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Baoliang Liu,1,* Haotian Zheng,1,* Guoyuan Ma,1,2 Hongchang Shen,3 Zhaofei Pang,3 Gemu Huang,4 Qingtao Song,4 Guanghui Wang,1,2 Jiajun Du1,2 1Department of Thoracic Surgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, People’s Republic of China; 2Department of Thoracic Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, People’s Republic of China; 3Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, People’s Republic of China; 4Research and Development Department, Amoy Diagnostics Co., LTD., Xiamen, Fujian, People’s Republic of China*These authors contributed equally to this workCorrespondence: Guanghui Wang; Jiajun Du, Email wangguanghui66@email.sdu.edu.cn; dujiajun@sdu.edu.cnBackground: Lung cancer is the most common type of cancer in the world. In lung adenocarcinoma (LUAD), studies on receptor tyrosine kinase ROS proto-oncogene 1 (ROS1) have mainly focused on the oncogenic effects of its fusion mutations, whereas ROS1 has been reported to be aberrantly expressed in a variety of cancers and can extensively regulate the growth, survival, and proliferation of tumor cells through multiple signaling pathways. The comprehensive analysis of ROS1 expression has not been fully investigated regarding its predictive value for LUAD patients.Methods: Gene expression profiles collected from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases were used to build and validate prognostic risk models. The association of ROS1 with overall survival and the immune landscape was obtained from the Tumor Immune Estimation Resource (TIMER) database. The following analyses were performed using the R package to determine the model’s validity: pathway dysregulation analysis, gene set enrichment analysis, Gene Oncology analysis, immune invasion analysis, chemotherapy, radiotherapy, and immunotherapy sensitivity analysis. Finally, we conducted a pan-cancer analysis and performed in vitro experiments to explore the regulatory role of intercellular adhesion molecule 5 (ICAM5) in the progression of LUAD.Results: We constructed a 17-gene model that categorized patients into two risk groups. The model had predictive accuracy for tumor prognosis and was specific for patients with high ROS1 expression. Comprehensive analysis showed that patients in the high-risk group were characterized by marked dysregulation of multiple pathways (eg, unfolded protein response), immune suppression of the tumor microenvironment, and poor benefit from immunotherapy and radiotherapy compared with patients in the low-risk group. PLX4720 may be a suitable treatment for the high-risk patient population. The ICAM5 gene has been demonstrated to inhibit the proliferation, cell cycle, invasion, and migration of LUAD cells.Conclusion: We constructed a 17-gene prognostic risk model and found differences in immune-related cells, biological processes, and prognosis among patients in different risk groups based on the correlation between ROS1 and immunity. Personalized therapy may play an essential role in treatment. We further investigated the role of ICAM5 in inhibiting the malignant bioactivity of LUAD cells.Keywords: ROS1, ICAM5, LUAD, immune, prognostic model

Jiayi Chen,1,* Yonghong Liu,2,* Shengsheng Huang,1 Zixiang Pang,1 Qian Wei,1 Yuzhen Liu,1 Hongyuan Qin,1 Yuanming Chen1 1Department of Spinal Orthopedics, The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 2Department of Preventive Health Care, The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuanming Chen, Department of Spinal Orthopedics, The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, People’s Republic of China, Email rkyygk@163.comIntroduction: This study evaluates the efficacy of metagenomic next-generation sequencing (mNGS) in diagnosing spinal infections and developing therapeutic regimens that combine mNGS, microbiological cultures, and pathological investigations.Methods: Data were collected from 108 patients with suspected spinal infections between January 2022 and December 2023. Lesion tissues were obtained via C-arm assisted puncture or open surgery for mNGS, conventional microbiological culture, and pathological analysis. Personalized antimicrobial therapies were tailored based on these findings, with follow-up evaluations 7 days postoperatively. The sensitivity and specificity of mNGS were assessed, along with its impact on treatment and prognosis.Results: mNGS showed a significantly higher positive detection rate (61.20%) compared to conventional microbiological culture (30.80%) and PCT (28%). mNGS demonstrated greater sensitivity (79.41%) and negative predictive value (63.16%) than cultures (25% and 22.58%, respectively), with no significant difference in specificity and positive predictive value. Seven days post-surgery, a significant reduction in neutrophil percentage (NEUT%) was observed, though decreases in white blood cell count (WBC), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were not statistically significant. At the last follow-up, significant improved in Visual Analogue Scale (VAS) scores, Oswestry Disability Index (ODI), and Japanese Orthopaedic Association (JOA) scores were noted.Conclusion: mNGS outperforms traditional microbiological culture in pathogen detection, especially for rare and critical pathogens. Treatment protocols combining mNGS, microbiological cultures, and pathological examinations are effective and provide valuable clinical insights for treating spinal infections.Keywords: spinal infection, metagenomic next-generation sequencing, sensitivity, specificity treatment

