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Pingping Xiang,1,2,* Qi Liu,1,3,* Wangwei Jing,1,4 Yaping Wang,1 Hong Yu1,5,6 1Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310009, People’s Republic of China; 2Key Laboratory of Multiple Organ Failure (Zhejiang University), Ministry of Education, Hangzhou, Zhejiang Province, People’s Republic of China; 3Department of Cardiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China; 4Department of Cardiology, Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, Hangzhou, Zhejiang, People’s Republic of China; 5State Key Laboratory of Transvascular Implantation Devices, Hangzhou, 310009, People’s Republic of China; 6Binjiang Institute of Zhejiang University, Hangzhou, 310053, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hong Yu; Yaping Wang, Tel +86-571-87783992, Fax +86-571-88002709, Email yuvascular@zju.edu.cn; yapingwang@zju.edu.cnBackground and Purpose: Growth hormone-releasing hormone (GHRH) agonist, a 29-amino acid peptide, shows significant potential in treating myocardial infarction (MI) by aiding the repair of injured heart tissue. The challenge lies in the effective on-site delivery of GHRH agonist. This study explores the use of a targetable delivery system employing ROS-responsive PEG-PPS-PEG polymers to encapsulate and deliver GHRH agonist MR409 for enhanced therapeutic efficacy.Methods: We synthesized a self-assembling poly (ethylene glycol)-poly (propylene sulfide)-poly (ethylene glycol) polymer (PEG-PPS-PEG) amphiphilic polymer responsive to reactive oxygen species (ROS). The hydrophilic peptide GHRH agonist MR409 was encapsulated within these polymers to form nano PEG-PPS-PEG@MR409 vesicles (NPs). Cardiomyocyte apoptosis was induced under hypoxia and serum-free culture condition for 24 hours, and their production of ROS was detected by fluorescence dye staining. The cellular uptake of PEG-PPS-PEG@MR409 NPs was observed using fluorescence-labeled MR409. Targeting ability and therapeutic efficacy were evaluated using a mouse MI model.Results: PEG-PPS-PEG@MR409 NPs were efficiently internalized by cardiomyocytes, reducing ROS levels and apoptosis. These NPs exhibited superior targeting to the infarcted heart compared to naked MR409 peptide. With a reduced injection frequency (once every three days), PEG-PPS-PEG@MR409 NPs significantly promoted cardiac function recovery post-MI, matching the efficacy of daily MR409 injections.Conclusion: ROS-responsive PEG-PPS-PEG polymers provide a novel and effective platform for the targeted delivery of GHRH agonist peptides, improving cardiac function and offering a new approach for peptide therapy in MI treatment. Keywords: myocardial infarction, targeted therapy, growth hormone-releasing hormone, reactive oxygen species

Primary cilia are microtubule-based sensory and signaling organelles on the surfaces of most eukaryotic cells. Despite their early description by microscopy studies, islet cilia had not been examined in the functional context until recent decades. In pancreatic islets as in other tissues, primary cilia facilitate crucial developmental and signaling pathways in response to extracellular stimuli. Many human developmental and genetic disorders are associated with ciliary dysfunction, some manifesting as obesity and diabetes. Understanding the basis for metabolic diseases in human ciliopathies has been aided by close examination of cilia action in pancreatic islets at cellular and molecular levels. In this article, we review the evidence for ciliary expression on islet cells, known roles of cilia in pancreas development and islet hormone secretion, and summarize metabolic manifestations of human ciliopathy syndromes. We discuss emerging data on primary cilia regulation of islet cell signaling and the structural basis of cilia-mediated cell crosstalk, and offer our interpretation on the role of cilia in glucose homeostasis and human diseases.

Qixiu Li,1,2 Wenhua Jing,3 Zhen Wei,1,2 Yifan Wang,1,2 Long Sun1,2 1Centre for Health Management and Policy Research, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, People’s Republic of China; 2National Health Commission of China (NHC) Key Laboratory of Health Economics and Policy Research (Shandong University), Jinan, Shandong, People’s Republic of China; 3Hospital Service Department, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of ChinaCorrespondence: Long Sun, Centre for Health Management and Policy Research, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhuaxi Road, Jinan, Shandong, 250012, People’s Republic of China, Email sunlong@sdu.edu.cnBackground: Workplace violence (WPV) had always been the focus of attention in all walks of lives, especially in the health fields. Previous studies had shown it adversely affected mental health for healthcare workers. In addition, both sleep quality and physical activity were supported to have impact on mental health. However, the mechanism of sleep quality and physical activity influence the association between WPV and mental health had not been explored, so the purpose of this paper was to explore this mechanistic link among workplace violence, sleep quality, physical activity, and mental health in Chinese health technicians.Methods: Cross-sectional study was conducted in 3 cities of China, totally 3426 valid questionnaires were collected. WPV, physical activity, and social-demographic variables were evaluated. The Pittsburgh Sleep Quality Index and the Kessler Psychological Distress Scale were used to measure sleep quality and mental health. Descriptive analysis, univariate analysis, Pearson correlation, and moderated mediation analysis were used to estimate prevalence of WPV, association between WPV and mental health, and the role of sleep quality and physical activity on association between WPV and mental health.Results: The prevalence of WPV was 52.2% among Chinese health technicians. After controlling social-demographic and work-related variables, sleep quality partially mediated the effect of WPV on mental health (indirect effect=0.829). Physical activity moderated the relationship between WPV and sleep quality (β=0.235, p=0.013), but not find the moderating role between WPV and mental health (β=0.140, p=0.474), and between sleep quality and mental health (β=0.018, p=0.550).Conclusion: The rate of WPV among health technicians remained at an alarming level. Sleep quality and physical activity could mitigate the adverse effect of WPV on mental health. In the future, we could improve sleep quality and encourage health technicians to engage physical activity to decrease the negative effect of WPV on mental health.Keywords: workplace violence, mental health, sleep quality, physical activity

