitter regulation, and long-term potentiation (LTP) in emodin-treated CCI model. Real-time quantitative fluorescence PCR (qRT-PCR) and Western blot analysis revealed that emodin decreased expression of calcium signaling related proteins, including calmodulin (CaM) dependent protein kinase II (CaMK II), phospholipase Cβ 1 (PLCβ 1), protein kinase C (PKC), protein kinase C (PKA), and tropomyosin-related kinase B (TrkB), compared with the CCI group.Conclusion: Overall, these findings indicated that emodin might alleviate NP by regulating the calcium signaling pathway.Keywords: neuropathic pain, emodin, CCI, iTRAQ, KEGG

Chenyu Song,1,* Bingqi Shen,1,* Zhi Dong,1,* Zhenzhen Fan,2 Ling Xu,3 Zi-Ping Li,1 Yin Li,4 Shi-Ting Feng1 1Department of Radiology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510080, People’s Republic of China; 2Department of Radiology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, Henan 471009, People’s Republic of China; 3Faculty of Medicine and Dentistry, University of Western Australia, Perth, Australia; 4Department of Gastroenterology Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, 510080, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shi-Ting FengDepartment of Radiology, The First Affiliated Hospital, Sun Yat-Sen University, 58th, the Second Zhongshan Road, Guangzhou, Guangdong, People’s Republic of ChinaTel +86 20-87755766 Extension 8471Fax +86 20-87615805Email fengsht@mail.sysu.edu.cnYin LiDepartment of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510080, People’s Republic of ChinaEmail liyin2736@163.comBackground: The purpose of this study was to investigate the feasibility of CT parameters to predict the presence of KRAS mutations in rectal cancer patients. The relationship between the presence of a KRAS mutation and pathological findings was evaluated simultaneously.Methods: Eighty-nine patients (29 females, 60 males, age 27– 90, mean 59.7± 12 years) with pathologically proven rectal cancer were enrolled. A KRAS mutation test was completed following surgery. Parameters evaluated on CT included the tumor location, the diameter of the superior rectal vein (SRV) and inferior mesenteric vein (IMV), the presence of calcification, ulceration, lymph node enlargement (LNE), distant metastasis, tumor shape (intraluminal polypoid mass, infiltrative mass, or bulky), circumferential extent (C0–C1/4, C1/4–C1/2, C1/2–C3/4, or C3/4–C1), enhanced pattern (homogeneous or heterogeneous), CT ratio, and the length of the tumor (LOT). Pathological findings included lymphovascular emboli, signet ring cell, peripheral fat interval infiltration, focal ulcer, lymph node metastasis, tumor pathological type, and differentiation extent. The correlations between KRAS status and CT parameters, and KRAS status and pathological findings were investigated. The accuracy of CT characteristics for predicting KRAS mutation was evaluated.Results: A KRAS mutation was detected in 42 cases. On CT image, the diameter of the SRV was significantly increased in the KRAS mutation group compared to in the KRAS wild-type group (4.6± 0.9 mm vs 4.2± 0.9 mm, p=0.02), and LNE was more likely to occur in the KRAS mutation group (73.3% vs 26.7%, p=0.03). There was no significant difference between the KRAS mutation group and the KRAS wild-type group on the other CT parameters (location, IMV, calcification, ulcer, distant metastasis, tumor shape, enhanced pattern, circumferential extent, CT ratio, and LOT). In the pathological findings, a KRAS mutation was more likely to occur in the middle differentiation group (p=0.03). No significant difference was found between the KRAS mutation group and the KRAS wild-type group in the presence of lymphovascular emboli, signet ring cell, peripheral fat interval infiltration, focal ulcer, lymph node metastasis, and tumor pathological type. With the best cut-off value of 4.07 mm, the AUC of the SRV to predict a KRAS mutation was 0.63 with a sensitivity of 76.2% and a specificity of 48.9%.Conclusion: It was feasible to use the diameter of the SRV to predict a KRAS mutation in rectal cancer patients, and LNE also can be regarded as an important clue on preoperative CT images.Keywords: rectal neoplasms, mutation, computed tomography, superior rectal vein

Ning Li,1 Ju-hua He,2 Chao Song,3 Li-chun Yang,4 Hong-jiang Zhang,1 Zhi-hai Li1 1Department of Ultrasound, Yunnan Kungang Hospital, Kunming, Yunnan Province, People’s Republic of China; 2Department of Function Examination, Yunnan Provincial Hospital of Traditional Chinese Medicine, Kunming, Yunnan Province, People’s Republic of China; 3Department of Radiology, Yunnan Kungang Hospital, Kunming, Yunnan Province, People’s Republic of China; 4Department of Ultrasound, Yunnan Cancer Hospital, Kunming, Yunnan Province, People’s Republic of ChinaCorrespondence: Chao SongDepartment of Radiology, Yunnan Kungang Hospital, Kunming, Yunnan Province 650302, People’s Republic of ChinaTel +86-13908848395Email songchaodr@aliyun.comLi-chun YangDepartment of Ultrasound, Yunnan Cancer Hospital, Kunming, Yunnan Province 650118, People’s Republic of ChinaTel +86-13888087336Email yangchunli321@126.comBackground: It is controversial whether contralateral prophylactic central neck dissection (PCND) should be performed for patients with solitary and clinical lymph node negative (cN0) papillary thyroid carcinoma (PTC) although routine ipsilateral PCND is required.Objective: The aim of this study was to develop an improved nomogram including clinical features, ultrasound, and acoustic radiation force impulse (ARFI) elastography for the prediction of contralateral central lymph node metastasis (CLNM) in patients with solitary and cN0 PTC in the preoperative period.Materials and Methods: A total of 340 patients were retrospectively included as the training cohort and 170 patients as the external validation cohort. Patients were grouped according to the pathological results of contralateral CLNM. The association between the clinical characteristics, ultrasound, and ARFI elastography and the risk for contralateral CLNM were analyzed. A nomogram was established based on the result of multivariable logistic analysis to predict the risk of contralateral CLNM, which was assessed by internal and external validation.Results: CLNM was found in 213 patients (41.8%), among whom 142 (27.8%) had ipsilateral CLNM and 95 (18.6%) had contralateral CLNM (including 68 (13.3%) with bilateral CLNM). Multivariable analysis revealed that patients with younger age, male gender, larger tumor size, closer distance from the capsule, microcalcification, and larger SWVmean were independent predictors associated with the contralateral CLNM (P < 0.05), which was served as the basis of the nomogram. It showed good discrimination (C-index: 0.856) and calibration (χ2 = 9.028, P = 0.340, Hosmer–Lemeshow test) in the training cohort, and good discrimination was maintained in the external validation cohort (C-index: 0.792).Conclusion: The nomogram utilizing the features of ultrasound combined with ARFI elastography in preoperatively predicting the risk of contralateral CLNM in patients with solitary and cN0 PTC was established, which showed superior performance both in internal and external validation.Keywords: prophylactic central neck dissection, papillary thyroid carcinoma, central lymph node metastasis, acoustic radiation force impulse, nomogram

