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Xiao-Fang Dong,1 Qiang Zhang,2 Jia-Ning Wei,2 Qian-Yu Zhou,3 Fan-Jia-Yi Yang,2 Yan-Jin Liu,4 Yu-Sheng Li,5 Chang-Qing Sun2,3 1Department of Endocrinology and Metabolic Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 2School of Nursing and Health, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 3Department of Epidemiology and Biostatistics, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 4Department of Nursing, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 5Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of ChinaCorrespondence: Chang-Qing Sun, School of Nursing and Health, Zhengzhou University, NO. 101 Kexue Road, High-Tech Development Zone of District, Zhengzhou, Henan, People’s Republic of China, Email suncq@zzu.edu.cnObjective: Our research explores how leisure-time sedentary behavior (SB) correlates with stroke risk. Additionally, we utilize the isotemporal substitution model (ISM) to examine how replacing brief durations of leisure-time SB with light physical activity (LPA), moderate physical activity (MPA), vigorous physical activity (VPA), and sleep might influence the risk of stroke.Methods: This investigation tracked 478,198 participants from the UK Biobank. Data regarding individual leisure-time SB and PA were collected through a standardized questionnaire. A Cox proportional hazards model, alongside an isotemporal substitution model (ISM), was utilized.Results: We identified 10,003 cases of incident stroke over 12.7 years. When compared to participants who engaged in leisure-time SB for less than 4 hours per day, the hazard ratios (HRs) for stroke incidence increased with more prolonged leisure-time SB: HRs were 1.06 (95% CI: 1.01 to 1.11) for 4– 6 h/d, 1.16 (95% CI: 1.10 to 1.23) for 6– 8 h/d, and 1.24 (95% CI: 1.15 to 1.33) for over 8 h/d. According to the ISM analysis, substituting leisure-time SB with various forms of PA could markedly reduce stroke risk. For individuals sleeping ≤ 8h/d, replacing one hour of leisure-time SB with an equivalent duration of LPA, VPA, or sleep corresponded to a 3.0%, 7.0%, and 22.0% decrease in stroke risk, respectively. Meanwhile, for those already sleeping more than 8h/d, substituting one hour of leisure-time SB with an equivalent duration of LPA or VPA resulted in a notable decrease in the risk of stroke by 6.0% and 18.0%, respectively.Conclusion: The findings demonstrate that leisure-time SB and unhealthy sleep durations are confirmed risk factors for stroke. For individuals sleeping 8 hours or less per day, and for those who sleep more than 8 hours, substituting SB with an adequate amount of sleep or engaging in VPA, respectively, emerges as an effective strategy for reducing stroke risk.Keywords: stroke, isotemporal substitution model, physical activity, sedentary behavior, sleep

Yuan-Jie Liu,1,* Jing-Xiao Li,1,* Jie-Pin Li,1 Yi-Dou Hu,2 Zhi-Bin Ma,3 Wei Huang,1 Shen-Lin Liu,1 Xi Zou1 1Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 3Nanjing YOUMENG Biology Science and Technology Co. Ltd, Nanjing, Jiangsu, 210029, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xi Zou; Shen-lin Liu, Email fsyy00670@njucm.edu.cn; lsljsszyy@126.comBackground: Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, underscoring the need for novel therapeutic targets. This study aimed to elucidate the role of endoplasmic reticulum membrane protein complex subunit 1 (EMC1) in HCC progression and its therapeutic potential.Methods: Publicly available sequencing data and biopsy specimens were analyzed to assess EMC’s clinical value and functions in HCC. In vitro experiments validated EMC functions, and multiplex immunofluorescence analysis examined EMC-associated sorafenib resistance mechanisms. EMC1 expression was knocked down in HCC cell lines, followed by cell viability, wound healing, and transwell migration assays. Tumor growth and response to sorafenib treatment were evaluated in mouse models. Metabolomic analysis assessed changes in the TCA cycle.Results: EMC genes were aberrantly expressed in HCC, and high EMC1 expression correlated with poorer survival rates. EMC1 disruption enhanced HCC cells’ sensitivity to sorafenib, reducing cell viability, increasing apoptosis, and decreasing tumor size and weight. EMC1 maintained cancer cell stemness and promoted M2 macrophage infiltration. Metabolomic analysis revealed significant changes in the TCA cycle, indicating EMC1’s role in HCC metabolic reprogramming. Importantly, EMC1 is highly associated with sorafenib resistance, potentially linked to CTNNB1 mutation or activation.Conclusion: EMC1 plays a critical role in regulating the sorafenib resistance in HCC. Targeting EMC1 may improve HCC treatment efficacy.Keywords: endoplasmic reticulum membrane complex subunit 1, catenin beta 1, tumor stemness, M2 macrophage, sorafenib resistance

Li-Ping Ou,* Yong-Jian Liu,* Shi-Tong Qiu,* Chen Yang, Ji-Xin Tang, Xiao-Yu Li, Hua-Feng Liu, Zhen-Nan Ye Institute of Nephrology, and Guangdong Provincial Key Laboratory of Autophagy and Major Chronic Non-Communicable Diseases, and Key Laboratory of Prevention and Management of Chronic Kidney Disease of Zhanjiang City, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhen-Nan Ye; Hua-Feng Liu, Email yezhennan12@mails.ucas.ac.cn; liuhf@gdmu.edu.cnAbstract: Metabolic reprogramming contributes to the progression and prognosis of various kidney diseases. Glutamine is the most abundant free amino acid in the body and participates in more metabolic processes than other amino acids. Altered glutamine metabolism is a prominent feature in different kidney diseases. Glutaminolysis converts glutamine into the TCA cycle metabolite, alpha-ketoglutarate, via a cascade of enzymatic reactions. This metabolic pathway plays pivotal roles in inflammation, maladaptive repair, cell survival and proliferation, redox homeostasis, and immune regulation. Given the crucial role of glutaminolysis in bioenergetics and anaplerotic fluxes in kidney pathogenesis, studies on this cascade could provide a better understanding of kidney diseases, thus inspiring the development of potential methods for targeted therapy. Emerging evidence has shown that targeting glutaminolysis is a promising therapeutic strategy for ameliorating kidney disease. In this narrative review, equation including keywords related to glutamine, glutaminolysis and kidney are subjected to an exhaustive search on Pubmed database, we identified all relevant articles published before 1 April, 2024. Afterwards, we summarize the regulation of glutaminolysis in major kidney diseases and its underlying molecular mechanisms. Furthermore, we highlight therapeutic strategies targeting glutaminolysis and their potential clinical applications.Keywords: glutamine, glutaminolysis, kidney diseases, therapeutic target

Wen Jing Sun,1,2 Yu Jin Liu1 1Department of Nursing Welfare, Changchun Humanities and Sciences College, Changchun, Jilin, 130117, People’s Republic of China; 2Department of Counseling Psychology, Dongshin University, Naju-si, Jeollanam, Republic of KoreaCorrespondence: Wen Jing Sun, Department of Nursing Welfare, Changchun Humanities and Sciences College, No. 1488, Boshuo Road, Jingyue National High-Tech Industrial Development Zone, Changchun City, Jilin, 130117, People’s Republic of China, Tel +8615904405614, Email 179403066@qq.comPurpose: The aim of this study was to investigate the sleep quality as well as the influence of social support on the sleep quality of elderly people in nursing homes in northeast China, and analyze the chain-mediating role of psychological adjustment and coping styles in social support and sleep quality, thereby to provide a scientific basis for the development of effective intervention measures in this direction.Patients and Methods: This study was conducted during January–March 2023 and adopted a cluster sampling method to select 5 elderly care institutions from across the Jilin, Liaoning, and Heilongjiang provinces in Northeast China. A questionnaire survey was conducted using the Self-mate General Situation Questionnaire, Pittsburgh Sleep Quality Index, Nursing Home Adjustment Scale for the Elderly, Social Support Rating Scale, and Medical Coping Modes Questionnaire. Statistical analysis methods, including ANOVA, logistic multi-factor regression, and Pearson’s correlation were employed in SPSS 26.0, while Amos 26.0 was used to build a structural equation model to analyze the interaction path and the mediating role between the variables.Results: The sleep quality of elderly individuals in elderly care institutions was relatively low 8.43(3.456). Social support of elderly individuals in elderly care institutions affected their sleep quality through i) both psychological adjustment and face-to-face coping style (B = 0.493, P < 0.001, 95% CI = 0.050– 0.122) and ii) both psychological adjustment and avoidance coping style (B = − 0.302, P < 0.001, 95% CI = − 0.119 to − 0.048). Psychological adjustment, confrontation coping, and avoidance coping played a mediating role in the sequential relationship between social support and the sleep quality of elderly individuals in elderly care institutions.Conclusion: Psychological adjustment and coping styles have a chain-mediating effect between social support and sleep quality of the elderly in northeast China’s elderly care institutions.Keywords: elderly care institutions, elderly people, sleep quality, psychological adjustment, social support, nursing