Diabetes is a growing pandemic affecting over ten percent of the U.S. population. Individuals with all types of diabetes exhibit glucose dysregulation due to altered function and coordination of pancreatic islets. Within the critical intercellular space in pancreatic islets, the primary cilium emerges as an important physical structure mediating cell-cell crosstalk and signal transduction. Many events leading to hormone secretion, including GPCR and second-messenger signaling, are spatiotemporally regulated at the level of the cilium. In this review, we summarize current knowledge of cilia action in islet hormone regulation and glucose homeostasis, focusing on newly implicated ciliary pathways that regulate insulin exocytosis and intercellular communication. We present evidence of key signaling proteins on islet cilia and discuss ways in which cilia might functionally connect islet endocrine cells with the non-endocrine compartments. These discussions aim to stimulate conversations regarding the extent of cilia-controlled glucose homeostasis in health and in metabolic diseases.

Primary cilia are slender cell-surface organelles that project into the intercellular space. In pancreatic beta cells, primary cilia coordinate a variety of cell responses including GPCR signaling, calcium influx, and insulin secretion, along with likely many underappreciated roles in islet development and differentiation. To study cilia function in islet biology, direct visualization of primary cilia by microscopic methods is often a necessary first step. Ciliary abundance, distribution, and morphology are heterogeneous among islet cells and are best visualized by fluorescence microscopy, the tools for which are readily accessible to most researchers. Here we present a collection of fluorescence imaging methods that we have adopted and optimized for the observation of primary cilia in mouse and human islets. These include conventional confocal microscopy using fixed islets and pancreas sections, live-cell imaging with cilia-targeted biosensors and probes, cilia motion recordings, and quantitative analysis of primary cilia waveform in the ex vivo environment. We discuss practical considerations and limitations of our approaches as well as new tools on the horizon to facilitate the observation of primary cilia in pancreatic islets.

Qi Liu,1,* Pingping Xiang,1,* Mingyao Chen,1 Yi Luo,1 Yun Zhao,1,2 Jinyun Zhu,1 Wangwei Jing,1 Hong Yu1 1Department of Cardiology, Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310009, People’s Republic of China; 2The Affiliated Cardiovascular Hospital of Qingdao University, Qingdao, Shandong Province, 266071, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hong YuDepartment of Cardiology, Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 JieFang Road, Hangzhou, Zhejiang Province, 310009, People’s Republic of ChinaTel +86-571-87783992Fax +86-571-88002709Email yuvascular@zju.edu.cnPurpose: The deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression.Methods: Transmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining.Results: We found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125.Conclusion: JNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.Keywords: vascular calcification, hydroxyapatite, apoptosis, mitochondria, osteogenic transformation, JNK

Primary cilia as a signaling organelle have garnered recent attention as a regulator of pancreatic islet function. These rod-like sensors exist on all major islet endocrine cell types and transduce a variety of external cues, while dysregulation of cilia function contributes to the development of diabetes. The complex role of islet primary cilia has been examined using genetic deletion targeting various components of cilia. In this review, we summarize experimental models for the study of islet cilia and current understanding of mechanisms of cilia regulation of islet hormone secretion. Consensus from these studies shows that pancreatic cilia perturbation can cause both endocrine and exocrine defects that are relevant to human disease. We discuss future research directions that would further elucidate cilia action in distinct groups of islet cells, including paracrine and juxtacrine regulation, GPCR signaling, and endocrine-exocrine crosstalk.

Ji Li,1,* Wang Jing,2,* Xiaoyang Zhai,2 Wenxiao Jia,2 Hui Zhu,2 Jinming Yu1 1Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, Hubei, 430060, People’s Republic of China; 2Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, 250117, Shandong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hui Zhu; Jinming Yu Tel +86-531-67626782; +86-531-67626947Email drzhuh@126.com; sdyujinming@163.comPurpose: A new tool based on clinical characteristics and molecular factors (Lung-molGPA) was developed to predict the survival of patients with non-small-cell lung cancer but was has not been validated. This study aims to validate the feasibility of the Lung-molGPA in NSCLC.Patients and Methods: Patients diagnosed NSCLC between Feb 2012 and July 2018 were retrospectively reviewed and scored using the Lung-molGPA tool to compare clinical outcomes. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated by Cox regression analyses.Results: A total of 618 patients (524 adenocarcinoma [ADC], 94 non-adenocarcinoma [non-ADC]) were collected. For all patients, the median survival time (MST) was 33.0 months (33.6 and 28 months in the ADC and non-ADC groups, respectively; p = 0.21). In the ADC group, the MST for patients with a Lung-molGPA score of 3.5 to 4 was more than 4 years, while the MST was only 25 months in patients scoring 0– 1, 30.0 months in patients scoring 1.5– 2, and 35.0 months for scores of 2.5– 3 (p = 0.048). For the non-ADC group, the MST for scores 0– 1, 1.5– 2, 2.5– 3, and 3.5– 4 were 12.0, 20.2, 29.0, and 33.0 months, respectively (p = 0.017).Conclusion: Our findings provided evidence validating the Lung-molGPA score as a useful tool to determine treatment strategies and to predict prognosis. The model is still exploratory and needs to be evaluated further in combination with additional prognostic markers.Keywords: NSCLC, GPA, brain metastasis, survival, prognosis

Abstract The separation of biomarkers from blood is straightforward in most molecular biology laboratories. However, separation in resource-limited settings, allowing for the successful removal of biomarkers for diagnostic applications, is not always possible. The situation is further complicated by the need to separate hydrophobic signatures such as lipids from blood. Herein, we present a microfluidic device capable of centrifugal separation of serum from blood at the point of need with a system that is compatible with biomarkers that are both hydrophilic and hydrophobic. The cross-flow filtration device separates serum from blood as efficiently as traditional methods and retains amphiphilic biomarkers in serum for detection.