Mohammadhossein Hajiebrahimi,1,2 Ci Song,3 David Hägg,1 Therese M-L Andersson,4 Reginald Villacorta,5 Marie Linder1 1Centre for Pharmacoepidemiology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 2Department of Statistics and Epidemiology, Public Health Faculty, Golestan University of Medical Sciences, Gorgan, Iran; 3Janssen GCSO, Stockholm, Sweden; 4Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; 5Market Access Department, Janssen Pharmaceuticals, Horsham, PA, USACorrespondence: Mohammadhossein Hajiebrahimi Email mohammadhossein.hajiebrahimi@synergusrwe.comPurpose: To assess the relative risk of metabolic risk factors in patients stratified by psoriasis severity compared with population controls.Patients and Methods: A retrospective cohort study was conducted using national Swedish registers. Adult patients with psoriasis were selected if they had a dispensing of anti-psoriasis prescription (2007– 2013) and at least one diagnosis within five years before the dispensing date. The patients with psoriasis were matched 1:10 to controls from the general population on birth year, sex, and county. The cohort was further divided into three disease severity groups (mild, moderate, or severe) based on their dispensed anti-psoriasis medication. Subjects were followed from the index date until censoring. We applied flexible parametric modeling to understand the risks of the incident comorbidities hypertension, hypercholesterolemia, and diabetes mellitus among patients with psoriasis from 6 months through 10 years. Hazard ratios and predicted risk (ie, 1 minus the survival probability) of comorbidities were reported.Results: The hazard of hypertension, hypercholesterolemia, and diabetes mellitus is higher among psoriasis patients compared with population controls, and the hazard ratio increases with psoriasis severity. For example, HRs of hypertension for patients with mild, moderate, and severe psoriasis are 1.29 (95% CI: 1.27– 1.32), 1.35 (95% CI: 1.32– 1.38), and 1.73 (95% CI: 1.64– 1.82), respectively. The predicted risk of hypertension, hypercholesterolemia, and diabetes mellitus among patients with severe psoriasis at year ten was 0.58 (95% CI: 0.56, 0.59), 0.33 (95% CI: 0.32, 0.35), and 0.21 (95% CI: 0.20, 0.23), respectively, while it was 0.42 (0.41, 0.43), 0.23 (0.22, 0.23), 0.11 (0.10, 0.11) among controls, respectively. The predicted risk at year ten was similar among patients with mild or moderate psoriasis.Conclusion: The HRs and predicted risks of metabolic risk factors are higher among patients with psoriasis compared with matched controls and are more prominent among the severe psoriasis group.Keywords: psoriasis severity, predicted risk, survival probability, population-based, metabolic risk factors

Can Liu,1 Chao Song,2 Jiaxi Li,3 Qing Sun1,3 1Department of Pathology, Shandong Provincial Qianfoshan Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong Province, People’s Republic of China; 2Department of Pathology, Zibo Central Hospital, Zibo, Shandong Province, People’s Republic of China; 3Department of Pathology, Shandong Provincial Qianfoshan Hospital, The First Hospital Affiliated with Shandong First Medical University, Jinan, Shandong Province, People’s Republic of ChinaCorrespondence: Qing SunDepartment of Pathology, Shandong Provincial Qianfoshan Hospital, Cheeloo College of Medicine, Shandong University, Jingshi Street, Jinan 250014, Shandong Province, People’s Republic of ChinaTel +86-531-89269710Email sun18653130364@163.comPurpose: Cystic fibrosis transmembrane conductance regulator (CFTR) was shown to be downregulated or silenced in carcinomas and acts as a candidate tumor suppressor gene. However, the function of CFTR gene in colorectal cancer (CRC) is still unclear. This aim of this study was to investigate the CFTR promoter methylation status and its impact on the expression and functional role of CFTR in CRC development.Patients and Methods: CFTR expression in CRC tissues and CRC cell lines was detected via quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The promoter methylation status of CFTR was measured using methylation-specific PCR (MSP). colony formation, transwell, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to evaluate the effect of CFTR overexpression in CRC cell lines.Results: qRT-PCR and IHC results indicated that CFTR expression was downregulated in the CRC tissues compared to the adjacent normal tissues. The promoter methylation status of CFTR was further analyzed in 70 CRC specimens. MSP validation showed methylation of CFTR promoter in 62.2% (45/70) of CRC tissues. The methylation of CFTR promoter was significantly associated with age (P=0.013) and lymph node metastasis (P=0.026) in CRC tissues. Results of transwell, MTT, and colony formation assays showed that CFTR overexpression inhibited the migration, invasion, and proliferation of CRC cells.Conclusion: CFTR expression was downregulated in CRC and promoter methylation may be responsible for this downregulation. Overexpression of CFTR may suppress CRC tumor growth by inhibiting the proliferation, migration, and invasion of CRC cells. CFTR promoter methylation was significantly correlated with lymph node metastasis; thus, CFTR may be a potential marker for lymph node metastasis of CRC.Keywords: CFTR, DNA methylation, colorectal cancer, lymph node metastasis