Shi-Yuan Fan,1,&ast; Zi-Chi Zhao,1,&ast; Xing-Lv Liu,1 Ying-Gang Peng,1 Hui-Min Zhu,1 Shi-Fan Yan,2 Yan-Juan Liu,2 Qin Xie,2 Yu Jiang,2 Sai-Zhen Zeng1 1The First Affiliated Hospital of Hunan Normal University (Hunan Provincial People’s Hospital), Changsha, Hunan, 410005, People’s Republic of China; 2Department of Emergency, Institute of Emergency Medicine, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha, Hunan, 410005, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Sai-Zhen Zeng, The First Affiliated Hospital of Hunan Normal University (Hunan Provincial People’s Hospital), 61 Jie-Fang West Road, Fu-Rong District, Changsha, Hunan, 410005, People’s Republic of China, Email 55445321@qq.com Yu Jiang, Department of Emergency, Institute of Emergency Medicine, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), 61 Jie-Fang West Road, Fu-Rong District, Changsha, 410005, People’s Republic of China, Email jiangyu@hunnu.edu.cnBackground: Globally, the subsequent complications that accompany sepsis result in remarkable morbidity and mortality rates. The lung is among the vulnerable organs that incur the sepsis-linked inflammatory storm and frequently culminates into ARDS/ALI. The metformin-prescribed anti-diabetic drug has been revealed with anti-inflammatory effects in sepsis, but the underlying mechanisms remain unclear. This study aimed to ascertain metformin’s effects and functions in a young mouse model of sepsis-induced ALI.Methods: Mice were randomly divided into 4 groups: sham, sham+ Met, CLP, and CLP+ Met. CLP was established as the sepsis-induced ALI model accompanied by intraperitoneal metformin treatment. At day 7, the survival state of mice was noted, including survival rate, weight, and M-CASS. Lung histological pathology and injury scores were determined by hematoxylin–eosin staining. The pulmonary coefficient was used to evaluate pulmonary edema. Furthermore, IL-1β, CCL3, CXCL11, S100A8, S100A9 and NLRP3 expression in tissues collected from lungs were determined by qPCR, IL-1β, IL-18, TNF-α by ELISA, caspase-1, ASC, NLRP3, P65, p-P65, GSDMD-F, GSDMD-N, IL-1β and S100A8/A9 by Western blot.Results: The data affirmed that metformin enhanced the survival rate, lessened lung tissue injury, and diminished the expression of inflammatory factors in young mice with sepsis induced by CLP. In contrast to sham mice, the CLP mice were affirmed to manifest ALI-linked pathologies following CLP-induced sepsis. The expressions of pro-inflammatory factors, for instance, IL-1β, IL-18, TNF-α, CXCL11, S100A8, and S100A9 are markedly enhanced by CLP, while metformin abolished this adverse effect. Western blot analyses indicated that metformin inhibited the sepsis-induced activation of GSDMD and the upregulation of S100A8/A9, NLRP3, and ASC.Conclusion: Metformin could improve the survival rate, lessen lung tissue injury, and minimize the expression of inflammatory factors in young mice with sepsis induced by CLP. Metformin reduced sepsis-induced ALI via inhibiting the NF-κB signaling pathway and inhibiting pyroptosis by the S100A8/A9-NLRP3-IL-1β pathway.Keywords: sepsis, pediatric, inflammation, pyroptosis, ALI

Jin Wan, Yu Jie Liu, Wen Jun Zhou, Si Yuan Wu School of Economics and Management, East China Jiaotong University, Nanchang, Jiangxi, 330013, People’s Republic of ChinaCorrespondence: Jin Wan, Email 244022935@qq.comIntroduction: To investigate the impact of interpersonal sensitivity on the subjective well-being of accompanying children of migrant workers and the role of perception of exclusion and peer support in the process.Methods: A questionnaire survey was conducted among 304 migrant workers’ accompanying children and 501 urban children in grades 4– 9 in seven schools in Jiangxi Province, China. Hierarchical regression and bootstrap analysis were used.Results: Interpersonal sensitivity not only had a significant direct negative effect on the subjective well-being of migrant workers’ accompanying children (β= − 0.27, 95% CI = [− 0.37, − 0.17]), but also had an indirect effect through perception of exclusion (β= − 0.06, 95% CI = [− 0.11, − 0.03]). Peer support negatively moderated the relationship between interpersonal sensitivity and perception of exclusion (β= − 0.18, 95% CI = [− 0.28, − 0.08]) and the mediating effect of perceptions of exclusion between interpersonal sensitivity and subjective well-being (β = 0.06, CI = [0.02, 0.11]).Conclusion: The subjective well-being of migrant children is indeed lower than that of urban children, and one of the most important reasons is their higher interpersonal sensitivity. Interpersonal sensitivity not only directly reduces their subjective well-being, but also reduces it by triggering their perception of exclusion, while peer support can effectively mitigate this negative effect. Therefore, one way to improve the subjective well-being of these children is to reduce their excessive interpersonal sensitivity. Their parents should help them to adapt to urban life, to develop correct professional values and to deal correctly with “occupational stigma”, to overcome feelings of inferiority, while communities can create specialized activity centers to provide more social opportunities and psychological counseling services for these children.Keywords: migrant workers’ accompanying children, interpersonal sensitivity, perception of exclusion, peer support, subjective well-being

Qian Zhang,1,2,&ast; Jia Liu,1,&ast; Dan Yao,1,2 Jian-Xin Shi,1 Yuan-Jie Liu,2– 4 Yue-Gang Wei,1 Shun Guo1,2 1Department of Dermatology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 4Key Laboratory of Tumor System Biology of Traditional Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Shun Guo, Email retinoid@163.comBackground: Psoriasis is a complex autoimmune disease. Frequent interactions between epidermal and immune cells are likely to be responsible for the strong heterogeneity of psoriasis. Therefore, our work aims to build on current knowledge and further search for new molecular mechanisms related to psoriasis pathogenesis in order to develop new targeted drugs.Methods: Data from psoriasis samples were obtained from the Gene Expression Omnibus (GEO) database, and batch effects were corrected using the “Combat” algorithm in the “SVA” package. Functional annotation of differential genes in psoriasis was performed by Gene set enrichment analysis (GSEA). Core functional modules were identified using the Multiscale Embedded Gene Co-Expression Network Analysis (MEGENA) algorithm for selection from the differential gene interaction network. The expression and potential function of Rh Family C Glycoprotein (RHCG) was predicted in single cell data by the “Seurat” package and validated in psoriasis samples by multiplex immunofluorescence. In addition, the regulatory function of HOP Homeobox (HOPX) on RHCG in keratinocytes was confirmed using RNA interference. Using immune infiltration analysis, RHCG and DC cells were analyzed for their association. Finally, the molecular mechanisms of treatment of psoriasis using Tripterygii Radix (TR) and Cinnamomi Ramulus (CR) were explored through network pharmacology and experimental validation.Results: Immune response (represented by C1_2) and collagen matrix formation (represented by C1_3) were identified as two important pathogenic factors in psoriasis and helped to define new biological subtypes of psoriasis. One important psoriasis hub gene, RHCG, was obtained and found to be closely associated with keratinocyte differentiation as well as DC cell maturation. And RHCG was regulated by HOPX in keratinocytes. In addition, the mechanism of action of CR and TR in the treatment of psoriasis was tentatively confirmed to be related to TRPV3, NFKB2, and YAP1.Conclusions: Our study identifies a new causal disease gene (RHCG) and offers potential alternatives for the treatment of psoriasis.Keywords: psoriasis, Rh family C glycoprotein, RHCG, DC cells, keratinocyte, traditional Chinese medicine