Ying Zhu,1–3,* Lei Fu,2,* Wang Jing,2 Li Kong,2,4 Jinming Yu2,4 1Department of Clinical Medicine, Weifang Medical University, Weifang, Shandong Province, People’s Republic of China; 2Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Jinan, People’s Republic of China; 3Heze Medical College Affiliated Hospital, Heze, Shandong Province, People’s Republic of China; 4Shandong Academy of Medical Sciences, Jinan, Shandong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinming YuDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, 440 Jinan Road, Jinan 250117, Shandong Province, People’s Republic of ChinaTel +86 05 318 798 4777Fax +86 05 318 798 4079Email sdyujinming@163.comIntroduction: Adjuvant radiotherapy in non–small-cell lung cancer (NSCLC) remains controversial,Whether the mutation status of epidermal growth factor receptor (EGFR) will affect the recurrence and survival of patients with resected NSCLC is rarely reported. Our purpose is to study the effect of postoperative radiotherapy on patients with stage IIIA(N2) NSCLC with EGFR mutation.Methods: Total of of 115 patients diagnosed with stage IIIA(N2) resected NSCLC were analyzed retrospectively. Their EGFR mutations were detected by real-time quantitative PCR and DNA sequencing technology together.Results: At a median follow-up of 34.2 months for the postoperative adjuvant radiotherapy (PORT) group and 31.0 months for the non-PORT group, PORT group significantly improved progression free survival (PFS) and overall survival (OS). The median PFS and OS in the EGFR mutant group were not significantly longer than those in the EGFR wild-type group. The number of chemotherapy cycles, postoperative radiotherapy and the number of metastatic lymph nodes were independent factors influencing long-term survival.Conclusion: Our retrospective analysis showed that PORT can improve survival of patients with stage IIIA(N2) NSCLC. EGFR-mutant group with stage IIIA(N2) NSCLC has a tendency of a higher survival than the wild-type EGFR group, but there was no significant difference for both groups. The EGFR mutation status was not associated with PFS or OS of stage IIIA(N2) NSCLC.Keywords: non-small cell lung cancer, N2-stage, adjuvant radiotherapy, EGFR mutations

Yaru Tian,1 Xiaoyang Zhai,2 Hairong Tian,1 Wang Jing,2 Hui Zhu,1,2 Jinming Yu1,2 1Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong University, Jinan, People’s Republic of China; 2Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Science, Jinan, People’s Republic of ChinaCorrespondence: Hui ZhuDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Science, 440 Jiyan Road, Jinan, Shandong Province 250117, People’s Republic of ChinaTel +86 531 6762 6112Email drzhuh@126.comJinming YuDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong University, 44 Wen Hua Xi Road, Jinan, Shandong Province 250012, People’s Republic of ChinaTel +86 531 6762 6971Email sdyujinming@163.comBackground: The study aims to evaluate the clinical efficacy and safety of bevacizumab in combination with the first-line pemetrexed-platinum (PP) in patients with advanced adenocarcinoma non-small-cell lung cancer (NSCLC) and brain metastases.Methods: The clinical data of patients with adenocarcinoma NSCLC and symptomatic or asymptomatic brain metastases were collected in our study. The basic chemotherapy regimen was pemetrexed-platinum (PP). According to whether combined with bevacizumab (B) or not, all enrolled patients were assigned to the B+PP group or the PP alone group.Results: A total of 71 patients were enrolled in the current study. Twenty-six patients were allocated to the B+PP group and 45 were allocated to the PP group. Overall response rates (ORRs), disease control rates (DCRs) of the thoracic tumors and intracranial metastases and overall survival (OS) were not significantly different between the 2 groups. However, progression-free survival (PFS) and intracranial PFS (iPFS) were significantly prolonged in the B+PP group compared with the PP group. The median PFS was 9.2 and 8.2 months, and the 1-year PFS rates were 47.1% and 15.9%, respectively, in the 2 groups (P=0.029). And, the median iPFS were 24.3 and 10.9 months, and the 1-year iPFS rates were 80.1% and 40.1%, respectively, in the 2 groups (P=0.008). Univariate and multivariate analyses suggested that maintenance therapy and bevacizumab therapy were independent favorable prognostic factors of PFS and iPFS.Conclusion: The addition of bevacizumab to the first-line pemetrexed and platinum significantly improved clinical outcomes of patients with advanced adenocarcinoma NSCLC and brain metastases.Keywords: bevacizumab, non-small cell lung cancer, brain metastases, clinical outcome, toxicity

Ning An,1 Haoyi Wang,2 Ji Li,3 Xiaoyang Zhai,3 Wang Jing,3 Wenxiao Jia,3 Li Kong,3 Hui Zhu,3 Jinming Yu3 1Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong University, Jinan, People’s Republic of China; 2Department of Hematology, Qilu Hospital, Shandong University, Jinan, People’s Republic of China; 3Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, People’s Republic of ChinaCorrespondence: Hui Zhu; Jinming YuDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, People’s Republic of ChinaTel/fax +86 531 6762 6782; +86 531 6762 6947Email drzhuh@126.com; sdyujingming@163.comPurpose: Non-small cell lung cancer (NSCLC) patients with EGFR mutation are suffering from a high incidence of brain metastasis (BM). It is still controversial whether cranial radiotherapy could be delayed when the EGFR-tyrosine kinase inhibitors (TKIs) used as first-line therapy for EGFR-positive patients with BM. This study aims to investigate the therapeutic effect of TKIs combined with concurrent cranial radiotherapy on BM.Patients and methods: NSCLC patients with EGFR mutation and BM were retrospectively analyzed from January 2013 to December 2016 in Shandong Cancer Hospital. Identified cases were treated with first-line EGFR-TKIs with or without concurrent cranial radiation.Results: A total of 64 eligible patients were enrolled in this study, while 35 patients received first-line EGFR-TKIs plus cranial radiotherapy (RT+TKI group) and 29 patients received first-line EGFR-TKIs only (TKI alone group). The intracranial progression-free survival (PFS) of the RT+TKI group was significantly longer than the TKI alone group (25 vs 16 months; p=0.019), but no significant differences were observed between the two groups on extracranial PFS (20 vs 17 months, p=0.660). The median overall survival was also longer in the RT+TKI group (31 vs 24 months, p=0.019).Conclusion: Our retrospective data suggest that first-line TKIs plus concurrent cranial radiotherapy is a promising therapeutic strategy that led to remarkable intracranial PFS improvement and survival benefits for EGFR-mutant NSCLC with BM. Hence, it should be considered as a crucial treatment method during clinical management.Keywords: non-small cell lung cancer, EGFR mutation, brain metastasis, radiotherapy, tyrosine kinase inhibitor