Chuanhui Song, 1, 2,* Chuanchao Tang, 1, 2,* Wenguang Xu, 2 Jianchuan Ran, 1, 2 Zheng Wei, 1, 3 Yufeng Wang, 1, 2 Huihui Zou, 1, 2 Wei Cheng, 4 Yu Cai, 1 Wei Han 1, 2 1Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing 210008, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing 210008, People’s Republic of China; 3Pediatric Dentistry, Nanjing Stomatology Hospital, Medical School of Nanjing University, Nanjing 210008, People’s Republic of China; 4Department of Oral Implantology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing 210008, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wei ChengDepartment of Oral Implantology, Nanjing Stomatological Hospital, Medical School of Nanjing University, No. 30 Zhongyang Road, Nanjing 210008, People’s Republic of ChinaTel +86-025-83620238Email dentist_nj@163.com Wei HanDepartment of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School of Nanjing University, No 30 Zhongyang Road, Nanjing 210008, People’s Republic of ChinaTel +86-025-83620349Email doctorhanwei@hotmail.comPurpose: Chemotherapy in head and neck squamous cell carcinoma (HNSCC) has many systemic side effects, as well as hypoxia-induced chemoresistance. To reduce side effects and enhance chemosensitivity are urgently needed.Methods: We synthesized a drug delivery system (named CECMa NPs) based on cisplatin (CDDP) and metformin (chemotherapeutic sensitizer), of which chlorin e6 (Ce6) and polyethylene glycol diamine (PEG) were synthesized as the shell, an anti-LDLR antibody (which can target to hypoxic tumor cells) was modified on the surface to achieve tumor targeting.Results: The NPs possessed a great synergistic effect of chemotherapy and phototherapy. After laser stimulation, both CDDP and metformin can be released in situ to achieve anti-tumor effects. Meanwhile, PDT and PTT triggered by a laser have anticancer effects. Furthermore, compared with free cisplatin, CECMa exhibits less systemic toxicity with laser irradiation in the xenograft mouse tumor model.Conclusion: CECMa effectively destroyed the tumors via hypoxia targeting multimodal therapy both in vitro and in vivo, thereby providing a novel strategy for targeting head and neck squamous cell carcinoma.Keywords: cisplatin, metformin, chlorin e6, head and neck squamous cell carcinoma, nano-delivery

Chengcheng Song,1–3 Peng Liu,3,4 Qi Zhao,1–3 Suqian Guo,1–3 Guolin Wang1–3 1Tianjin Research Institute of Anesthesiology, Tianjin, China; 2Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin, China; 3Tianjin Medical University, Tianjin, China; 4Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China Background: Remifentanil is widely used in general anesthesia due to its reliability and rapid onset. However, remifentanil-induced postoperative hyperalgesia might be a challenge nowadays. Accumulating evidence suggests that the transient receptor potential vanilloid 1 (TRPV1) was involved in the development of neuropathic pain and hyperalgesia. However, the contribution of TRPV1 in modulating remifentanil-induced postoperative hyperalgesia is still unknown. The aim of this study is the contribution of TRPV1 to the surface expression of N-methyl-d-aspartate (NMDA) receptors in remifentanil-induced postoperative hyperalgesia.Methods: The hot plate test and the Von Frey test were performed to evaluate thermal and mechanical hyperalgesia. Capsazepine (CPZ) was administrated intrathecally to confirm our results. TRPV1, NMDA receptors, CaMKII (calcium/calmodulin-dependent kinase II), and protein kinase C (PKC) in the dorsal root ganglion (DRG) were detected by Western blotting. Immunofluorescence assay was applied to analyze the distribution of TRPV1 and the relationship between TRPV1 and NMDA receptor subunit 1 (NR1).Results: Remifentanil-induced both thermal and mechanical postoperative hyperalgesia. Here, we found the membrane trafficking of NR1, possibly due to the activation of TRPV1 in DRG neurons after remifentanil infusion. Furthermore, intrathecal injection of CPZ was able to relieve remifentanil-induced postoperative hyperalgesia according to a behavioral test and CPZ confirmed that TRPV1 is involved in NR1 trafficking. In addition, CaMKII/PKC but not protein kinase A (PKA) contributed to remifentanil-induced postoperative hyperalgesia.Conclusion: Our study demonstrates that TRPV1 receptors are involved in remifentanil-induced postoperative hyperalgesia. TRPV1 contributes to the persistence of remifentanil-induced postoperative hyperalgesia through the trafficking of NMDA receptors via the activation of CaMKII-PKC signaling pathways in DRG neurons. Keywords: opioid, pain, TRPV1, NMDA receptor, CaMKII

Siegfried Wahl,1,2 Chengyang Song,1 Arne Ohlendorf1,2 1Institute for Ophthalmic Research, Eberhard Karls University Tuebingen, Tuebingen, Baden-Wuerttemberg, Germany; 2Technology and Innovation, Carl Zeiss Vision International GmbH, Aalen, Baden-Wuerttemberg, Germany Introduction: The simulation of vision with intraocular lenses (IOLs) prior to the clinical validation is of great interest for manufacturers as well as clinicians. We have tested the influence of different IOLs on the perception of contrast using different devices.Materials and methods: A Rassow telescope and the VirtIOL have been used to assess the contrast sensitivity function (CSF) through monfocal IOL (CT Asphina 409 MP, IOL 1), multifocal IOL (AT LISA tri 839 MP, IOL 2) and extended-depth-of-focus-IOL (TECNIS Symfony ZXR00, IOL3) in 21 participants, aged between 22 and 29 years. Contrast sensitivity (CS) was tested at various spatial frequencies (3–30 cycles per degree), using the Tuebingen contrast sensitivity test.Results: All tested IOL reduced the CS when compared to measurements with a trial lens correction of refractive errors. The analysis of the area under the curve of the contrast sensitivity function (AUC-CSF) revealed a significant reduction compared to the trial lens correction in case the Rassow telescope was used (IOL 1: P=0.008; IOL2: P