Xiao-Yan Li,1 Xin-Guang Liu,2 Zhi-Ling Dong,1 Liang-Ting Chai,3 Yan-Jun Liu,4 Jie Qi,3 Jie Zhao1 1Department of Clinical Laboratory, Handan Central Hospital, Handan, Hebei, People’s Republic of China; 2Department of Clinical Laboratory, Children’s Hospital of Hebei, Shijiazhuang, Hebei, People’s Republic of China; 3Department of Clinical Laboratory, Handan First Hospital, Handan, Hebei, People’s Republic of China; 4Department of General Surgery, Handan Central Hospital, Handan, Hebei, People’s Republic of ChinaCorrespondence: Jie Zhao, No. 59 Congtai North Road, Congtai District, Handan City, Hebei Province, 057150, People’s Republic of China, Tel +86-18003205055, Email zjlxy8160@163.comBackground: Drug-resistant Pseudomonas aeruginosa infections rapidly increased and contributed to life-threatening nosocomial infections; however, the distribution, species, drug susceptibility and dynamic trends of P. aeruginosa infection in China remained unclear. This study was conducted to better understand the epidemiological data of increased P. aeruginosa infections from 2016 to 2022 in a hospital in China.Methods: This study involved 3301 patients infected with P. aeruginosa, diagnosed using a nosocomial infection surveillance system in a tertiary hospital between 2016 and 2022. The P. aeruginosa infections from 2016 to 2022 were assessed according to the hospital department and species, and the drug susceptibility was evaluated using 16 antimicrobial agents.Results: The P. aeruginosa infection prevalence in the hospital department was: Neurosurgery (14.30%), Emergency (13.30%), and Critical Care Medicine (11.69%). Samples for P. aeruginosa infection identification were from sputum (72.52%) and other secreta (9.91%). The P. aeruginosa infections demonstrated a greater sensitivity to amikacin (AMK, 91.82%), tobramycin (TOB, 82.79%), and gentamycin (GEN, 82.01%); however, P. aeruginosa infection demonstrated greater resistance to ticarcillin (22.57%), levofloxacin (21.63%), and ciprofloxacin (18.00%).Conclusion: The P. aeruginosa infections were commonly observed in the Neurosurgery, Emergency, and Critical Care Medicine departments and demonstrated greater sensitivity to AMK, TOB, and GEN than the other drugs.Keywords: Pseudomonas aeruginosa, distribution, drug susceptibility, dynamic trend

Pan Huang,1,&ast; Yi-dou Hu,1,&ast; Yuan-jie Liu,2,3 Jie-pin Li,1,2,4 Yong-hua Zhang1 1Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 2No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 4Key Laboratory of Tumor System Biology of Traditional Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yong-hua Zhang; Jie-pin Li, Email zjgzy043@njucm.edu.cn; zjgzy027@njucm.edu.cnBackground: The nuclear pore complex (NPC) is the main mediator of nuclear and cytoplasmic communication, and delaying or blocking nuclear RNA export and protein shuttling can inhibit cell proliferation and induce apoptosis. Although NPC is a research hotspot in structural biology, relevant studies in hepatocellular carcinoma are scarce, especially in terms of translation into clinical practice.Methods: This study used a bioinformatics approach combining validation experiments to investigate the biological mechanisms that may be related with NPC. A series of experiments performed to explore the function of the Targeting protein for Xenopus kinesin-like protein 2 (TPX2) in HCC.Results: Patients with HCC can be divided into two NPC clusters. Patients with high NPC levels (C1) had a shorter survival time than those with low NPC levels (C2) and are characterised by high levels of proliferative signals. We demonstrated that TPX2 regulates HCC growth and inhibits apoptosis in an NPC-dependent manner and contributes to the maintenance of HCC stemness. We developed the NPCScore to predict the prognosis and degree of differentiation in HCC patients.Conclusion: NPC plays an important role in the malignant proliferation of HCC. Assessing NPC expression patterns could help enhance our understanding of tumor cell proliferation and could guide more effective chemotherapeutic strategies.Keywords: nuclear pore complex, proliferation, hepatocellular carcinoma, bioinformatics, TPX2

Yan Sha,1,&ast; An-qi Mao,1,&ast; Yuan-jie Liu,2 Jie-pin Li,2 Ya-ting Gong,3 Dong Xiao,1 Jun Huang,1 Yan-wei Gao,1 Mu-yao Wu,3 Hui Shen1 1Departments of Dermatology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, People’s Republic of China; 2Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, People’s Republic of China; 3Departments of Rehabilitation, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Hui Shen; Mu-yao Wu, Email zjgzy046@njucm.edu.cn; wumuyao0613@163.comBackground: The incidence of cutaneous melanoma continues to rise rapidly and has an extremely poor prognosis. Immunotherapy strategies are the most effective approach for patients who have developed metastases, but not all cases have been successful due to the complex and variable mechanisms of melanoma response to immune checkpoint inhibition.Methods: We synthesized collagen-coding gene expression data (second-generation and single-cell sequencing) from public Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis was performed using R software and several database resources such as Metascape database, Gene Set Cancer Analysis (GSCA) database, and Cytoscape software, etc., to investigate the biological mechanisms that may be related with collagens. Immunofluorescence and immunohistochemical staining were used to validate the expression and localization of Nidogen-2 (NID2).Results: Melanoma patients can be divided into two collagen clusters. Patients with high collagen levels (C1) had a shorter survival than those with low collagen levels (C2) and were less likely to benefit from immunotherapy. We demonstrated that NID2 is a potential key factor in the collagen phenotype, is involved in fibroblast activation in melanoma, and forms a barrier to limit the proximity of CD8+ T cells to tumor cells.Conclusion: We clarified the adverse effects of collagen on melanoma patients and identified NID2 as a potential therapeutic target.Keywords: Skin Cutaneous Melanoma, collagen molecules, NID2, bioinformatics, immunotherapy

Wei Huang,1,2,&ast; Jia Mei,3,&ast; Yuan-Jie Liu,1,4 Jie-Pin Li,1,4,5 Xi Zou,1,6 Xiao-Ping Qian,2,7 Yu Zhang8 1Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2Comprehensive Cancer Center, Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210008, People’s Republic of China; 3Department of Pathology, Nanjing Jinling Hospital, Nanjing, Jiangsu, 210001, People’s Republic of China; 4No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 5Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 6Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor, Nanjing, 210023, People’s Republic of China; 7The Comprehensive Cancer Center of Nanjing Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, Jiangsu, 210008, People’s Republic of China; 8Department of Oncology, Nanjing Jinling Hospital, Nanjing, Jiangsu, 210001, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yu Zhang; Xiao-ping Qian, Email zyzyzhangyu1986@163.com; xiaopingqian@nju.edu.cnBackground: The Proteasome (PSM) is a large multi-catalytic protease complex consisting of a 20S core particle and a 19S regulatory particle whose main function is to accept and degrade ubiquitinated substrates, are now considered as one of the potential regulators of tumor proliferation, and stemness maintenance. However, to date, studies on the relationship between PSM and hepatocellular carcinoma (HCC) are limited.Methods: This study used a bioinformatics approach combining validation experiments to investigate the biological mechanisms that may be related with PSM. A series of experiments in vivo and in vitro were performed to explore the function of the 26S proteasome non-ATPase regulatory subunit 13 (PSMD13) in HCC.Results: HCC patients can be divided into two clusters. Cluster 1 (C1) patients having a significantly worse prognosis than Cluster (C2). Two subtypes had significant differences in proliferation-related signaling. In particular, the frequency of TP53 mutation was significantly higher in C1 than in C2. In addition, PSM-associated genes were highly consistent with the expression of DNA repair-related signatures, suggesting a potential link between PSM and genomic instability. We also found that downregulation of PSMD13 expression significantly inhibited stemness of tumor cells and impaired the Epithelial mesenchymal transition (EMT) process. Finally, the correlation between the PSMD13 and Ki67 was found to be strong.Conclusion: PSM is a valid predictor of prognosis and therapeutic response in patients with HCC disease. Furthermore, PSMD13 may be a potential therapeutic target.Keywords: hepatocellular carcinoma, proteasome, PSMD13, cell stemness, proliferation