Ning An,1,* Haoyi Wang,2,* Hui Zhu,3 Weiwei Yan,3 Wang Jing,3 Li Kong,3 Yan Zhang,3 Jinming Yu3 1Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong University, Jinan, People’s Republic of China; 2Department of Hematology, Qilu Hospital, Shandong University, Jinan, People’s Republic of China; 3Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yan Zhang; Jinming YuDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, People’s Republic of ChinaTel +86 5 316 762 6782; +86 5 316 762 6947Fax +86 53 167 62 6782; +86 5 316 762 6947Email Zhangyan0681@163.com; sdyujingming@163.comAbstract: EGFR)-targeted drugs have been the first-line treatment for patients with EGFR-mutant non-small cell lung cancer (NSCLC), especially exon 19 deletions and L858R mutation in exon 21. However, there is insufficient evidence for other less common types of EGFR mutations, such as delE709_T710insD (del 18). Recent studies have revealed that these rare genotypes could be targetable if appropriate mutations, such as delE709_T710insD (del 18). Recent studies have revealed that these rare genotypes could be targetable if appropriate EGFR tyrosine kinase inhibitors are selected. Here we reported a stage Ⅳ NSCLC patient with delE709_T710insD mutation who responded well to afatinib, a second-generation TKI. Afatinib had taken good control of the patient’s brain metastasis with a progression-free survival of 11 months and an overall survival exceeded 21 months, although he had received multi-line therapy. This case demonstrates EGFR delE709_T710insD is a rare but potentially afatinib responsive mutation in NSCLC, which may contribute to changes in clinical practice and further research into the precise detection and treatment of rare mutations in EGFR.Keywords: non-small-cell lung cancer, epidermal growth factor receptor, molecular targeted therapy, tyrosine kinase inhibitor, afatinib, EGFR rare mutation

Wei Jing,1 Na Song,2,3 Yun-Peng Liu,2,3 Xiu-Juan Qu,2,3 Ya-Fei Qi,4 Ce Li,2,3 Ke-Zuo Hou,2,3 Xiao-Fang Che,2,3 Xiang-Hong Yang41The First Department of Oncology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of China; 2Department of Medical Oncology, The First Hospital of China Medical University, Shenyang, People’s Republic of China; 3Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province, The First Hospital of China Medical University, Shenyang, People’s Republic of China; 4Department of Pathology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of ChinaBackground: Although aberrant DNA methyltransferase 3a (DNMT3a) expression is important to the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC), the role of DNMT3a in PDAC prognosis is not clarified yet due to the limited studies and lacking of underlying molecular mechanism.Methods: The expression of DNMT3a was examined by immunohistochemistry in PDAC tissues. Gene expression profiles assays were conducted to explore the impact of DNMT3a on biological processes and signal pathways. Cell cycle and apoptosis were measured by flow cytometry. Western blotting and real-time qPCR assays were used to explore the impact of DNMT3a on expression of protein and mRNA related to cell cycle, STAT3 signaling pathway and apoptosis.Results: DNMT3a was overexpressed and closely associated with poor outcomes of PDAC. DNMT3a knockdown restrained PDAC cell proliferation, induced cell cycle arrest and promoted apoptosis in vitro. Affymetrix GeneChip Human Transcriptome Array identified that the cell cycle-related process was most significantly associated with DNMT3a. DNMT3a knockdown induced G1-S phase transition arrest by decreasing the expression of cyclin D1, which was mediated by the reduction of IL8 and the subsequent inactivation of STAT3 signaling pathway. Furthermore, exogenous apoptosis was also promoted after DNMT3a knockdown, probably via up-regulation of DNA transcription and expression in CASP8.Conclusion: These findings indicate that DNMT3a plays an important role in PDAC progression. DNMT3a may serve as a prognostic biomarker and a therapeutic strategy candidate in PDAC.Keywords: DNA methyltransferase 3a, pancreatic ductal adenocarcinoma, prognosis, proliferation