Qianqian Zhou,1,* Chao Song,1,* Xiaoqiu Liu,2,3,* Hao Qin,1 Lixia Miao,1 Xuesen Zhang1 1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China; 2Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, China; 3Department of Microbiology, Nanjing Medical University, Nanjing, China *These authors contributed equally to this work Background: Adriamycin (ADR) is widely used in the clinical chemotherapy against breast cancer. But its efficacy is strongly limited due to the acquisition of multidrug resistance (MDR). Therefore, acquisition of the resistance to ADR is still a major cause of chemotherapy failure in breast cancer patients. Peptidylarginine deiminase IV (PAD4) is reported to target non-histone proteins for citrullination, regulate their substrate activities, and thereby play critical roles in maintaining cell phenotype in breast cancer cells. However, whether PAD4 is involved in the development of MDR in breast cancer is poorly understood.Materials and methods: We examined the expression of PAD family members, including PAD4 in ADR-resistant MCF-7 cells compared with the parental control cells by real-time PCR and Western blotting analyses. Rescue of PAD4 expression in MCF-7/ADR cells was performed to assess whether PAD4 could restore the sensitivity of MCF-7/ADR cells to ADR treatment with cell counting kit-8, flow cytometry, TUNEL, nuclear and cytoplasmic extract preparations, and immunofluorescence staining analyses.Results: Both PAD2 and PAD4 were significantly decreased in ADR-resistant cells. However, only PAD4 overexpression can increase the sensitivity of MCF-7/ADR cells to ADR treatment and decrease MDR1 gene expression. Overexpression of PAD4 in MCF-7/ADR cells inhibited cell proliferation by inducing cell apoptosis. Under ADR treatment, overexpression of PAD4 promoted nuclear accumulation of glycogen synthase kinase-3β and p53, which further activated proapoptotic gene expression and downregulated MDR1 expression. Moreover, PAD4 activity was required for activating proapoptotic gene transcripts.Conclusion: We demonstrate the previously unappreciated role of PAD4 in reversing ADR resistance in MCF-7/ADR cells and help establish PAD4 as a candidate biomarker of prognosis and chemotherapy target for MDR in breast cancers. Keywords: PAD4, apoptosis, MDR, breast cancer, GSK3β

Abstract: A Perturbed Parameter Ensemble (PPE) with the Community Atmosphere Model version 6 (CAM6) is used to better understand the sensitivity of aerosol forcing and cloud feedbacks to changes in model processes. Aerosol forcing through aerosol‐cloud interactions is mostly negative (a cooling) due to shortwave radiation, while feedbacks are positive or negative in different regions due to contrasting longwave and shortwave effects. Both forcing and feedbacks are related to the mean climate state. Higher magnitude cloud radiative effects generally mean larger magnitude net negative forcing and larger magnitude net positive feedback. Aerosol forcing is broadly related to the susceptibility of clouds to drop number. Feedbacks also related to susceptibility, but to a lesser extent and in different regions to aerosol forcing. Aerosol forcing and cloud feedbacks are anti‐correlated in the CAM6 PPE such that stronger negative forcing is associated with stronger positive feedbacks. Even the processes governing forcing and feedback sensitivity in the PPE are similar. These include the warm rain formation process, ice loss processes and deep convective intensity.

Christian Song,1,2 Connor J Baharozian,3 Kathryn M Hatch,1,2 Jonathan H Talamo1,2 1Department of Opthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA, USA; 2Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; 3Department of Opthalmology, Boston University School of Medicine, Boston, MA, USA Purpose: To evaluate the collective user experience with an image-guided femtosecond laser (FSL) for cataract surgery in a high-volume, multi-surgeon, ambulatory surgical center. Subjects and methods: A detailed online survey was distributed to all surgeons in a single ambulatory surgical center who had performed cataract surgery using a FSL since its acquisition in December 2012. Information collected included the number of cases performed, typical surgical techniques and parameters, satisfaction with individual features of the laser (rated on a scale from 1=completely unsatisfied to 10=extremely satisfied) and commentary on ease of use and suggested improvements. Results: Seventeen of 30 surgeons (56.7%) completed the survey, representing a case volume of 1,967 eyes. Fourteen surgeons (82.4%) felt they required ≤10 cases with the FSL to operate with the same safety and control as in standard phacoemulsification surgery. Satisfaction was highest for capsulotomies, lens fragmentation, lens softening, arcuate incisions and the graphic user interface (mean scores 9.4, 8.7, 8.7, 7.2 and 8.9, respectively). Preferred capsulotomy diameter was 4.8–5.2 mm (64.7% of respondents). About half (52.9%) of respondents centered the capsulotomy on the pupil and the other 47.1% centered the capsulotomy using optical coherence tomography. Most respondents (81.3%) preferred transepithelial arcuate incisions compared to intrastromal incisions. Satisfaction was lowest with FSL-created, main, clear corneal incisions and paracenteses (mean scores 4.4 and 4.2, respectively). Conclusion: Laser-assisted cataract surgery has a short learning curve and a high rate of user satisfaction. Further software and hardware development is warranted to improve user satisfaction with peripheral and clear corneal incisions. Keywords: survey, satisfaction, learning curve, performance, Catalys, FLACS, capsulorhexis

Linhua Zhang,1 Yanqing Ren,2 Yong Wang,3 Yingna He,2 Wei Feng,2 Cunxian Song1 1Key Laboratory of Biomedical Material of Tianjin, Institute of Biomedical Engineering, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin, China; 2Hebei Key Laboratory of Chinese Medicine Research on Cardio-Cerebrovascular Disease, Pharmaceutical College, Hebei University of Chinese Medicine, Shijiazhuang City, Hebei Province, China; 3Department of Physics and Chemistry, College of Medicine, Hebei University, Baoding City, Hebei Province, China Background: A previous study developed a novel luteinizing hormone-releasing hormone (LHRH) receptor-targeted liposome. The aim of this study was to further assess the pharmacokinetics, biodistribution, and anti-tumor efficacy of LHRH receptor-targeted liposomes loaded with the anticancer drug mitoxantrone (MTO). Methods: Plasma and tissue distribution profiles of LHRH receptor-targeted MTO-loaded liposomes (LHRH-MTO-LIPs) were quantified in healthy mice or a xenograft tumor nude mouse model of MCF-7 breast cancer, and were compared with non-targeted liposomes and a free-drug solution. Results: The LHRH-MTO-LIPs demonstrated a superior pharmacokinetic profile relative to free MTO. The first target site of accumulation is the kidney, followed by the liver, and then the tumor; maximal tumor accumulation occurs at 4 h post-administration. Moreover, the LHRH-MTO-LIPs exhibited enhanced inhibition of MCF-7 breast cancer cell growth in vivo compared with non-targeted MTO-loaded liposomes (MTO-LIPs) and free MTO. Conclusion: The novel LHRH receptor-targeted liposome may become a viable platform for the future targeted treatment of cancer. Keywords: liposome, mitoxantrone, luteinizing hormone-releasing hormone receptor, tumor targeting, gonadorelin