Zhong-Han Fang,1,2 Hong-Lin Liao,1,2 Qing-Feng Tang,1,2 Ya-Jing Liu,1,2 Yan-Yan Zhang,1,2 Jiu Lin,1,2 Hao-Peng Yu,3 Cheng Zhou,4 Chun-Jie Li,1,5 Fei Liu,1,2,&ast; Jie-Fei Shen1,2,&ast; 1State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, National Center for Stomatology, West China School of Stomatology, Sichuan University, Chengdu, People’s Republic of China; 2Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, People’s Republic of China; 3West China Biomedical Big Data Center, West China Hospital, Sichuan University, Chengdu, People’s Republic of China; 4Laboratory of Anesthesia and Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, People’s Republic of China; 5Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Fei Liu; Jie-Fei Shen, Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, People’s Republic of China, Email liufei.hxkq@scu.edu.cn; shenjiefei@scu.edu.cnBackground: Recent studies have demonstrated the contribution of non-coding RNAs (ncRNAs) to neuropathic pain. However, the expression profile of ncRNAs in the trigeminal ganglion (TG) and their functional mechanism underlying trigeminal neuropathic pain are still unclear.Methods: In the present study, the trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve (CCI-ION) was used to study the expression profile and potential regulatory mechanism of miRNAs, lncRNAs, circRNAs, and mRNAs in the TG by RNA-sequencing (RNA-seq) and bioinformatics analysis. CCI-ION mice suffered from mechanical allodynia from 3 days to 28 days after surgery.Results: The RNA-seq results discovered 67 miRNAs, 216 lncRNAs, 14 circRNAs, 595 mRNAs, and 421 genes were differentially expressed (DE) in the TG of CCI-ION mice 7 days after surgery. And 39 DEGs were known pain genes. Besides, 5 and 35 pain-related DE mRNAs could be targeted by 6 DE miRNAs and 107 DE lncRNAs, respectively. And 23 pain-related DEGs had protein–protein interactions (PPI) with each other. GO analysis indicated membrane-related cell components and binding-related molecular functions were significantly enriched. KEGG analysis showed that nociception-related signaling pathways were significantly enriched for DE ncRNAs and DEGs. Finally, the competing endogenous RNA (ceRNA) regulatory network of DE lncRNA/DE circRNA-DE miRNA-DE mRNA existed in the TG of mice with trigeminal neuropathic pain.Conclusion: Our findings demonstrate ncRNAs are involved in the development of trigeminal neuropathic pain, possibly through the ceRNA mechanism, which brings a new bright into the study of trigeminal neuropathic pain and the development of novel treatments targeting ncRNAs.Keywords: trigeminal neuropathic pain, non-coding RNAs, RNA-sequencing, trigeminal ganglion, trigeminal nerve injury, bioinformatics

Yi Yin,1,2,&ast; Zi-Yuan Xu,1,2,&ast; Yuan-jie Liu,1,2 Wei Huang,1,2 Qian Zhang,1,2 Jie-pin Li,1– 3 Xi Zou1,2,4 1Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 4Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor, Nanjing, Jiangsu, 210029, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xi Zou; Jie-pin Li, Email zxvery@126.com; zjgzy027@njucm.edu.cnBackground: Helicases have been classified as a class of enzymes that determine the stability of the cellular genome. There is growing evidence that helicases can help tumor cells resist drug killing by repairing Deoxyribose Nucleic Acid (DNA) or stabilizing transcription, which may contribute to the understanding of drug resistance. Currently, identifying cancer biomarkers among helicases and stratifying patients according to helicase activity will be able to guide treatment well.Methods: We clustered 371 hepatocellular carcinoma (HCC) patients from The Cancer Genome Atlas (TCGA) by consensus clustering based on helicase expression profiles to identify potential molecular subtypes. The Multiscale Embedded Gene Co-Expression Network Analysis (MEGENA) algorithm was used to find core differential gene modules between different molecular subtypes, and single-cell analysis was utlized to explore the potential function of hub gene. Immunohistochemical (IHC) staining was used to verify the diagnostic value of DDX56 and its ability to reflect the proliferation efficiency of cancer cells.Results: We identified two subtypes associated with helicase. High helicase subtype was associated with poor clinical outcome, massive M0 macrophage infiltration, and highly active cell proliferation features. In addition, we identified a new biomarker, DDX56, which has not been reported in HCC, was highly expressed in HCC tissues, associated with poor prognosis, and was also shown to be associated with high cell proliferative activity.Conclusion: In conclusion, based on helicase expression profiles, we have developed a new classification system for HCC, which is a proliferation-related system, and has clinical significance in evaluating prognosis and treating HCC patients, including immunotherapy and chemotherapy. In addition, we identified a new biomarker, DDX 56, which is overexpressed in HCC tissues, predicts a poor prognosis and is a validated index of tumor cell proliferation.Keywords: helicases, hepatocellular carcinoma, DDX56, bioinformatics

Qian Zhang,1,2,&ast; Yuan-Jie Liu,1,2,&ast; Jie-Pin Li,1– 3 Shu-Hong Zeng,1,2 Hui Shen,3 Mei Han,1 Shun Guo,1,2 Shen-Lin Liu,1,2 Xi Zou1,2,4 1Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 4Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor, Nanjing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xi Zou; Shun Guo, Email zxvery@126.com; retinoid@163.comBackground: As one of the most immunogenic malignancies, skin cutaneous melanoma (SKCM) is mainly characterized by a high prevalence in immune-compromised patients and a brisk lymphocyte infiltration in the tumor microenvironment (TME). However, to date, studies on deubiquitination in SKCM are still very limited.Methods: Public data with regard to this study in SKCM patients were acquired from The Cancer Genome Atlas (TCGA) and the Gene-Expression Omnibus (GEO) databases. We stratified TCGA-SKCM cases using consensus clustering and identified independent prognostic factors in deubiquitinating enzymes encoding genes (DECGs) by LASSO-Cox analysis. USP35 transcriptome level was examined using public data and validated by Immunohistochemical (IHC) staining at the protein level. Enrichment analysis was used to explore the potential functions of USP35, and the TISCH database, providing further evidence at the single-cell level. The CIBERSORT algorithm was used to assess the relationship between USP35 and the immune microenvironment, and IHC was used to further evaluate the relationship between USP35 and immunotherapy response. Finally, we used the cBioPortal and the Methsurv database to analyze the significance of genomic alterations of USP35 in melanoma.Results: Our results showed that DECGs can be effectively used to stratify SKCM patients, suggesting their potential significance in the development of SKCM. Furthermore, USP35 overexpression was significantly associated with an unfavorable prognosis. We further revealed that USP35 may be involved in the activation of TORC1 signaling. Most importantly, USP35 was found to be significantly associated with an immunosuppressive TME, both in terms of negative correlation with the abundance of infiltrating CD8+ T cells and in terms of the fact that patients with high USP35 expression may benefit less from immunotherapy than those with low USP35 expression.Conclusion: Deubiquitinating enzymes are of great importance in the diagnosis and treatment of SKCM, and USP35 is an extremely promising target for immunotherapy.Keywords: skin cutaneous melanoma, deubiquitinating enzymes, USP35, immunotherapy