Jing Wang,1* Yu Pang,2* Wei Jing,1* Wei Chen,1* Ru Guo,1* Xiqin Han,1 Limin Wu,3 Guangxu Yang,4 Kunyun Yang,5 Cong Chen,6 Lin Jiang,7 Chunkui Cai,8 Zhi Dou,9 Lijuan Diao,10 Hongqiu Pan,11 Jianyun Wang,12 Feifei Du,13 Tao Xu,14 Lixia Wang,15 Renzhong Li,16 Naihui Chu1 1Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing 101149, P.R. China; 2National Clinical Laboratory on Tuberculosis, Beijing Chest Hospital affiliated to Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing 101149, P.R. China; 3Tuberculosis Prevention Institute, Hangzhou Center for Disease Control and Prevention, Zhejiang 310021, P.R. China; 4Tuberculosis Prevention Institute, Changchun Infectious Disease Hospital, Jilin 130123, P.R. China; 5Department of Tuberculosis Resistance, Hunan Chest Hospital, Hunan 410013, P.R. China; 6Multidrug Resistant Tuberculosis Project Office, Wuhan Pulmonary Hospital, Hubei 430030, P.R. China; 7Department of Internal Medicine, The Fifth People’s Hospital of Ganzhou, Jiangxi 341000, P.R. China; 8Department of Drug-Resistant Tuberculosis, Dalian Tuberculosis Hospital, Liaoning, 116033, P.R. China; 9Department of Tuberculosis, Qiqihar Tuberculosis Prevention and Control Institute, Heilongjiang 161006, P.R. China; 10Department of Tuberculosis, Nanyang Tuberculosis Control Institute, Henan 473000, P.R. China; 11Department of Tuberculosis, The Third People’s Hospital of Zhenjiang, Jiangsu 212000, P.R. China; 12Department of Infection, Lanzhou Pulmonary Hospital, Gansu 730046, P.R. China; 13Department of Respiratory, The Fourth People’s Hospital of Ningxia Hui Autonomous Region, Ningxia 750021, P.R. China; 14Department of Epidemiology and Biostatistics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, P.R. China; 15National Center for Tuberculosis Control and Prevention, Chinese Center For Disease Control and Prevention, Beijing 102206, P.R. China; 16Department of Tuberculosis Resistance Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, P.R. China *These authors contributed equally to this work Background: Our aim was to assess whether the use of cycloserine (CS) would bring additional benefit for multidrug-resistant tuberculosis (MDR-TB) patients, and to estimate the incidence and associated risk factors of adverse drug reactions (ADRs) from CS.Patients and methods: In this study, we retrospectively reviewed the clinical outcomes and ADRs of MDR-TB patients treated with CS containing regimens between January 2012 and June 2015 in China.Results: A total of 623 MDR-TB cases enrolled in this study received regimens containing CS. Of these cases, in 411 of the patients 374 (66.0%) were “cured” and 37 (5.9%) “complete treatment” by the end of the study. The elderly, patients with prolonged previous exposure to and history of anti-TB drugs, and pre-existing co-morbidity were more likely to be associated with adverse outcomes of MDR-TB patients (P

Bin Liang,1,* Na Li,2,* Shuofei Zhang,3 Aihua Qi,4 Jianhua Feng,1 Weiwei Jing,5 Changcan Shi,2 Zhaipu Ma,6 Shenmeng Gao7 1Department of Hematology, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, Zhejiang 325000, China; 2Wenzhou Institute of Biomaterials and Engineering, CNITECH, CAS, Wenzhou, Zhejiang 325000, China; 3Department of Orthodontics, Stomatological Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325000, China; 4Department of Internal Medicine, Zaoqiang People’s Hospital, Zaoqiang, Hebei 053100, China; 5Department of Obstetrics and Gynecology, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, Wenzhou, Zhejiang 325000, China; 6Department of Bioinformatics, College of Life Science, Hebei University, Baoding, Hebei 071002, China; 7Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, Zhejiang 325000, China *These authors contributed equally to this work Purpose: Nanoparticle (NP)-based drug delivery approaches have tremendous potential for enhancing treatment efficacy and decreasing doses of chemotherapeutics. Idarubicin (IDA) is one of the most common chemotherapeutic drugs used in the treatment of acute myeloid leukemia (AML). However, severe side effects and drug resistance markedly limit the application of IDA.Methods: In this study, we encapsulated IDA in polymeric NPs and validated their antileukemia activity in vitro and in vivo.Results: NPs with an average diameter of 84 nm was assembled from a methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide) (mPEG-PLGA). After loading of IDA, IDA-loaded mPEG-PLGA NPs (IDA/mPEG-PLGA NPs) were formed. The in vitro release data showed that the IDA/mPEG-PLGA NPs have excellent sustained release property. IDA/mPEG-PLGA NPs had exhibited the lower IC50 than pure IDA. Moreover, IDA/mPEG-PLGA NPs in the same concentration substantially induced apoptosis than did pure IDA. Most importantly, IDA/MPEG-PLGA NPs significantly decreased the infiltration of leukemia blasts and improved the overall survival of MLL-AF9-induced murine leukemia compared with free IDA. However, the blank NPs were nontoxic to normal cultured cells in vitro, suggesting that NPs were the safe carrier.Conclusion: Our data suggest that IDA/mPEG-PLGA NPs might be a suitable carrier to encapsulate IDA. Low dose of IDA/mPEG-PLGA NPs can be used as a conventional dosage for antileukemia therapy to reduce side effect and improve survival. Keywords: idarubicin, mPEG-PLGA, acute myeloid leukemia, nanoparticles