Jingjing Wang,1 Lin Wang,1 Zhe Pang,1 Qingmiao Ge,1 Yonggui Wu,1,2 Xiangming Qi1 1Department of Nephropathy, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230022, People’s Republic of China; 2Center for Scientific Research of Anhui Medical University, Hefei, Anhui, 230022, People’s Republic of ChinaCorrespondence: Yonggui Wu; Xiangming Qi, Department of Nephropathy, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, People’s Republic of China, Tel +86 551 6292 2111, Email wuyonggui@medmail.com.cn; qxm119@126.comPurpose: Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease (CKD) worldwide. Elucidation of the molecular mechanisms underlying ferroptosis and immunity in DKD could aid the development of potentially effective therapeutics. This study aimed to perform an integrated analysis of ferroptosis and immune-related differentially expressed mRNAs (DEGs) in DKD.Materials and Methods: Gene expression profiles of samples obtained from patients with DKD and controls were downloaded from the Gene Expression Omnibus (GEO) database. The potential differentially expressed genes (DEGs) were screened using R software, and ferroptosis immune-related differentially expressed genes (FIRDEGs) were extracted from the DEGs. We performed functional enrichment analyses, and constructed protein–protein interaction (PPI) networks, transcription factor (TFs)-gene networks, and gene-drug networks to explore their potential biological functions. Correlation analysis and receiver operating characteristic curves were used for evaluating the FIRDEGs. We used the CIBERSORT algorithm to examine the composition of immune cells and determine the relationship between FIRDEG signatures and immune cells. Finally, the RNA expression of six FIRDEGs was validated in animal kidney samples using RT-PCR.Results: We identified 80 FIRDEGs and performed their functional analyses. We identified six hub genes (Ccl5, Il18, Cybb, Fcgr2b, Myd88, and Ccr2) using PPI networks and predicted potential TF gene networks and gene-drug pairs. Immune cells, including M2 macrophages, resting mast cells, and gamma-delta T cells, were altered in DKD; the FIRDEGs (Fcgr2b, Cybb, Ccr2, and Ccl5) were closely correlated with the infiltration abundance of M2 macrophages and gamma-delta T cells. Finally, the hub genes were verified in mouse kidney samples.Conclusion: We identified six hub FIRDEGs (Ccl5, Il18, Cybb, Fcgr2b, Myd88, and Ccr2) in DKD, and predicted the potential transcription factor gene networks and possible treatment targets for future research.Keywords: diabetic kidney disease, ferroptosis, immunity, bioinformatics