Chanyoung Song,1,* Hathaichanok Phuengkham,1,* Sun-Young Kim,1 Min Sang Lee,2 Ji Hoon Jeong,2 Sung Jae Shin,3 Yong Taik Lim1 1SKKU Advanced Institute of Nanotechnology, School of Chemical Engineering, 2Department of Pharmacy, Sungkyunkwan University, Suwon, 3Department of Microbiology and Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: In this study, we suggest a designer vaccine adjuvant that can mimic the drainage of pathogens into lymph nodes and activate innate immune response in lymph nodes. By the amination of multivalent carboxyl groups in poly-(γ-glutamic acid) (γ-PGA) nanomicelles, the size was reduced for rapid entry into lymphatic vessels, and the immunologically inert nanomicelles were turned into potential activators of inflammasomes. Aminated γ-PGA nanomicelles (aPNMs) induced NLRP3 inflammasome activation and the subsequent release of proinflammatory IL-1β. The NLRP3-dependent inflammasome induction mechanism was confirmed through enzyme (cathepsin B and caspase-1) inhibitors and NLRP3 knockout mice model. After the aPNMs were combined with a clinically evaluated TLR3 agonist, polyinosinic–polycytidylic acid sodium salt (aPNM-IC), they triggered multiple arms of the innate immune response, including the secretion of pro-inflammatory cytokines by both inflammasomes and an inflammasome-independent pathway and the included type I interferons. Keywords: vaccine adjuvant, nanoparticles, innate immunity, antigen-presenting cells, lymphatic vessel, type I interferon

Connor J Baharozian,1 Christian Song,2,3 Kathryn M Hatch,2,3 Jonathan H Talamo2,3 1Boston University School of Medicine, 2Massachusetts Eye and Ear Infirmary, 3Department of Ophthalmology, Harvard Medical School, Boston, MA, USA Purpose: The purpose of this study was to determine an arcuate incision (AI) nomogram to treat astigmatism during femtosecond laser-assisted cataract surgery. Methods: This is a retrospective, cohort study. Femtosecond laser (FSL)-assisted transepithelial AIs were created at a 9.0 mm optical zone, 80% depth, centered on the limbus. We modified the manual Donnenfeld limbal relaxing incision nomogram to 70% for with-the-rule (WTR), 80% for oblique (OBL), and 100% for against-the-rule (ATR) astigmatism. The correction index (CI) equaled AI-induced astigmatism/target-induced astigmatism. Measures included preoperative keratometric corneal cylinder (Pre Kcyl), postoperative Kcyl (Post Kcyl), and postoperative residual refractive astigmatism (Post RRA). Results: Mean Pre Kcyl and 1–2 months Post RRA in 161 eyes of 116 patients were 0.626±0.417 diopters (D) (range 0.5–2 D), and 0.495±0.400 D (range 0–1.5 D), respectively. Mean absolute astigmatic changes (Pre Kcyl–Post Kcyl) without accounting for axis change in the WTR, ATR, and OBL groups were 0.165±0.383 D (P

Shengnan Huang,1,* Chunming Li,2,* Weiping Wang,1 Huanjie Li,1 Zhi Sun,3 Chengzhi Song,4 Benyi Li,5 Shaofeng Duan,6,7 Yurong Hu1,8,9 1Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, People’s Republic of China; 2Department of Pharmacy, Chongqing Cancer Institute & Hospital & Cancer Center, Chongqing, People’s Republic of China; 3Department of Pharmacy, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 4School of Physical Sciences, University of Science and Technology of China, Hefei, People’s Republic of China; 5Department of Urology and Cancer Center, the University of Kansas Medical Center, Kansas City, KS, USA; 6College of Pharmacy, Henan University, Kaifeng, People’s Republic of China; 7Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 8Key Laboratory of Key Technology of Drug Preparation, Ministry of Education, Institute of Drug Discovery & Development, Zhengzhou University, Zhengzhou, People’s Republic of China; 9Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou, People’s Republic of China *These authors contributed equally to this work Abstract: The combination of photothermal therapy and chemotherapy (photothermal–­chemotherapy) is a promising strategy for cancer therapy. Gold nanocages (AuNCs), with hollow and porous structures and unique optical properties, have become a rising star in the field of drug delivery. Here, we designed a novel targeted drug delivery system based on functionalized AuNCs and evaluated their therapeutic effects in vitro and in vivo. We then loaded doxorubicin into this promising system, designated as DHTPAuNCs consisting of hyaluronic acid-grafted and A54 peptide-targeted PEGylated AuNCs. Its formation was corroborated by ultraviolet–visible spectroscopy, transmission electron microscopy and dynamic light scattering. This delivery platform needed hyaluronidase to release encapsulated drugs, meanwhile the acidic pH and near-infrared irradiation could accelerate the release. In addition, the results of cellular uptake demonstrate that this system could bind specifically with BEL-7402 cells. In vitro, we evaluated therapeutic effects of the DHTPAuNCs in BEL-7402 cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay. Moreover, in BEL-7402 tumor-bearing nude mice, its therapy effect in vivo was also evaluated. As expected, DHTPAuNCs exhibited excellent therapeutic effect by photothermal–chemotherapy, both in vitro and in vivo. In short, DHTPAuNCs with low toxicity showed great potential as a drug delivery system for cancer therapy. Keywords: hepatocellular carcinoma, nanocarriers, targeted delivery, chemotherapy, photothermal therapy