Yanzhe Liu,1,* Lijuan Shao,2,* Kuang Chen,1 Zizheng Wang,1 Jin Wang,1 Wei Jing,3 Minggen Hu1 1Department of Hepatobiliary and Pancreatic Surgical Oncology, Chinese PLA General Hospital, Beijing, People’s Republic of China; 2Translational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University, Shenzhen, People’s Republic of China; 3Department of General Surgery, Changhai Hospital, The Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Growth differentiation factor (GDF) acted as a factor that regulated proliferation, apoptosis and differentiation in several tumors. However, the effects of growth differentiation factor (GDF11) in pancreatic cancer remain unclear.Purpose: To investigate the expression and significance of GDF11 in pancreatic cancer.Patients and methods: Pancreatic cancer and corresponding paracancerous tissues (n=28) were collected from the Department of Hepatobiliary and Pancreatic Surgical Oncology of Chinese PLA General Hospital. Tissue microarray was obtained from Outdo Biotech Co., Ltd. (Shanghai, People’s Republic of China). GDF11 mRNA and protein expressions in pancreatic cancer samples and cell lines were detected using qRT-PCR, Western-Blot and immunohistochemistry. Overexpression and knockdown of GDF11 were performed with lentiviral transduction system and siRNA technique in PANC-1 cell line and CFPAC-1 cell line. Proliferation, migration and invasion of pancreatic cancer cell lines were examinated by MTS and transwell assay, respectively. Flow cytometry was used for cell apoptosis analysis.Results: The results of this study indicated that GDF11 was significantly down-regulated in pancreatic cancer tissues compared with adjacent tissues of pancreatic cancer. GDF11 was also associated with low expression in pancreatic cancer cell lines when compared with normal pancreatic cell line. In a cohort of 63 pancreatic cancer patients, high GDF11 expression levels was associated with favorable perineural invasion, T classification, N classification and overall survival (OS). Cox proportional hazards model revealed that high GDF11 expression was an independent predictor of favorable prognosis (HR: 0.496; 95% CI: 0.255–0.967; P=0.040). Overexpression of GDF11 in PANC-1 cells repressed the proliferation, migration and invasion abilities in vitro. Inhibition of GDF11 in CFPAC-1 showed inverse results. Furthermore, enhanced GDF11 expression promoted apoptosis and down-regulated GDF11 expression inhibited apoptosis in pancreatic cancer cell lines. Conclusion: These findings suggested that GDF11 acted as a tumor suppressor gene for pancreatic cancer. Keywords: GDF11, pancreatic cancer, proliferation, apoptosis, prognosis

Abstract Maintenance of functional β‐cell mass is critical to preventing diabetes, but the physiological mechanisms that cause β‐cell populations to thrive or fail in the context of obesity are unknown. High fat‐fed SM/J mice spontaneously transition from hyperglycemic‐obese to normoglycemic‐obese with age, providing a unique opportunity to study β‐cell adaptation. Here, we characterize insulin homeostasis, islet morphology, and β‐cell function during SM/J’s diabetic remission. As they resolve hyperglycemia, obese SM/J mice dramatically increase circulating and pancreatic insulin levels while improving insulin sensitivity. Immunostaining of pancreatic sections reveals that obese SM/J mice selectively increase β‐cell mass but not α‐cell mass. Obese SM/J mice do not show elevated β‐cell mitotic index, but rather elevated α‐cell mitotic index. Functional assessment of isolated islets reveals that obese SM/J mice increase glucose‐stimulated insulin secretion, decrease basal insulin secretion, and increase islet insulin content. These results establish that β‐cell mass expansion and improved β‐cell function underlie the resolution of hyperglycemia, indicating that obese SM/J mice are a valuable tool for exploring how functional β‐cell mass can be recovered in the context of obesity.

Dong Guo,1,2,* Wang Jing,2,3,* Hui Zhu,2 Minghuan Li,2 Bing Zou,2 Yan Zhang,2 Lei Fu,2 Li Kong,2 Jinbo Yue,2,4 Jinming Yu2,4 1Department of Oncology, Clinical College, Weifang Medical University, Weifang, Shandong Province, People’s Republic of China; 2Department of Radiotherapy, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong Province, People’s Republic of China; 3Department of Radiotherapy, The First Affiliated Hospital to Zhengzhou University, Zhengzhou, Henan Province, People’s Republic of China; 4Shandong Academy of Medical Sciences, Jinan, Shandong Province, People’s Republic of China *These authors contributed equally to this work Background: Although the role of prophylactic cranial irradiation (PCI) in the treatment of small cell lung cancer (SCLC) has been confirmed, the occurrence of brain metastases (BM) in patients remains a major problem. We designed this study to evaluate the clinical value of carcinoembryonic antigen (CEA) for predicting the incidence of BM and survival in SCLC patients who received PCI.Materials and methods: The records of 128 consecutive SCLC patients, who underwent PCI in our institute between 2005 and 2015, were analyzed. The collected data included clinicopathological features and the levels of CEA, neuron-specific enolase (NSE), cytokeratin 19 fragments (CYFRA21-1), and albumin. Kaplan–Meier and Cox regression analyses were used to determine the factors that affect BM and survival in SCLC patients after PCI.Results: In total, 128 patients were identified, with a median (range) age of 62 (30–83) years. Thirty-two patients developed BM at some time during follow-up. The median levels of CEA, NSE, CYFRA21-1, and albumin were 7.6 ng/mL, 44 ng/mL, 4.6 ng/mL, and 42.1 g/L, respectively. In the multivariate analysis, CEA level (HR: 2.479, 95% CI: 1.101–5.581; P=0.028), advanced clinical stage (HR: 2.929, 95% CI: 1.338–6.413; P=0.007), and NSE level (HR: 3.021, 95% CI: 1.226–7.442; P=0.016) were significantly correlated with BM. CEA (HR: 1.903, 95% CI: 1.133–3.195; P=0.015) and advanced clinical stage (HR: 2.002, 95% CI: 1.227–3.267; P=0.005) were independently associated with worse overall survival in SCLC patients.Conclusion: CEA is an independent predictive factor for the incidence of BM after PCI in SCLC and can be used as a predictor of BM in SCLC. In addition, a high level of CEA indicates a poor prognosis in SCLC patients after PCI. Prospective randomized clinical studies are required to confirm these findings. Keywords: carcinoembryonic antigen, brain metastases, predicting, survival, small cell lung cancer