Zhiya Yang,1,* Fangfang Zhang,2,* Dongmei Li,3 Sisi Wang,1 Zhiping Pang,1 Liu Chen,1 Renzhe Li,4 Dongmei Shi1,5 1The Laboratory of Medical Mycology, Jining No.1 People’s Hospital, Jining, Shandong, 272111, People’s Republic of China; 2Department of Dermatology, Jining Dermatosis Prevention and Treatment Hospital, Jining, Shandong, 272000, People’s Republic of China; 3Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC, 20057 USA; 4The Laboratory of Clinical Medicine, Jining No.1 People’s Hospital, Jining, Shandong, 272111, People’s Republic of China; 5Department of Dermatology, Jining No.1 People’s Hospital, Jining, Shandong, 272001, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dongmei Shi, The Laboratory of Medical Mycology and Dermatology Department of Jining No.1 People’s Hospital, Shandong, 272011, China, Tel +86 537-6051008, Email shidongmei28@163.com Renzhe Li, Clinical Laboratory of Jining No.1 People’s Hospital, 272111, People’s Republic of China, Tel +86 13563704987, Email 552080646@qq.comPurpose: This article aims to provide a theoretical basis for new or adjuvant strategies to facilitate the early diagnosis and treatment of candidiasis and to determine if drug-resistant Candida would affect virulence.Patients and Methods: Our strains were collected from patients diagnosed with candidiasis in our hospital. The strains were identified by MALDI-TOF system and ITS sequencing. Antifungal sensitivity testing in vitro was performed to evaluate susceptibility of these isolates to current widely used antifungal drugs. The Galleria mellonella larvae model infected by Candida spp. was used to compare the virulence of drug-resistant and susceptible Candida spp.Results: A total of 206 Candida strains were collected from clinical specimens. Candida albicans was the most common species among them, and was predominantly isolated from male patients aged over 40 years in ICU environments suffering from pulmonary and/or cerebral conditions. The accuracy rate of MALDI TOF-MS identification was 92.72% when compared with ITS sequencing as the standard method. Most Candida species, except for C. tropicalis which showed high resistance to micafungin, showed high susceptibilities to voriconazole, itraconazole, amphotericin B and micafungin but were highly resistant to terbinafine. For each specific Candida species, the G. mellonella larvae model revealed that the virulence of drug-resistant Candida isolates did not markedly differ from that of the drug-susceptible isolates, however, the virulence was dose-dependent on inoculated fungal cells in this model.Conclusion: The possibility of Candida infection should not be neglected in patients at critical care hospital settings and C. albicans is the most common causative agent. MALDI-TOF MS has the advantages of rapidity and high accuracy, and should be a preferred method for identification of Candida spp. in a clinical laboratory. Voriconazole, itraconazole, amphotericin B and micafungin can still be recommended as the first line antifungals to treat candidiasis.Keywords: Candida, antifungal susceptibility testing, fungal infection, MALDI-TOF MS

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

Xiaoyun Liu,1,* Liling Yang,1,* Yuxiang Han,1 Jingjing Xu,2 Zaiying Pang,1 Yifeng Du,1 Yabo Feng,1,* Youting Lin1,* 1Department of Neurology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan City, Shandong Province, 250021, People’s Republic of China; 2Department of Geriatrics, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan City, Shandong Province, 250021, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yabo Feng; Youting LinDepartment of Neurology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, No. 324, Jingwu Road, Huaiyin Zone, Jinan City, Shandong Province, 250021, People’s Republic of ChinaTel/Fax +86-531‐68776354Email fyb-sd@hotmail.com; youtinglin2019@126.comObjective: The purpose of this study was to illustrate the electrophysiological features of sleep disturbances in patients with anti-leucine-rich glioma-inactivated protein 1 (anti-LGI1) encephalitis in both active and recovery stages.Methods: Retrospectively filed video electroencephalogram (VEEG) and polysomnography (PSG) data in 24 patients with anti-LGI1 encephalitis were analyzed in comparison with that in 20 individuals without sleep disorders as control group.Results: Sleep efficiency (SE) and total sleep time involving REM and NREM sleep were significantly decreased in patients with anti-LGI1 encephalitis during the active stage compared to that during the recovery stage and in the control group. Imbalanced sleep structure was found, demonstrated by elevated N1, decreased N3 and REM components, as well as abnormal N2 structure characterized with significantly lower spindle duration and density during the active stage. These findings were independent of the presence of nocturnal episodic events or sleep hyperkinetic movements (HMs). HMs were present in 11/23 patients throughout NREM and REM sleep (nonspecific in sleep stages) during the active stage. During the recovery stage, SE and sleep structures were dramatically improved, including the percentage of N3 and REM sleep, spindle duration and density. Ten of 11 patients with HMs were followed up. HMs were totally remitted in 3 patients and still persistent in 1, while evolved into REM sleep behavior disorder (RBD) in 4 with comorbid periodic limb movement syndrome (PLMS) in 3/4, and only PLMS in 2.Conclusion: Sleep disturbances were remarkable and intrinsic features in active anti-LGI1 encephalitis, marked by overall disruptions of both NREM and REM sleep, as well as the presence of HMs, which tend to evolve into RBD or PLMS during the recovery stage. Long-term follow-up with PSG is needed, especially for those patients with severe sleep disturbances during the active phase.Keywords: anti-leucine-rich glioma-inactivated protein 1 encephalitis, video electroencephalogram, polysomnography, sleep disturbances, REM sleep behavior disorder, periodic limb movement in sleep