Gyoyeon Hwang,1,2,* Hyeonhye Kim,1,* Hojong Yoon,1 Chiman Song,1 Dong-Kwon Lim,3 Taebo Sim,1,3 Jiyeon Lee1,2 1Chemical Kinomics Research Center, Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul, 2Bio-Med, Korea University of Science and Technology, Daejeon, 3KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Fibroblast growth factor receptors (FGFRs) play an important role in determining cell proliferation, differentiation, migration, and survival. Although a variety of small-molecule FGFR inhibitors have been developed for cancer therapeutics, the interaction between FGFRs and FGFR inhibitors has not been well characterized. The FGFR–inhibitor interaction can be characterized using a new imaging probe that has strong, stable signal properties for in situ cellular imaging of the interaction without quenching. We developed a kinase–inhibitor-modified quantum dot (QD) probe to investigate the interaction between FGFR and potential inhibitors. Especially, turbo-green fluorescent protein-FGFR3s were overexpressed in HeLa cells to investigate the colocalization of FGFR3 and AZD4547 using the QD-AZD4547 probe. The result indicates that this probe is useful for investigating the binding behaviors of FGFR3 with the FGFR inhibitor. Thus, this new inhibitor-modified QD probe is a promising tool for understanding the interaction between FGFR and inhibitors and for creating future high-content, cell-based drug screening strategies. Keywords: quantum dot, fibroblast growth factor 3, AZD4547, kinase–inhibitor, in situ imaging

Yang Liu,1–3 Dan Li,2 Xinhong Guo,2 Haiwei Xu,2 Zhi Li,2 Yanling Zhang,2 Chuanjun Song,4 Ruhan Fan,2 Xing Tang,1,* Zhenzhong Zhang2,* 1Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 2Department of Pharmaceutics, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 3Department of Pharmaceutics, Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan, Zhengzhou, 4Department of Organic Chemistry, College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, People’s Republic of China *These authors contributed equally to this work Abstract: We synthesized a pH-responsive conjugate of 10-hydroxycamptothecin-thiosemicarbazide-linear polyethylene glycol 2000 (PEG2000). The conjugate was confirmed by matrix-assisted laser desorption time of flight mass spectrometry, 1H NMR, and 13C NMR. The water solubility of the prodrug was increased by over 3,000 times; much longer body circulation time, higher tumor-targeting ability, and reduced toxicity were observed, compared with commercial 10-HCPT injection. The linker contains a pH-sensitive hydrazone bond, which breaks under low pH conditions in the tumor microenvironment. The conjugates showed good stability in phosphate-buffered saline (pH 7.4) and rat plasma. This amphiphilic conjugate could self-assemble into nanosized micelles of 80–100 nm. Cytotoxicity assay results indicate significantly higher efficacy of the conjugate (IC50 [half maximal inhibitory concentration] =0.117 µM on SW180 cells) than 10-HCPT solution (IC50 =0.241 µM on SW480 cells). Cellular uptake analysis suggested its rapid internalization and nuclear transport. Pharmacokinetic analysis of the conjugates demonstrated that the conjugate circulated for a longer time in the blood circulation system (T2/1 =10.516±1.158 h) than did 10-HCPT solution (T2/1 =1.859±1.385 h), and that it also enhanced the targeting and mean residence time (MRT0–inf =39.873±4.549 h) in the tumor site, compared with 10-HCPT (MRT0–inf =9.247±1.026 h). Finally, the conjugate demonstrated an increased tumor growth inhibition effect (TIR =82.66%±7.175%) in vivo and lower side effects than 10-HCPT (TIR =63.85%±5.233%). This prodrug holds great promise in improving therapeutic efficacy and overcoming multidrug resistance. Keywords: 10-hydroxycamptothecin, conjugate, controlled release, prodrug, pH-responsive, hydrazone

Yinbo Peng,1 Chenlu Song,1 Chuanfeng Yang,1 Qige Guo,1 Min Yao1,2 1Department of Burns and Plastic Surgery, Shanghai Ninth People’s Hospital, Institute of Traumatic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 2Department of Dermatology, Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Abstract: Silver nanoparticles (AgNPs) are being widely applied as topical wound materials; however, accumulated deposition of silver in the liver, spleen, and other main organs may lead to organ damage and dysfunction. We report here that low molecular weight chitosan-coated silver nanoparticles (LMWC-AgNPs) are effective against methicillin-resistant Staphylococcus aureus (MRSA), have better biocompatibility, and have lower body absorption characteristics when compared with polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) and silver nanoparticles without surface stabilizer (uncoated-AgNPs) in a dorsal MRSA wound infection mouse model. LMWC-AgNPs were synthesized by reducing silver nitrate with low molecular weight chitosan as a stabilizer and reducing agent, while PVP-AgNPs were synthesized using polyvinylpyrrolidone as a stabilizer and ethanol as a reducing agent. AgNPs with different surface stabilizers were identified by UV-visible absorption spectrometry, and particle size was determined by transmission electron microscopy. UV-visible absorption spectra of LMWC-AgNPs, PVP-AgNPs and uncoated-AgNPs were similar and their sizes were in the range of 10–30 nm. In vitro experiments showed that the three types of AgNPs had similar MRSA-killing effects, with obvious effect at 4 µg/mL and 100% effect at 8 µg/mL. Bacteriostatic annulus experiments also showed that all the three types of AgNPs had similar antibacterial inhibitory effect at 10 µg/mL. Cell counting kit-8 assay and Hoechst/propidium iodide (PI) staining showed that LMWC-AgNPs were significantly less toxic to human fibroblasts than PVP-AgNPs and uncoated-AgNPs. Treatment of mice with MRSA wound infection demonstrated that the three types of AgNPs effectively controlled MRSA wound infection and promoted wound healing. After continuous application for 14 days, LMWC-AgNPs-treated mice showed significantly reduced liver dysfunction as demonstrated by the reduced alanine aminotransferase and aspartate aminotransferase levels and liver deposition of silver, in comparison to mice treated with uncoated-AgNPs or PVP-AgNPs. Our results demonstrated that LMWC-AgNPs had good anti-MRSA effects, while harboring a better biocompatibility and lowering the body’s absorption characteristics. Keywords: silver nanoparticles, LMWC-AgNPs, MRSA, PVP-AgNPs, PVP, biocompatibility