Xianwei Zhang,1 Ping Luo,1 Wei Jing,1 Hu Zhou,2 Chunzi Liang,1 Jiancheng Tu1 1Department of Clinical Laboratory Medicine, Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, People’s Republic of China; 2Department of Blood Transfusion, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China Background: Circular RNAs (circRNAs) are a class of widely distributed non-coding RNAs, which drew little attention for decades. Recent studies show that circRNAs are involved in cancer progression.Methods: The circSMAD2 expression in HCC and adjacent non-tumor tissues was measured by quantitative real-time polymerase chain reaction, and the biological function of circSMAD2 was explored by proliferation, apoptosis, migration, invasion, and Western blot assays. Next, the dual-luciferase reporter assay was performed to identify the target miRNA of circSMAD2. Finally, circSMAD2 and its target miRNA were co-transfected in HCC cells to investigate their relationship to HCC progression.Results: In this study, we found that circRNA SMAD2 (circSMAD2) expression was downregulated in hepatocellular carcinoma (HCC) tissues (P = 0.014) compared to the adjacent non-tumor tissues and markedly associated with the differentiation degree of the HCC tissues (P < 0.001). The in vitro experiments showed that overexpressed circSMAD2 inhibited the migration, invasion, and epithelial–mesenchymal transition (EMT) in HCC cells. Bioinformatics predicted that miR-629 is a potential target of circSMAD2, and the dual-luciferase reporter assay verified that miR-629 directly bound circSMAD2. In addition, we found that overexpression of circSMAD2 suppressed the expression of miR-629 in HCC cells, whereas knockdown of circSMAD2 upregulated the expression of miR-629. Furthermore, co-transfection of miR-629 mimics with circSMAD2 reversed the circSMAD2 effects of inhibiting the migration, invasion, and EMT of HCC cells.Conclusion: Altogether, our data support that circSMAD2 inhibits the migration, invasion, and EMT of HCC cells by targeting miR-629. Keywords: circular RNA, circSMAD2, hepatocellular carcinoma, epithelial–mesenchymal transition, microRNA-629

Background & Aims: Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. Methods: To analyze the evolution of the buoyant density of in vivoâderived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. Results: Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Conclusions: Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet. Keywords: HCV, Lipoprotein, Mouse Model, Human Liver Chimeric Mice

Ge Song,1,2,* Wang Jing,3,* Song Xue,1,2 Hongbo Guo,4 Jinming Yu1,2 1Department of Radiation Oncology, School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences; 2Department of Radiation Oncology, Shandong Cancer Hospital Affiliated with Shandong University, Jinan, 3Department of Radiation Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 4Department of Thoracic Surgery, Shandong Cancer Hospital Affiliated with Shandong University, Jinan, China *These authors contributed equally to the work Purpose: This study aimed to investigate the incidence of nodal skip metastasis (NSM) to identify the risk factors that influence NSM and to assess the prognostic value of NSM in patients with thoracic esophageal squamous cell carcinoma (ESCC).Patients and methods: Between January 2009 and December 2013, 285 patients with ESCC with positive lymph nodes who underwent complete resection were enrolled.Results: For the entire group, NSM occurred in 32.3% (92/285) of patients. The median survival time and 5-year survival rate in the NSM group were 28 months and 12.0%, respectively, compared with 36.3 months and 25.0%, respectively, in the non-NSM group (P=0.008). Both N stage (P=0.001) and T stage (P=0.014) were associated with the incidence of NSM. NSM (P=0.008), T stage (P=0.000), and N stage (P=0.000) were independent prognostic factors for survival. In the NSM group, T stage (P=0.014) and N stage (P=0.000) were independent prognostic factors for survival.Conclusion: It was concluded that NSM is common in ESCC and is associated with poor survival. Keywords: esophageal squamous cell carcinoma, surgery, nodal skip metastasis, incidence, risk factors, prognosis

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host–microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

Wang Jing,1,2,* Miaomiao Li,3,* Yan Zhang,2 Feifei Teng,2 Anqin Han,2 Li Kong,2 Hui Zhu2 1Weifang Medical University, Weifang, Shandong Province, People’s Republic of China; 2Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan, Shandong Province, People’s Republic of China; 3Shandong Medical College, Jinan, Shandong Province, People’s Republic of China *Both these authors contributed equally to the work Abstract: Lung cancer is the leading cause of cancer death in males and the second leading cause of death in females worldwide. Non-small-cell lung cancer (NSCLC) is the main pathological type of lung cancer, and most newly diagnosed NSCLC patients cannot undergo surgery because the disease is already locally advanced or metastatic. Despite chemoradiotherapy and targeted therapy improving clinical outcomes, overall survival remains poor. Immune checkpoint blockade, especially blockade of programmed death-1 (PD-1) receptor and its ligand PD-L1, achieved robust responses and improved survival for patients with locally advanced/metastatic NSCLC in preclinical and clinical studies. However, with regard to PD-1/PD-L1 checkpoint blockade as monotherapy or in combination with other antitumor therapies, such as chemotherapy, radiotherapy (including conventional irradiation and stereotactic body radiotherapy), and target therapy, there are still many unknowns in treating patients with NSCLC. Despite this limited understanding, checkpoint blockade as a novel therapeutic approach may change the treatment paradigm of NSCLC in the future. Here we review the main results from completed and ongoing studies to investigate the feasibility of PD-1/PD-L1 inhibitors, as monotherapy or combinatorial agents in patients with locally advanced and metastatic NSCLC, and explore optimal strategy in such patients. Keywords: immunotherapy, checkpoint, PD-1, PD-L1, NSCLC

Several techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations.

Abstract Objective Acquired clitoromegaly is rare and may result from hormonal and nonhormonal causes, and evaluation of the pregnant patient with clitoromegaly invokes a specific set of differential diagnoses. Methods Case report. Results We describe the case of a young woman with pregnancy-associated clitoral enlargement whose hormonal evaluation proved negative. Further investigation concluded that an epidermoid cyst was the culprit of her pseudoclitoromegaly. The patient underwent successful surgical resection and has had no recurrence at her subsequent pregnancy. Conclusion We review the differential diagnosis of clitoromegaly, including hormonal and nonhormonal causes, with focus on the evaluation of pregnancy-associated clitoromegaly.