Chunxiao Liao,1 Wenjing Gao,1 Weihua Cao,1 Jun Lv,1 Canqing Yu,1 Shengfeng Wang,1 Zengchang Pang,2 Liming Cong,3 Hua Wang,4 Xianping Wu,5 Liming Li1 1Department of Epidemiology and Biostatistics, School of Public Health, Peking University, Beijing, 100191, People’s Republic of China; 2Qingdao Center for Diseases Control and Prevention, Qingdao, 266033, People’s Republic of China; 3Zhejiang Center for Disease Control and Prevention, Hangzhou, 310051, People’s Republic of China; 4Jiangsu Center for Disease Control and Prevention, Nanjing, 210009, People’s Republic of China; 5Sichuan Center for Disease Control and Prevention, Chengdu, 610041, People’s Republic of ChinaCorrespondence: Liming Li; Wenjing GaoDepartment of Epidemiology and Biostatistics, School of Public Health, Peking University, 38 XueYuan Road, HaiDian District, Beijing, 100191, People’s Republic of ChinaTel +86 10 82801528Fax +86 10 82801528 Ext. 322Email lmlee@vip.163.com; wenjinggao@bjmu.edu.cnBackground: Obesity is a heterogeneous condition in terms of metabolic status. Different obesity phenotypes have various health risks. The aim of this work was to define different subtypes of obesity and investigate their relationship with inflammatory-cardiometabolic abnormalities among Chinese adult twins.Methods: The analyses used data from 1113 adult twins in 4 provinces (Shandong, Zhejiang, Jiangsu and Sichuan) from Chinese National Twin Registry (CNTR) which collected detailed information. We defined those with 0 or 1 metabolic syndrome (MetS) components excluding waist circumference as metabolically healthy, and those with waist circumference ≥ 90 cm (for men) and ≥ 85 cm (for women) as obese. The two-category obesity status and metabolic states are combined to generate four metabolic/obesity phenotypes. High sensitivity C reactive protein (hsCRP) was measured to assess underlying inflammation and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as surrogate measure of insulin resistance. Mixed-effect linear regression models and fixed-effect linear regression models were used to analyse the correlation between HOMA-IR, hsCRP and different metabolic/obesity phenotypes.Results: In cross-sectional analyses of 1113 individuals (mean [SD] age, 46.6 [12.9] years; 463 obese [41.6%]), 20.3% obese twins were metabolic healthy and 64.2% non-obese twins were metabolic unhealthy. Serum HOMA-IR level was higher in metabolically unhealthy non-obesity (MUNO) (β=0.42, 95% CI: 0.21– 0.64), metabolically healthy obesity (MHO) (β=0.68, 95% CI: 0.36– 1.00) and metabolically unhealthy obesity (MUO) (β=0.69, 95% CI: 0.46– 0.91) twins, compared with their metabolically healthy non-obesity (MHNO) counterparts. HsCRP was similar between MHO and MUO, which differed significantly to metabolic healthy non-obesity (MHNO).Conclusion: MHO and MUNO phenotypes were common in Chinese twin population. Both phenotypes were associated with elevated IR and hsCRP which may not be benign and need to be concerned.Keywords: metabolic status, insulin resistance, chronic inflammation, twin study