Yuan-jie Liu,1,2,&ast; Mei Han,1,&ast; Jie-pin Li,1– 3 Shu-hong Zeng,1,2 Qian-wen Ye,1,2 Zhong-hua Yin,1,2 Shen-lin Liu,1,2 Xi Zou1,2,4 1Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, People’s Republic of China; 2No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China; 3Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, People’s Republic of China; 4Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor, Nanjing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xi Zou; Shen-lin Liu, Email zxvery@126.com; lsljsszyy@126.comBackground: Gap junctions, as one of the major ways to maintain social connections between cells, are now considered as one of the potential regulators of tumor metastasis. However, to date, studies on the relationship between gap junctions and colorectal cancer (CRC) are limited.Methods: We synthesized connexins-coding gene expression data from public Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis was performed using R software and several database resources such as MEXPRESS database, Gene Set Cancer Analysis (GSCA) database, Human Protein Atlas (HPA) database, Tumor Immune Single Cell Hub (TISCH) database, Search Tool for Retrieval of Gene Interaction Relationships (STRING), and Cytoscape software, etc., to investigate the biological mechanisms that may be involved in connexins. Immunofluorescence and immunohistochemical staining were used to validate the expression and localization of GJA4.Results: We found that CRC patients can be divided into two connexin clusters and that patients in cluster C1 had shorter survival than in cluster C2. The infiltration of M1 macrophages and NK cells was lower in cluster C1, while the levels of M2 macrophages and immune checkpoints were higher, indicating an immunosuppressed state in cluster C1. In addition, the epithelial–mesenchymal transition (EMT) phenotype was significantly activated in cluster C1. We observed that GJA4 was up-regulated in colorectal cancer tissues, which was related to poor prognosis. It was mainly expressed in fibroblasts, but the expression levels in normal intestinal epithelial cells were low. Finally, we found that GJA4 was associated with M2 macrophages and may be a potential immunosuppressive factor.Conclusion: We found that there is a significant correlation between abnormal connexins expression and patients’ prognosis, and connexins play an important role in stromal-tumor interactions. Connexins, especially GJA4, can help enhance our understanding of tumor microenvironment (TME) and may guide more effective immunotherapeutic strategies.Keywords: colorectal cancer, gap junction protein alpha 4, cancer-associated fibroblasts, M2 macrophage, bioinformatics

Yuan-Jie Liu,1,2,&ast; Shu-hong Zeng,2,3,&ast; Wei-hua Qian,1 Min-xian Tao,1 Ying-ying Zhu,4 Jie-pin Li1– 3 1Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 2Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 3No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 4Department of Oncology, Affiliated Aoyang Hospital of Jiangsu University, Zhangjiagang, Jiangsu, 215600, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jie-pin Li, Email zjgzy027@njucm.edu.cnPurpose: Low-grade gliomas (LGG) are primary brain tumors that often affect predominantly young adults, which usually have a painless course, and have a longer survival period compared to patients with high-grade gliomas. Relatively established treatment options include surgery, radiotherapy, chemotherapy or combination therapy, as well as individualized management based on tumor location, histology, molecular features and patient characteristics. Due to the rapid development of targeted therapies, the development of new molecular targets is now a very promising research direction.Methods: We explored the diagnostic value, clinical relevance, and molecular function of deoxynucleotidyl transferase terminal-interacting proteins 2 (DNTTIP2) in LGG using MethSurv, MEXPRESS, STRING, cBioPortal, Tumor Immunity Estimation Resource (TIMER) database, Gene Expression Profiling Interactive Analysis (GEPIA) databases. Besides, the “CIBERSORT” algorithm was conducted to estimate immune cells infiltration abundance, with “ggplot2” package visualizing the results. In vivo and vitro experiments were used to verify the speculations of bioinformatics analysis.Results: In LGG patients, DNTTIP1/2 were over-expressed at mRNA levels and high DNTTIP1/2 levels correlated with poor survival in LGG patients. We confirmed that DNTTIP2 significantly promotes M2 macrophage activation and angiogenesis, which may be related to the IL6/JAK/STAT3 signaling pathway. In addition, we found that DNTTIP2 amplification was associated with an unfavorable prognosis in LGG patients. We demonstrated, finally, a correlation between DNTTIP2 gene hypermethylation and a poor prognosis in LGG.Conclusion: This study demonstrated that DNTTIP1/2 had diagnostic and prognostic value in LGG patients. The biological mechanisms of DNTTIP2 regarding angiogenesis and macrophage activation may provide new insights into the treatment of glioma.Keywords: low-grade glioma, DNTTIP2, angiogenesis, M2 macrophages infiltration, bioinformatics

Bo-Wen Jiang,1,2,* Xue Ji,1,2,* Zhong-Qing Lyu,3 Bing Liang,1,2 Jian-Hang Li,3 Ling-Wei Zhu,1,2 Xue-Jun Guo,1,2 Jun Liu,1,2 Yang Sun,1,2 Yan-Jing Liu3 1Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, People’s Republic of China; 2Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Changchun, Jilin, People’s Republic of China; 3Third Affiliated Clinical Hospital to Changchun University of Chinese Medicine, Changchun, Jilin, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yang Sun; Yan-Jing Liu, Tel +86 431-86986933, Email sunyang10@hotmail.com; 214953156@qq.comPurpose: To elucidate the contribution of a transferable plasmid harboring the blaNDM-1 gene in an Escherichia coli clinical isolate to the spread of resistance determinants.Methods: Nine extended-spectrum β-lactamase-producing E. coli were collected from diarrhea samples from a pediatric patient and genetic linkage was investigated through enterobacteriaceae repetitive intragenic consensus polymerase chain reaction (PCR). Bacterial species were identified by 16s rRNA sequencing, susceptibility testing with the use of a BD PhoenixTM-100 Automated Microbiology System, and assessment of virulence genes by PCR. The transferability of blaNDM-1 in E. coli strain TCM3e1 was confirmed by conjugation experiments. Complete sequencing of E. coli strain TCM3e1 was determined with the PacBio and Illumina NovaSeq platforms and the characteristics were analyzed with bioinformatics software.Results: The results showed that all nine E. coli strains were the same clone. E. coli strain TCM3e1 was resistant to 12 antimicrobial agents and carried the virulence gene EAST-1. Conjugation transfer analysis showed that blaNDM-1 was carried on a self-transmissible plasmid. Two copies of the blaNDM-1 gene were present on an IncC plasmid and some resistance genes with two or three copies were located downstream of the blaNDM-1 gene and formed a tandem repeat fragment (blaDNM-1-bleo-sul1- aadA17- dfrA12).Conclusion: A transmissible plasmid harboring two copies of the blaNDM-1 gene, including clonal dispersions of the blaNDM-1 gene, was identified in clinical isolates. These findings emphasized the necessity of surveillance of the plasmid-borne blaNDM-1 to prevent dissemination.Keywords: diarrheagenic Escherichia coli, multi-drug-resistant, New Delhi metallo-β-lactamase, blaDNM-1 gene

Ling-Wang An,1 Xiang-Lan Li,1 Lin-Hui Chen,2 Hong Tang,3 Qun Yuan,4 Yan-Jun Liu,5 Yu Ji,6 Ju-Ming Lu1,7 1Department of Endocrinology, Beijing Ruijing Diabetes Hospital, Beijing, 100079, People’s Republic of China; 2Department of Endocrinology, Taiyuan Diabetes Hospital, Taiyuan, 030013, People’s Republic of China; 3Department of Share-Care Center, Chengdu Ruien Diabetes Hospital, Chengdu, 610000, People’s Republic of China; 4Department of Endocrinology, Heilongjiang Ruijing Diabetes Hospital, Harbin, 150009, People’s Republic of China; 5Department of Endocrinology, Lanzhou Ruijing Diabetes Hospital, Lanzhou, 730000, People’s Republic of China; 6Department of Endocrinology, Beijing Aerospace General Hospital, Beijing, 100076, People’s Republic of China; 7Department of Endocrinology, The General Hospital of the People’s Liberation Army, Beijing, 100853, People’s Republic of ChinaCorrespondence: Ju-Ming LuDepartment of Endocrinology, The General Hospital of the People’s Liberation Army, No. 28 of Fuxing Road, Haidian District, Beijing, 100853, People’s Republic of ChinaTel +86 10 8822 9999Email lujm666_1@21cn.comObjective: To analyse diabetes treatment, treatment change and self-management behaviours in association with 2-year glycaemic trajectories in patients with non-newly diagnosed type 2 diabetes mellitus in Chinese primary care.Methods: This was an observational, multi-centre, longitudinal, retrospective cohort study. Clinical data of 4690 subjects were extracted from electronic medical records, including serial glycated haemoglobin A1c (HbA1c) measurements, antidiabetic medication records and compliance to exercise, diet, medications and self-monitoring of blood glucose (SMBG). Patterns of longitudinal HbA1c trajectories were identified using the percentage of HbA1c measurements < 7.5% from the second available HbA1c measurement. Clinical relevance of the clusters was assessed through multivariable analysis.Results: Approximately half of the participants demonstrated good glycaemic control; of these, 34.5% demonstrated stable, good control, and 13.7% demonstrated relatively good control. About 16.2% demonstrated moderate control, and 35.6% demonstrated poor control. From the good to poor control groups, the percentage of subjects treated with insulin at baseline and during the follow-up period increased gradually, while the percentage of subjects adhering to exercise, diet, medications and SMBG decreased gradually. Compared with baseline, the adherence to exercise, diet, medications and SMBG improved significantly. Approximately 50% and 26% of subjects in the two poorest control groups, respectively, experienced treatment changes. After multivariable adjustments, baseline HbA1c ≥ 7.5%, HbA1c change ≥− 0.5% from baseline to visit 1, insulin treatment, treatment change, poor adherence to diet, exercise, SMBG during the follow-up period and HbA1c measurements < 3 per year were significantly associated with poorer glycaemic control.Conclusion: We identified four longitudinal HbA1c trajectories in patients with non-newly diagnosed type 2 diabetes. Even if baseline HbA1c is suboptimal, aggressive treatment changes, good adherence during the follow-up period, ≥ 3 HbA1c measurements per year and reducing HbA1c levels to a certain extent by the first follow-up visit were important for good, stable, long-term glycaemic control.Keywords: haemoglobin A1c, self-monitoring of blood glucose, diabetes, glycaemic trajectories, glycaemic control