Chanyoung Song,* Young-Woock Noh,* Yong Taik Lim SKKU Advanced Institute of Nanotechnology (SAINT), School of Chemical Engineering, Sungkyunkwan University, Suwon, South Korea *These authors contributed equally to this work Abstract: Effective induction of an antigen-specific cytotoxic T lymphocyte (CTL) immune response is one of the key goals of cancer immunotherapy. We report the design and fabrication of polyethylenimine (PEI)-coated polymer nanoparticles (NPs) as efficient antigen-delivery carriers that can induce antigen cross-presentation and a strong CTL response. After synthesis of poly(d,l-lactide-co-glycolide) (PLGA) NPs containing ovalbumin (OVA) by the double-emulsion solvent-evaporation method, cationic-charged PLGA NPs were generated by coating them with PEI. In a methyl tetrazolium salt assay, no discernible cytotoxic effect of PEI-coated PLGA (OVA) NPs was observed. The capacity and mechanism of PEI-coated PLGA (OVA) NPs for antigen delivery and cross-presentation on dendritic cells (DCs) were determined by fluorescence microscopy and flow cytometry. PEI-coated PLGA (OVA) NPs were internalized efficiently via phagocytosis or macropinocytosis in DCs and induced efficient cross-presentation of the antigen on MHC class I molecules via both endosome escape and a lysosomal processing mechanism. The DCs treated with PEI-coated PLGA (OVA) NPs induced a release of IL-2 cytokine from OVA-specific CD8-OVA1.3 T cells more efficiently than DCs treated with PLGA (OVA) NPs. Therefore, the PEI-coated PLGA (OVA) NPs can induce antigen cross-presentation and are expected to be used for induction of a strong CTL immune response and for efficient anticancer immunotherapy. Keywords: antigen delivery, dendritic cells, polymer NPs, vaccine, cross-presentation

Bin Zhang,1 Cunfeng Song,1 Bo Feng,1 Weibing Fan2 1Department of Neurology, The Third Hospital of Liaocheng, Liaocheng, Shandong, 2Department of Neurology, The Third Hospital of Changsha, Changsha, Hunan, People’s Republic of China Abstract: Triptolide, an active compound extracted from the Chinese herb thunder god vine (Tripterygium wilfordii Hook F.), has potent antitumor activity. Recently, triptolide was found to have protective effects against acute cerebral ischemia/reperfusion (I/R) injury through inhibition of cell apoptosis. However, the regulatory mechanism of the effect remains unclear. We hypothesize that the regulatory mechanisms of triptolide are mediated by nuclear factor κB (NF-κB) and p53-upregulated-modulator-of-apoptosis signal inhibition. To verify this hypothesis, we occluded the middle cerebral artery in male rats to establish focal cerebral I/R model. The rats received triptolide or vehicle at the onset of reperfusion following middle cerebral artery occlusion. At 24 hours after reperfusion, neurological deficits, infarct volume, and cell apoptosis were evaluated. The expression levels of NF-κBp65, PUMA, and caspase-3 were determined by Western blot. Real-time polymerase chain reaction was used to determine the levels of NF-κBp65 mRNA, PUMA mRNA, and caspase-3 mRNA. NF-κB activity was determined by electrophoretic mobility shift assay. Apoptotic cells were detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In I/R group, neurological deficit scores, cerebral infarct volume, expression of NF-κBp65, PUMA, caspase-3, NF-κB activity, and TUNEL-positive cells were found to be increased at 24 hours after I/R injury. The I/R/triptolide rats showed significantly better neurological deficit scores, decreased neural apoptosis, and reduced cerebral infarct volume. In addition, the expression of NF-κBp65, PUMA, caspase-3, and NF-κB activity was suppressed in the I/R/triptolide rats. These results indicate that the neuroprotective effects of triptolide during acute cerebral I/R injury are possibly related to the inhibition of apoptosis through suppression of NF-κB/PUMA signaling pathway. Keywords: ischemia/reperfusion, apoptosis, triptolide, neuroprotection

Linhua Zhang,1 Dunwan Zhu,1 Xia Dong,1 Hongfan Sun,1 Cunxian Song,1 Chun Wang,2 Deling Kong1 1Tianjin Key Laboratory of Biomaterials, Institute of Biomedical Engineering, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin, People’s Republic of China; 2Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA Abstract: The purpose of this study was to develop a novel lipid–polymer hybrid drug carrier comprised of folate (FA) modified lipid-shell and polymer-core nanoparticles (FLPNPs) for sustained, controlled, and targeted delivery of paclitaxel (PTX). The core-shell NPs consist of 1) a poly(ε-caprolactone) hydrophobic core based on self-assembly of poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCL-PEG-PCL) amphiphilic copolymers, 2) a lipid monolayer formed with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), 3) a targeting ligand (FA) on the surface, and were prepared using a thin-film hydration and ultrasonic dispersion method. Transmission electron microscopy and dynamic light scattering analysis confirmed the coating of the lipid monolayer on the hydrophobic polymer core. Physicochemical characterizations of PTX-loaded FLPNPs, such as particle size and size distribution, zeta potential, morphology, drug loading content, encapsulation efficiency, and in vitro drug release, were also evaluated. Fluorescent microscopy proved the internalization efficiency and targeting ability of the folate conjugated on the lipid monolayer for the EMT6 cancer cells which overexpress folate receptor. In vitro cytotoxicity assay demonstrated that the cytotoxic effect of PTX-loaded FLPNPs was lower than that of Taxol®, but higher than that of PTX-loaded LPNPs (without folate conjugation). In EMT6 breast tumor model, intratumoral administration of PTX-loaded FLPNPs showed similar antitumor efficacy but low toxicity compared to Taxol®. More importantly, PTX-loaded FLPNPs showed greater tumor growth inhibition (65.78%) than the nontargeted PTX-loaded LPNPs (48.38%) (P