Hao Wang,1,* Zengxin Jiang,1,* Zhiying Pang,1 Guobin Qi,1 Bingxuan Hua,1 Zuoqin Yan,1 Hengfeng Yuan2 1Department of Orthopaedic Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Department of Orthopaedic Surgery, Shanghai Sixth People’s Hospital, Shanghai Jiaotong University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zuoqin YanDepartment of Orthopaedic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Xuhui District, Shanghai, 200032, People’s Republic of ChinaTel +86 13818009668Email yan.zuoqin@zs-hospital.sh.cnHengfeng YuanDepartment of Orthopaedic Surgery, Shanghai Sixth People’s Hospital, Shanghai Jiaotong University, 600 Yishan Road, Xuhui District, Shanghai, 200233, People’s Republic of ChinaTel +86 13917398796Email yuan.hengfeng@hotmail.comPurpose: Osteoarthritis (OA) is a progressive disease characterized by pain and impaired joint functions. Engeletin is a natural compound with anti-inflammatory and antioxidant effects on other diseases, but the effect of engeletin on OA has not been evaluated. This study aimed to elucidate the protective effect of engeletin on cartilage and the underlying mechanisms.Methods: Chondrocytes were isolated from rat knee cartilage, and TNF-α was used to simulate OA in vitro. After treatment with engeletin, the expression of extracellular matrix (ECM) components (collagen II and aggrecan) and matrix catabolic enzymes (MMP9 and MMP3) was determined by Western blotting and qPCR. Chondrocyte apoptosis was evaluated using Annexin V-FITC/PI and flow cytometry. Apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) were evaluated by Western blotting. The mitochondrial membrane potential of chondrocytes was measured with JC-1, and intracellular reactive oxygen species (ROS) levels were determined with DCFH-DA. Changes in signaling pathways (Nrf2, NF-κB and MAPK) were evaluated by Western blotting. In vivo, anterior cruciate ligament transection (ACLT) was used to induce the rat OA model, and engeletin was administered intraarticularly. The therapeutic effect of engeletin was analyzed by histopathological analysis.Results: Pretreatment with engeletin alleviated TNF-α-induced inhibition of ECM components (collagen II and aggrecan) and upregulation of matrix catabolic enzymes (MMP9 and MMP3). Engeletin ameliorated chondrocyte apoptosis by inhibiting Bax expression and upregulating Bcl-2 expression. Engeletin maintained the mitochondrial membrane potential of chondrocytes and scavenged intracellular ROS by activating the Nrf2 pathway. The NF-κB and MAPK pathways were inhibited by treatment with engeletin. In vivo, ACLT-induced knee OA in rats was alleviated by intraarticular injection of engeletin.Conclusion: Engeletin ameliorated OA in vitro and in vivo. It may be a potential therapeutic drug for OA.Keywords: engeletin, osteoarthritis, OA, apoptosis, reactive oxygen species, ROS, mitochondrial membrane potential

The sintering pot test was used to investigate the effect of MgO/Al2O3 ratio in the range of 1.2 to 2.2 on the sintering behavior of iron ore. The main characterization methods of X-ray diffraction (XRD) and scanning electron microscopyenergy disperse spectroscopy (SEM-EDS) were employed. The results showed that the strength, yield, and reducibility of sinter initially increased and then decreased with increasing MgO/Al2O3 ratio from 1.2 to 2.2. The index would reach the peak value with the strength of 70.6% and the yield of 83.81%, respectively, when MgO/Al2O3 ratio was 1.8. The reduction index would reach the peak value of 88.54% with MgO/Al2O3 ratio of 1.6. The FeO content, solid fuel consumption, and the RDI+3.15 of sinter increased dramatically with increasing MgO/Al2O3 ratio. When MgO/Al2O3 ratio was up to 2.2, the solid fuel consumption and RDI+3.15 reached a maximum value of 61.93 kg/t and 96.1%, respectively. The form of SFCA was acicular when MgO/Al2O3 ratio was less than 1.6 and then transformed to plate-like which content also reduced. The magnetite increased while hematite decreased with enhancing MgO/Al2O3 ratio.

Zhiying Pang,1,* Zhangyi Pan,1,* Min Ma,1 Zhiyan Xu,2 Shiqi Mei,2 Zengxin Jiang,3 Feng Yin1 1Department of Joint Surgery, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai 200092, People’s Republic of China; 2Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China; 3Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zengxin Jiang Tel +86 21-64041990Email Dr_Jiangzx@163.comFeng Yin Tel +86 21-64041990Email 001yinfeng@sina.comPurpose: As a dental material, polyetheretherketone (PEEK) is bioinert that does not induce cellular response and bone/gingival tissues regeneration. This study was to develop bioactive coating on PEEK and investigate the effects of coating on cellular response.Materials and Methods: Tantalum pentoxide (TP) coating was fabricated on PEEK surface by vacuum evaporation and responses of rat bone marrow mesenchymal stem (RBMS) cells/human gingival epithelial (HGE) were studied.Results: A dense coating (around 400 nm in thickness) of TP was closely combined with PEEK (PKTP). Moreover, the coating was non-crystalline TP, which contained many small humps (around 10 nm in size), exhibiting a nanostructured surface. In addition, the roughness, hydrophilicity, surface energy, and protein adsorption of PKTP were remarkably higher than that of PEEK. Furthermore, the responses (adhesion, proliferation, and osteogenic gene expression) of RBMS cells, and responses (adhesion and proliferation) of HGE cells to PKTP were remarkably improved in comparison with PEEK. It could be suggested that the nanostructured coating of TP on PEEK played crucial roles in inducing the responses of RBMS/HGE cells.Conclusion: PKTP with elevated surface performances and outstanding cytocompatibility might have enormous potential for dental implant application.Keywords: polyetheretherketone, tantalum pentoxide, nanostructured coating, cytocompatibility, dental implant applications