Yuan-Jie Liu,1– 3,&ast; Sheng-Yan Yin,2,3,&ast; Shu-Hong Zeng,2,3 Yi-Dou Hu,1 Meng-Qi Wang,2,3 Pan Huang,1 Jie-Pin Li1– 3 1Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China; 2Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 3No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jie-Pin Li; Pan HuangDepartment of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Chang ‘an South Road No. 77, Zhangjiagang, Jiangsu, 215600, People’s Republic of ChinaTel +8615150224526; +8613921976647Email zjgzy027@njucm.edu.cn; yezipan2020@126.comPurpose: The identification of biomarkers and effective therapeutic targets for gastric cancer (GC), the most common cause of cancer-related deaths around the world, is currently a major focus in research. Here, we examined the utility of LHFPL6 as a prognostic biomarker and therapeutic target for GC.Methods: We explored the clinical relevance, function, and molecular role of LHFPL6 in GC using the MethSurv, cBioPortal, TIMER, Gene Expression Profiling Interactive Analysis, ONCOMINE, MEXPRESS, and EWAS Atlas databases. The GSE118919, GSE29272, and GSE13861 datasets were used for differential expression analysis. Using The Cancer Genome Atlas, we developed a Cox regression model and assessed the clinical significance of LHFPLs. In addition, we used the “CIBERSORT” algorithm to make reliable immune infiltration estimations. Western blot and immunohistochemistry were used to examine protein expression. Cell migration and invasion were assessed using transwell experiments. THP-1-derived macrophages and GC cells were co-cultured in order to model tumor–macrophage interactions in vitro. The levels of CD206 and CD163 were measured using immunofluorescence assays. The results were visualized with the “ggplot2” and “circlize” packages.Results: Our results showed that in GC, LHFPL6 overexpression was significantly associated with a poor prognosis. Our findings also suggested that LHFPL6 may be involved in the activation of the epithelial–mesenchymal transition. Furthermore, LHFPL6 expression showed a positive correlation with the abundance of M2 macrophages, which are potent immunosuppressors.Conclusion: LHFPL6 could be a prognostic biomarker and therapeutic target for GC.Keywords: gastric cancer, LHFPL6, biomarker, EMT, M2 macrophages

Yuan-Jie Liu,1,2,&ast; Jie-Pin Li,1– 3,&ast; Shu-Hong Zeng,1,2 Mei Han,1 Shen-Lin Liu,1,2 Xi Zou1 1Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2Department of No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xi Zou; Shen-lin Liu Email zxvery@126.com; lsljsszyy@126.comPurpose: Gastric cancer (GC) is a common type of cancer worldwide. It can relapse and metastasize even after standard treatment; therefore, it has a poor prognosis. Moreover, sensitive biomarkers for prognosis prediction in GC are lacking. In this study, using a bioinformatics approach, we aimed to examine the value of DAZ Interacting Protein 1 (DZIP1) as a prognostic predictor and therapeutic target in GC.Methods: We explored the clinical relevance, function, and molecular role of DZIP1 in GC using MethSurv, cBioPortal, TIMER, Gene Expression Profiling Interactive Analysis, IMEx, ONCOMINE, MEXPRESS, and EWAS Atlas databases. The GSE118919 dataset was used to plot receiver operating characteristic curves. Using The Cancer Genome Atlas, we developed a Cox regression model and assessed the clinical significance of DZIPs. In addition, we used the “xCELL” algorithm to make reliable immune infiltration estimations. Western blot and immunohistochemistry were used to examine protein expression. The results were visualized with the ‘ggplot2ʹ and “circlize” packages.Results: In GC patients, DZIP1 was over-expressed at both the mRNA and protein levels. High levels of DZIP1 were found to be associated with poor survival in patients with GC. Our results indicated that DZIP1 could be involved in multiple cancer-related pathways such as the PI3K-Akt signaling pathway, WNT signaling pathway, and RAS signaling pathway, and its expression was correlated with the infiltration of activated myeloid dendritic cells, naive CD4+ T cells, and naive CD8+ T cells. Furthermore, we found that mutations in DZIP1 were correlated with a good prognosis in GC patients. Finally, we demonstrated a correlation between hypomethylation of the DZIP1 gene promoter and a poor prognosis in GC.Conclusion: This study is the first to demonstrate a significant correlation between high levels of DZIP1 and a poor prognosis in GC patients. Our results clarify multiple potential mechanisms that could contribute to this correlation and may thus provide novel insights into the clinical diagnosis and treatment of GC.Keywords: gastric cancer, expression, DZIP, methylation, mutation, epithelial–mesenchymal transition, immune infiltration

Ai-Ling Liang,1,2,3,* Shen-Lin Du,4,* Bin Zhang,1,2,3,* Jing Zhang,5 Xuan Ma,1–3 Cui-Yun Wu,5 Yong-Jun Liu1–31Medical Molecular Diagnostics Key Laboratory of Guangdong, Guangdong Medical University, Dongguan, Guangdong 523808, People’s Republic of China; 2Department of Biochemistry and Molecular Biology, Guangdong Medical University, Dongguan, Guangdong 523808, People’s Republic of China; 3Department of Clinical Biochemistry, Guangdong Medical University, Dongguan, Guangdong 523808, People’s Republic of China; 4Department of Blood Transfusion, Dongguan Tung Wah Hospital, Dongguan, Guangdong 523210, People’s Republic of China; 5The Clinical Laboratory of Shunde Hospital, Southern Medical University, Shunde 528300, People’s Republic of China*These authors contributed equally to this workPurpose: To establish a gemcitabine-resistant lung adenocarcinoma cell line, A549/G+, and to screen the differences of miRNA expression in exosomes from A549 and A549/G+ cells.Methods: A549 cells were exposed in gemcitabine until they were resistant to gemcitabine, and extracted exosomes from A549 and A549/G+. The RNAs from exosomes were subjected to miRNA expression microarray experiments.Results: After 39 weeks of continuous induction, we induced drug resistance in A549 cells. The resistance index was 6. Via GeneChip miRNA 4.0 analysis, there were 446 differential miRNAs between A549 and A549/G+. Target gene prediction and pathway analysis discovered the microRNAs in the intersections may participate in drug resistance.Conclusion: These differential miRNAs help to do in-depth research to elucidate the mechanism of resistance to gemcitabine in non-small cell lung cancer.Keywords: non-small cell lung cancer, NSCLC, exosomes, miRNA, microarray, drug resistance, gemcitabine