The internal state of an organism influences its perception of attractive or aversive stimuli and thus promotes adaptive behaviors that increase its likelihood of survival. The mechanisms underlying these perceptual shifts are critical to our understanding of how neural circuits support animal cognition and behavior. Starved flies exhibit enhanced sensitivity to attractive odors and reduced sensitivity to aversive odors. Here, we show that a functional remodeling of the olfactory map is mediated by two parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion at the level of the first synapse. Short neuropeptide F sensitizes an antennal lobe glomerulus wired for attraction, while tachykinin (DTK) suppresses activity of a glomerulus wired for aversion. Thus we show parallel neuromodulatory systems functionally reconfigure early olfactory processing to optimize detection of nutrients at the risk of ignoring potentially toxic food resources.

Acquisition of distinct neuronal identities during development is critical for the assembly of diverse functional neural circuits in the brain. In both vertebrates and invertebrates, intrinsic determinants are thought to act in neural progenitors to specify their identity and the identity of their neuronal progeny. However, the extent to which individual factors can contribute to this is poorly understood. We investigate the role of orthodenticle in the specification of an identified neuroblast (neuronal progenitor) lineage in the Drosophila brain. Loss of orthodenticle from this neuroblast affects molecular properties, neuroanatomical features, and functional inputs of progeny neurons, such that an entire central complex lineage transforms into a functional olfactory projection neuron lineage. This ability to change functional macrocircuitry of the brain through changes in gene expression in a single neuroblast reveals a surprising capacity for novel circuit formation in the brain and provides a paradigm for large-scale evolutionary modification of circuitry.

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host-pathogen interactions.

Animals must set behavioural priority in a context-dependent manner and switch from one behaviour to another at the appropriate moment1-3. Here we probe the molecular and neuronal mechanisms that orchestrate the transition from feeding to courtship in Drosophila melanogaster. We find that feeding is prioritized over courtship in starved males, and the consumption of protein-rich food rapidly reverses this order within a few minutes. At the molecular level, a gut-derived, nutrient-specific neuropeptide hormone-Diuretic hormone 31 (Dh31)-propels a switch from feeding to courtship. We further address the underlying kinetics with calcium imaging experiments. Amino acids from food acutely activate Dh31+ enteroendocrine cells in the gut, increasing Dh31 levels in the circulation. In addition, three-photon functional imaging of intact flies shows that optogenetic stimulation of Dh31+ enteroendocrine cells rapidly excites a subset of brain neurons that express Dh31 receptor (Dh31R). Gut-derived Dh31 excites the brain neurons through the circulatory system within a few minutes, in line with the speed of the feeding-courtship behavioural switch. At the circuit level, there are two distinct populations of Dh31R+ neurons in the brain, with one population inhibiting feeding through allatostatin-C and the other promoting courtship through corazonin. Together, our findings illustrate a mechanism by which the consumption of protein-rich food triggers the release of a gut hormone, which in turn prioritizes courtship over feeding through two parallel pathways.

In their pioneering study on dopamine release, Romo and Schultz speculated "...that the amount of dopamine released by unmodulated spontaneous impulse activity exerts a tonic, permissive influence on neuronal processes more actively engaged in preparation of self-initiated movements...."1 Motivated by the suggestion of "spontaneous impulses," as well as by the "ramp up" of dopaminergic neuronal activity that occurs when rodents navigate to a reward,2-5 we asked two questions. First, are there spontaneous impulses of dopamine that are released in cortex? Using cell-based optical sensors of extrasynaptic dopamine, [DA]ex,6 we found that spontaneous dopamine impulses in cortex of naive mice occur at a rate of ∼0.01 per second. Next, can mice be trained to change the amplitude and/or timing of dopamine events triggered by internal brain dynamics, much as they can change the amplitude and timing of dopamine impulses based on an external cue?7-9 Using a reinforcement learning paradigm based solely on rewards that were gated by feedback from real-time measurements of [DA]ex, we found that mice can volitionally modulate their spontaneous [DA]ex. In particular, by only the second session of daily, hour-long training, mice increased the rate of impulses of [DA]ex, increased the amplitude of the impulses, and increased their tonic level of [DA]ex for a reward. Critically, mice learned to reliably elicit [DA]ex impulses prior to receiving a reward. These effects reversed when the reward was removed. We posit that spontaneous dopamine impulses may serve as a salient cognitive event in behavioral planning.

Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). Here, we find that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket 25 (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK25 mediates Ca2+-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK25 expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance our understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species.

Critical to evolutionary fitness, animals regulate social behaviors by integrating signals from both their external environments and internal states. Here, we find that population density modulates the courtship behavior of male Drosophila melanogaster in an age-dependent manner. In a competitive mating assay, males reared in a social environment have a marked advantage in courting females when pitted against males reared in isolation. Group housing promotes courtship in mature (7-day) but not immature (2-day) males; this behavioral plasticity requires the Or47b pheromone receptor. Using single-sensillum recordings, we find that group housing increases the response of Or47b olfactory receptor neurons (ORNs) only in mature males. The effect of group housing on olfactory response and behavior can be mimicked by chronically exposing single-housed males to an Or47b ligand. At the molecular level, group housing elevates Ca2+ levels in Or47b ORNs, likely leading to CaMKI-mediated activation of the histone-acetyl transferase CBP. This signaling event in turn enhances the efficacy of juvenile hormone, an age-related regulator of reproductive maturation in flies. Furthermore, the male-specific Fruitless isoform (FruM) is required for the sensory plasticity, suggesting that FruM functions as a downstream genomic coincidence detector in Or47b ORNs-integrating reproductive maturity, signaled by juvenile hormone, and population density, signaled by CBP. In all, we identify a neural substrate and activity-dependent mechanism by which social context can directly influence pheromone sensitivity, thereby modulating social behavior according to animals' life-history stage.