Hao Wang,1,* Zengxin Jiang,1,* Zhiying Pang,1 Tianyao Zhou,1 Yutong Gu1,2 1Department of Orthopaedic Surgery, Zhongshan Hospital Affiliated to Fudan University, Shanghai 200032, People’s Republic of China; 2Department of Orthopaedic Surgery, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yutong GuDepartment of Orthopaedic Surgery, Zhongshan Hospital Fudan University, Shanghai 200032, People’s Republic of ChinaTel +86 13816256848Email 447574313@qq.comPurpose: Intervertebral disc degeneration (IDD) is one of the most prevalent musculoskeletal disorders. The nucleus pulposus is the major component of the intervertebral disc, and nucleus pulposus cells (NPCs) play a significant role in the normal functioning of the intervertebral disc. Reactive oxygen species (ROS) generation, inflammation and extracellular matrix degradation in NPCs contribute to the degeneration of intervertebral discs. Acacetin is a drug that exerts antioxidant and anti-inflammatory effects on many types of cells. However, whether acacetin can relieve the degeneration of NPCs remains unknown.Methods: NPCs were extracted from rat intervertebral discs. The NPCs were treated with tert-butyl peroxide (TBHP) to simulate a high-ROS environment, and acacetin was subsequently added. The contents of ROS, inflammatory mediators (COX-2, iNOS) and extracellular matrix components (aggrecan, collagen II, MMP13, MMP9, MMP3) were measured. Components of related signaling pathways (Nrf2, MAPK) were also evaluated. To determine the effect of acacetin in vivo, we simulated disc degeneration via needle puncture. Acacetin was then applied intraperitoneally, and the degenerative status was evaluated using MRI and histopathological analysis.Results: In vitro, acacetin alleviated TBHP-induced ROS generation and upregulated the expression of antioxidant proteins, including HO-1, NQO1, and SOD. In addition, acacetin relieved the TBHP-induced generation of inflammatory mediators (COX-2, iNOS) and degradation of the extracellular matrix (aggrecan, collagen II, MMP13, MMP9, and MMP3). Acacetin exerted its effect by activating the Nrf2 pathway and inhibiting p38, JNK and ERK1/2 phosphorylation. In vivo, acacetin ameliorated puncture-induced disc degeneration in a rat tail model, which was evaluated using MRI and histopathological analysis.Conclusion: Acacetin alleviated IDD in vitro and in vivo and may have the potential to be developed as an effective treatment for IDD.Keywords: acacetin, intervertebral disc degeneration, IDD, reactive oxygen species, ROS, inflammation