Xing-Xi Pan,1 Li-Hua Tong,1 Yong-Fa Chen,1 Fei-Long Li,1 Wu-Bing Tang,1 Ya-Jie Liu,2 Wen Yang11Department of Oncology, Nanhai Hospital Affiliated to Southern Medical University, Foshan, Guangdong 528200, People’s Republic of China; 2Department of Oncology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, People’s Republic of ChinaObjective: This study aimed to establish a simplified T classification based on the 8th edition of the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) staging system for nasopharyngeal carcinoma (NPC).Methods: In total, 325 patients with NPC were included in this study. All patients underwent magnetic resonance imaging, and the staging criteria were recorded. These patients were subjected to staging with the 8th edition of the UICC/AJCC staging system for NPC.Results: Involvement of the oropharynx, nasal cavity, adjacent soft tissue (medial pterygoid, lateral pterygoid, and prevertebral muscles), cervical vertebra, orbit, and hypopharynx were always accompanied by other equivalently or more advanced T-stage classifications. All cases with involvement of the paranasal sinuses showed skull base erosion. The majority of cases with involvement of the pterygoid structure showed skull base erosion.Conclusion: According to the simplification principle, the following new T classification based on the 8th edition of the UICC/AJCC staging system was established: T1, tumor confined to nasopharynx, or beyond the nasopharynx without parapharyngeal involvement; T2, tumor with extension to the parapharyngeal space; T3, tumor with infiltration to bony structures at the skull base; T4, tumor with intracranial extension, involvement of the cranial nerves or parotid gland, and/or extensive soft tissue infiltration beyond the lateral surface of the lateral pterygoid muscle. Validation with a large series of patients is needed.Keywords: nasopharyngeal cancer, TNM staging system, T classification

Chunxi Liu,1,* Tingxian Liu,2,* Yongjun Liu,2 Na Zhang2 1Department of Pharmacy, Qilu Hospital, Shandong University, Ji’nan, China; 2School of Pharmaceutical Science, Shandong University, Ji’nan, China *These authors contributed equally to this work Background: We previously developed a simple effective system based on oligodeoxynucleotides with CGA repeating units (CGA-ODNs) for Dox and siRNA intracellular co-delivery. Methods: In the present study, the in vitro cytotoxicity, gene transfection and in vivo safety of the co-delivery system were further characterized and discussed. Results: Compared with poly(ethyleneimine) (PEI), both CGA-ODNs and the pH-sensitive targeted coating, o-carboxymethyl-chitosan (CMCS)-poly(ethylene glycol) (PEG)-aspargine-glycine-arginine (NGR) (CMCS-PEG-NGR, CPN) showed no obvious cytotoxicity in 72 h. The excellent transfection capability of CPN coated Dox and siRNA co-loaded nanoparticles (CPN-PDR) was confirmed by real-time PCR and Western blot analysis. It was calculated that there was no significant difference in silencing efficiency among Lipo/siRNA, CPN-modified siRNA-loaded nanoparticles (CPN-PR) and CPN-PDR. Furthermore, CPN-PDR was observed to be significantly much more toxic than Dox- and CPN-modified Dox-loaded nanoparticles (CPN-PD), implying their higher antitumor potential. Both hemolysis tests and histological assessment implied that CPN-PDR was safe for intravenous injection with nontoxicity and good biocompatibility in vitro and in vivo. Conclusion: The results indicated that CPN-PDR could be a potentially promising co-delivery carrier for enhanced antitumor therapy. Keywords: co-delivery, doxorubicin, VEGF, cytotoxicity, transfection

Yongjun Liu,1 Xiaoyun Wu,1 Xiaohe Sun,1 Dan Wang,1 Ying Zhong,1 Dandan Jiang,1 Tianqi Wang,1 Dexin Yu,2 Na Zhang1 1School of Pharmaceutical Science, Shandong University, 2Department of Radiology Medicine, Qilu Hospital, Jinan, People’s Republic of China Abstract: Developing magnetic resonance imaging (MRI) contrast agents with high relaxivity and specificity was essential to increase MRI diagnostic sensitivity and accuracy. In this study, the MRI contrast agent, vascular endothelial growth factor receptor (VEGFR)-targeted poly (l-lysine) (PLL)-diethylene triamine pentacetate acid (DTPA)-gadolinium (Gd) (VEGFR-targeted PLL-DTPA-Gd, VPDG), was designed and prepared to enhance the MRI diagnosis capacity of tumor. Biotin-PLL-DTPA-Gd was synthesized first, then, VEGFR antibody was linked to biotin-PLL-DTPA-Gd using biotin–avidin reaction. In vitro cytotoxicity study results showed that VPDG had low toxicity to MCF-7 cells and HepG2 cells at experimental concentrations. In cell uptake experiments, VPDG could significantly increase the internalization rates (61.75%±5.22%) in VEGFR-positive HepG2 cells compared to PLL-DTPA-Gd (PDG) (25.16%±4.71%, P

Bo Zhang,1 Yunmei Song,2 Tianqi Wang,1 Shaomei Yang,1 Jing Zhang,1 Yongjun Liu,1 Na Zhang,1 Sanjay Garg2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, People’s Republic of China; 2Centre for Pharmaceutical Innovation and Development (CPID), School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia Abstract: Combinational nanomedicine is becoming a topic of much interest in cancer therapy, although its translation into the clinic remains extremely challenging. One of the main obstacles lies in the difficulty to efficiently co-deliver immiscible hydrophilic/hydrophobic drugs into tumor sites. The aim of this study was to develop co-loaded lipid emulsions (LEs) to co-deliver immiscible hydrophilic/hydrophobic drugs to improve cancer therapy and to explore the co-delivery abilities between co-loaded LEs and mixture formulation. Multiple oxaliplatin/irinotecan drug–phospholipid complexes (DPCs) were formulated. Co-loaded LEs were prepared using DPC technique to efficiently encapsulate both drugs. Co-loaded LEs exhibited uniform particle size distribution, desired stability and synchronous release profiles in both drugs. Co-loaded LEs demonstrated superior anti-tumor activity compared with the simple solution mixture and the mixture of single-loaded LEs. Furthermore, co-loaded nanocarriers could co-deliver both drugs into the same cells more efficiently and exhibited the optimized synergistic effect. These results indicate that co-loaded LEs could be a desired formulation for enhanced cancer therapy with potential application prospects. The comparison between co-loaded LEs and mixture formulation is significant for pharmaceutical designs aimed at co-delivery of multiple drugs. Keywords: cancer, combination therapy, co-delivery, lipid emulsions, drug–phospholipid complex

Xiaofang Dong,1 Yanjin Liu,2 Aixia Wang,1 Min Wang11Neurology Department, 2Nursing Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, People’s Republic of ChinaObjective: To test the Chinese version of the SCI Exercise Self-Efficacy Scale (C-ESES) in stroke patients and evaluate its validity and reliability.Background: Physical inactivity is a well established and changeable risk factor for stroke, and regular exercise of 3–7 days per week is essential for stroke survivors and the general population. Though regular exercise is beneficial, it has been proved that duration, frequency, and intensity of exercise are generally low in stroke survivors.Methods: The performance of the instrument was assessed in 350 Chinese stroke survivors and repeated in 50 patients to examine test–retest reliability. Questionnaires included a form on demographic and socioeconomic characteristics, C-ESES, and the Chinese version of the General Self-Efficacy Scale. The AMOS 20.0 and SPSS 17.0 were chosen to evaluate their validity and reliability.Results: Even though 350 participants answered the questionnaires in the present study, useful data were obtained from 321 participants (response rate: 91.71%). Correlation between item and the total scale score (Item–Total Correlation) ranged from 0.551 to 0.718, indicating that no item needed to be omitted; two factors, with factor loading 0.620 and 0.806, were obtained from an exploratory principal components analysis, assuming 59.745% of the total variance. The two factors were named internal motivation and external motivation. A confirmatory factor analysis supported the results with a suitable model (χ2=291.157; df=185; P