Yingna He,1 Linhua Zhang,2 Dunwan Zhu,2 Cunxian Song2 1Laboratory of Chinese Medicine Pharmacology, College of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang, Hebei, People’s Republic of China; 2Key Laboratory of Biomedical Material of Tianjin, Institute of Biomedical Engineering, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin, People’s Republic of China Abstract: Tumor-targeting multifunctional liposomes simultaneously loaded with magnetic iron oxide nanoparticles (MIONs) as a magnetic resonance imaging (MRI) contrast agent and anticancer drug, mitoxantrone (Mit), were developed for targeted cancer therapy and ultrasensitive MRI. The gonadorelin-functionalized MION/Mit-loaded liposome (Mit-GML) showed significantly increased uptake in luteinizing hormone–releasing hormone (LHRH) receptor overexpressing MCF-7 (Michigan Cancer Foundation-7) breast cancer cells over a gonadorelin-free MION/Mit-loaded liposome (Mit-ML) control, as well as in an LHRH receptor low-expressing Sloan-Kettering HER2 3+ Ovarian Cancer (SK-OV-3) cell control, thereby leading to high cytotoxicity against the MCF-7 human breast tumor cell line. The Mit-GML formulation was more effective and less toxic than equimolar doses of free Mit or Mit-ML in the treatment of LHRH receptors overexpressing MCF-7 breast cancer xenografts in mice. Furthermore, the Mit-GML demonstrated much higher T2 enhancement than did Mit-ML controls in vivo. Collectively, the study indicates that the integrated diagnostic and therapeutic design of Mit-GML nanomedicine potentially allows for the image-guided, target-specific treatment of cancer. Keywords: multifunctional liposome, magnetic resonance imaging, theranostic nanomedicine, mitoxantrone, gonadorelin

Yongxia Chen,1,3,* Ziying Yang,1,* Chao Liu,1 Cuiwei Wang,1 Shunxin Zhao,2 Jing Yang,1 Hongfan Sun,1 Zhengpu Zhang,2 Deling Kong,1 Cunxian Song1 1Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, 2Institute of Polymer Chemistry, Nankai University, 3Center for Medical Device Evaluation of Tianjin, Tianjin, People's Republic of China *These authors contributed equally to this work Background: Star-shaped polymers provide more terminal groups, and are promising for application in drug-delivery systems. Methods: A new series of six-arm star-shaped poly(lactic-co-glycolic acid) (6-s-PLGA) was synthesized by ring-opening polymerization. The structure and properties of the 6-s-PLGA were characterized by carbon-13 nuclear magnetic resonance spectroscopy, infrared spectroscopy, gel permeation chromatography, and differential scanning calorimetry. Then, paclitaxel-loaded six-arm star-shaped poly(lactic-co-glycolic acid) nanoparticles (6-s-PLGA-PTX-NPs) were prepared under the conditions optimized by the orthogonal testing. High-performance liquid chromatography was used to analyze the nanoparticles' encapsulation efficiency and drug-loading capacity, dynamic light scattering was used to determine their size and size distribution, and transmission electron microscopy was used to evaluate their morphology. The release performance of the 6-s-PLGA-PTX-NPs in vitro and the cytostatic effect of 6-s-PLGA-PTX-NPs were investigated in comparison with paclitaxel-loaded linear poly(lactic-co-glycolic acid) nanoparticles (L-PLGA-PTX-NPs). Results: The results of carbon-13 nuclear magnetic resonance spectroscopy and infrared spectroscopy suggest that the polymerization was successfully initiated by inositol and confirm the structure of 6-s-PLGA. The molecular weights of a series of 6-s-PLGAs had a ratio corresponding to the molar ratio of raw materials to initiator. Differential scanning calorimetry revealed that the 6-s-PLGA had a low glass transition temperature of 40°C–50°C. The 6-s-PLGA-PTX-NPs were monodispersed with an average diameter of 240.4±6.9 nm in water, which was further confirmed by transmission electron microscopy. The encapsulation efficiency of the 6-s-PLGA-PTX-NPs was higher than that of the L-PLGA-PTX-NPs. In terms of the in vitro release of nanoparticles, paclitaxel (PTX) was released more slowly and more steadily from 6-s-PLGA than from linear poly(lactic-co-glycolic acid). In the cytostatic study, the 6-s-PLGA-PTX-NPs and L-PLGA-PTX-NPs were found to have a similar antiproliferative effect, which indicates durable efficacy due to the slower release of the PTX when loaded in 6-s-PLGA. Conclusion: The results suggest that 6-s-PLGA may be promising for application in PTX delivery to enhance sustained antiproliferative therapy. Keywords: PTX, polymer, drug delivery, nanoparticles, PLGA, antiproliferative therapy

Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles) onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP), thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules) of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol)-propionate (SPDP) as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein) were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle-immobilized PAA-BP-modified stents were successful as a gene delivery system. Gene delivery using DAC micelle-tethered stent-based PAA-BP functionalization should be suitable for a wide array of single or multiple therapeutic gene strategies, and could be used on cardiovascular metallic implants for achieving efficient gene therapy. Keywords: stent, polyallylamine bisphosphonate, plasmid DNA, micelles, gene delivery

Microarrays fabricated with oligonucleotides longer than 40 bp have been introduced for monitoring whole genome expression but have not been evaluated with environmental samples. To determine the potential of this type of microarray for environmental studies, a 50-mer oligonucleotide microarray was constructed using 763 genes involved in nitrogen cycling: nitrite reductase (nirS and nirK), ammonia monooxygenase (amoA), nitrogenase (nifH), methane monooxygenase (pmoA), and sulfite reductase (dsrAB) from public databases and our own sequence collections. The comparison of the sequences from pure cultures indicated that the developed microarrays could provide species-level resolution for analyzing microorganisms involved in nitrification, denitrification, nitrogen fixation, methane oxidation, and sulfite reduction. Sensitivity tests suggested that the 50-mer oligonucleotide arrays could detect dominant populations in the environments, although sensitivity still needs to be improved. A significant quantitative relationship was also obtained with a mixture of DNAs from eight different bacteria. These results suggest that the 50-mer oligonucleotide array can be used as a specific and quantitative parallel tool for the detection of microbial populations in environmental samples.