Kai Liu,1 Xiquan Zhang,1 Guangqing Cao,1 Yongjun Liu,2 Chuanzhen Liu,1 Hourong Sun,1 Xinyan Pang1 1Cardiovascular Surgery Department, Qilu Hospital of Shandong University, 2Pharmacy College of Shandong University, Jinan, People’s Republic of China Purpose: To investigate whether inhalation of ethyl pyruvate (EP) encapsulated with poly(ethylene glycol)-block-lactide/glycolide copolymer nanoparticles (EP-NPs) can prevent the development of shunt-flow-induced hyperkinetic pulmonary arterial hypertension (PAH) in a rat model.Materials and methods: Rats were separated into five groups: blank (ie, no treatment after shunt flow), normal control (ie, no shunt flow or treatment), EP-NP instillation, EP-only instillation, and vehicle. The animals received intratracheal instillation of EP-NPs or other treatments immediately after a shunt flow, and treatment continued weekly until the end of the experiment. Hemodynamic data were recorded, pulmonary arterial remodeling was assessed, and levels of inflammatory mediators and ET1 expression in the lung and serum were analyzed. In addition, retention of EP in the lungs of rats in the EP-NP and EP-only groups was measured using high-performance liquid chromatography.Results: After 12 weeks, hemodynamic abnormalities and pulmonary arterial remodeling were improved in the EP-NP instillation group, compared with the blank, EP-only, and vehicle groups (P

Xiao-fang Dong,1 Yan-jin Liu,2 Ai-xia Wang,1 Pei-hua Lv1 1Neurology Department, 2Nursing Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China Background: It has been reported that stroke has a higher incidence and mortality rate in the People’s Republic of China compared to the global average. These conditions can be managed by proper medication use, but ensuring medication adherence is challenging.Objective: To translate the Self-Efficacy for Appropriate Medication Use Scale into Chinese and test its validity and reliability in patients with stroke.Methods: Instrument performances were measured from January 15, 2015 to April 28, 2015 on a convenience sample of 400 patients with stroke recruited at four neurology departments of the First Affiliated Hospital of Zhengzhou University. Questionnaires included the Chinese versions of the Self-Efficacy for Appropriate Medication Use Scale (C-SEAMS) and the General Self-Efficacy Scale (C-GSE). Construct validity, convergent validity, internal consistency, and test–retest reliability were measured.Results: Item analysis showed that item-to-total correlations were in the range of 0.362–0.672. Exploratory factor analysis revealed two factors (which accounted for 60.862% of total variance), with factor loading ranging from 0.534 to 0.756. Confirmatory factor analysis was performed to support the results, with an acceptable fit (χ2=73.716; df=64; P

Ai-Ling Liang,1–3,* Hai-Li Qian,4,* Ting-Ting Zhang,1–3 Ning Zhou,1–3 Hai-Juan Wang,4 Xi-Ting Men,4 Wei Qi,5 Ping-Ping Zhang,6 Ming Fu,4 Xiao Liang,4 Chen Lin,4 Yong-Jun Liu1–3 1Medical Molecular Diagnostics Key Laboratory of Guangdong, 2Department of Biochemistry and Molecular Biology, 3Department of Clinical Biochemistry, Guangdong Medical University, Dongguan, Guangdong, 4State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, 5Electroencephalogram Room, 6Department of Orthopedics, Guangdong Medical University Affiliated Hospital, Zhanjiang, Guangdong, People’s Republic of China *These authors contributed equally to this study Abstract: Breast cancer is the most common cancer and the leading cause of cancer-related death among women worldwide, with urgent need to develop new therapeutics. Targeted therapy is a promising strategy for breast cancer therapy. Stromal-derived factor-1/CXC chemokine receptor 4 (CXCR4) has been implicated in the metastasis of breast cancer, which renders it to be therapeutic target. This study aimed to evaluate the anticancer effect of fused TAT–DV1–BH3 polypeptide, an antagonist of CXCR4, and investigate the underlying mechanism for the cancer cell-killing effect in the treatment of breast cancer in vitro and in vivo. This results in a potent inhibitory effect of fused TAT–DV1–BH3 polypeptide on tumor growth and metastasis in nude mice bearing established MDA-MB-231 tumors. Fused TAT–DV1–BH3 polypeptide inhibited the proliferation of MDA-MB-231 and MCF-7 cells but did not affect that of HEK-293 cells. The fused TAT–DV1–BH3 polypeptide colocalized with mitochondria and exhibited a proapoptotic effect through the regulation of caspase-9 and -3. Furthermore, the fused TAT–DV1–BH3 polypeptide suppressed the migration and invasion of the highly metastatic breast cancer cell line MDA-MB-231 in a concentration-dependent manner. Notably, the DV1-mediated inhibition of the stromal-derived factor-1/CXCR4 pathway contributed to the antimetastasis effect, evident from the reduction in the level of phosphoinositide 3 kinase and matrix metalloproteinase 9 in MDA-MB-231 cells. Collectively, these results indicate that the apoptosis-inducing effect and migration- and invasion-suppressing effect explain the tumor regression and metastasis inhibition in vivo, with the involvement of caspase- and CXCR4-mediated signaling pathway. The data suggest that the fused TAT–DV1–BH3 polypeptide is a promising agent for the treatment of breast cancer, and more studies are warranted to fully elucidate the therapeutic targets and molecular mechanism. Keywords: bifunctional, fused polypeptide, CXCR4, breast cancer, apoptosis, transfer

Yong-Jun Liu, Miguel Arreola, Cassandra M Coleman, David C Lyon Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA, USA Abstract: Neurons in primary visual cortex (V1) integrate across the representation of the visual field through networks of long-range projecting pyramidal neurons. These projections, which originate from within V1 and through feedback from higher visual areas, are likely to play a key role in such visual processes as low contrast facilitation and extraclassical surround suppression. The extent of the visual field representation covered by feedback is generally much larger than that covered through monosynaptic horizontal connections within V1, and, although it may be possible that multisynaptic horizontal connections across V1 could also lead to more widespread spatial integration, nothing is known regarding such circuits. In this study, we used injections of the CVS-11 strain of rabies virus to examine disynaptic long-range horizontal connections within macaque monkey V1. Injections were made around the representation of 5° eccentricity in the lower visual field. Along the opercular surface of V1, we found that the majority of connected neurons extended up to 8 mm in most layers, consistent with twice the typically reported distances of monosynaptic connections. In addition, mainly in layer 6, a steady presence of connected neurons within V1 was observed up to 16 mm away. A relatively high percentage of these connected neurons had large-diameter somata characteristic of Meynert cells, which are known to project as far as 8 mm individually. Several neurons, predominantly in layer 6, were also found deep within the calcarine sulcus, reaching as far as 20° of eccentricity, based on estimates, and extending well into the upper visual field representation. Thus, our anatomical results provide evidence for a wide-ranging disynaptic circuit within V1, mediated largely through layer 6, that accounts for integration across a large region of the visual field. Keywords: visual cortex, horizontal connections, long-range lateral connections, Meynert cells, surround suppression, feedback

Peng Li, Donghua Liu, Lei Miao, Chunxi Liu, Xiaoli Sun, Yongjun Liu, Na ZhangSchool of Pharmaceutical Science, Shandong University, Jinan, Shandong, People’s Republic of ChinaBackground: The success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol.Methods: Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency.Results: CMCS-CLDPD had ellipsoidal shapes and showed “core-shell” structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS-CLDPD from serum interaction but also enhance gene transfection efficiency.Conclusion: These results demonstrated that multifunctional CMCS-CLDPD had pH-sensitivity, which may provide a new approach for the antitumor gene delivery.Keywords: layer-by-layer, multifunctional nanovector, pH-sensitivity, gene delivery

We document economically large cross-sectional differences in the before- and after-fee returns earned by speculative traders by analyzing day traders in Taiwan from 1992 to 2006. We sort day traders based on their returns in year y and analyze their performance in year y+1; the 500 top-ranked day traders go on to earn daily before-fee (after-fee) returns of 61.3 (37.9) bps per day; bottom-ranked day traders go on to earn daily before-fee (after-fee) returns of -11.5 (-28.9). bps per day. Less than 1% of the day trader population is able to predictably and reliably earn positive abnormal returns net of fees. © 2013 Elsevier B.V.

Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1-mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4-dependent manner. Thus, it is important to understand how the development of IL-4- versus interferon (IFN)-gamma-producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500-70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-gamma, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4(+)IFN-gamma(-) NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4(-)IFN-gamma(+) NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4(+)IFN-gamma(-) NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-gamma.