Yuxiang Han, Liling Yang, Xiaoyun Liu, Yabo Feng, Zaiying Pang, Youting Lin Departments of Neurology, Shandong Provincial Hospital Affiliated to Shandong University, Ji’nan 250021, People’s Republic of ChinaCorrespondence: Youting Lin Tel/Fax +86 531 68776354Email youtinglin2019@126.ComPurpose: Late-onset epilepsy due to autoimmune dysfunction has been reported. However, definitive diagnosis requires positive antibody results. As a result, patients with negative antibody results, but presenting with classical manifestation of autoimmune epilepsy, may be managed as suspected cases. In this study, we aim to isolate and profile the concentration of cytokines/chemokines in the cerebrospinal fluid (CSF) and the serum to ascertain if they could act as alternative diagnostic biomarkers.Patients and Methods: Twenty patients aged ≥ 50 years were considered in this study. Ten patients were diagnosed with suspected autoimmune epilepsy (sAE) based on clinic manifestation, electroencephalogram, magnetic resonance imaging, and with negative antibody results of the serum and the CSF. The equivalent control group exhibited neurological disorders due to non-inflammatory pathologies. Serum and CSF were analyzed for cytokines/chemokines concentration, including interleukin (IL)-6, IL-10, IL-17, chemokine (C-X-C motif) ligand (CXCL)12 and CXCL13, as well as high-mobility group box protein 1 (HMGB1) and B cell activation factor (BAFF)).Results: The CSF levels of IL-6, IL-17, HMGB1, and CXCL12 were significantly higher in the sAE group than in the control group. There was no difference in the CSF levels of IL-10, CXCL13 and BAFF. The serum levels of HMGB1 and CXCL12 were elevated in the sAE group compared with the control group, and there was no statistical difference in the serum levels of IL-6, IL-10, IL-17, CXCL13, and BAFF between the two groups.Conclusion: Our study shows that cytokines/chemokines may act as alternative biomarkers for diagnosis of sAE. The activation of both HMGB1/CXCL12-mediated immunity and T helper cells 17 (Th17) cells may be playing a central role in the pathogenesis of sAE. We suggest that cytokines/chemokines be treated as adjuvant biomarkers, instead of solely relying on antibody screening test. However, a larger cohort in a prospective approach is required to validate our findings.Keywords: seizure, autoimmune, cytokine, chemokine, epilepsy

Hongbo Yang, 1,* Yanan Song, 1,* Jing Chen, 1,* Zhiqing Pang, 2 Ning Zhang, 1 Jiatian Cao, 1 Qiaozi Wang, 1 Qiyu Li, 1 Feng Zhang, 1 Yuxiang Dai, 1 Chenguang Li, 1 Zheyong Huang, 1 Juying Qian, 1 Junbo Ge 1 1Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Diseases, Shanghai 200032, People’s Republic of China; 2School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zheyong Huang; Juying QianDepartment of Cardiology, Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Diseases, Shanghai 200032, People’s Republic of ChinaTel +86-21-6404 1990 ext 2153Fax +86-21-6422 3006Email zheyonghuang@126.com; qian.juying@zs-hospital.sh.cnBackground: Aortic valve disease is the most common valvular heart disease leading to valve replacement. The efficacy of pharmacological therapy for aortic valve disease is limited by the high mechanical stress at the aortic valves impairing the binding rate. We aimed to identify nanoparticle coating with entire platelet membranes to fully mimic their inherent multiple adhesive mechanisms and target the sclerotic aortic valve of apolipoprotein E-deficient (ApoE−/−) mice based on their multiple sites binding capacity under high shear stress.Methods: Considering the potent interaction of platelet membrane glycoproteins with components present in sclerotic aortic valves, platelet membrane-coated nanoparticles (PNPs) were synthetized and the binding capacity under high shear stress was evaluated in vitro and in vivo.Results: PNPs demonstrated effectively adhering to von Willebrand factor, collagen and fibrin under shear stresses in vitro. In an aortic valve disease model established in ApoE−/− mice, PNPs exhibited good targeting to sclerotic aortic valves by mimicking platelet multiple adhesive mechanisms.Conclusion: PNPs could provide a promising platform for the molecular diagnosis and targeting treatment of aortic valve disease.Keywords: aortic valve, atherosclerosis, platelet membrane, nanoparticle, targeting

Zheyong Huang,1,* Yanan Song,1,* Zhiqing Pang,2 Bo Zhang,3 Hongbo Yang,1 Hongtao Shi,1 Jing Chen,1 Hui Gong,1,4 Juying Qian,1 Junbo Ge1,4 1Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 2School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery, Ministry of Education, Shanghai, 3Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei, 4Institute of Biomedical Science, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this work Purpose: Thymosin beta 4 (Tβ4) has multiple beneficial facets for myocardial injury, but its efficiency is limited by the low local concentration within the infarct. Here, we established a Tβ4 delivery system for cardiac repair based on the interaction between the abundant fibrin in the infarct zone and the fibrin-targeting moiety clot-binding peptide cysteine–arginine–glutamic acid–lysine–alanine (CREKA). Methods and results: CREKA and Tβ4 were conjugated to nanoparticles (CNP–Tβ4). In vitro binding test revealed that CNP–Tβ4 had a significant binding ability to the surface of fibrin clots when compared to the control clots (NP–Tβ4). Based on the validation of fibrin expression in the early stage of ischemia injury, CNP–Tβ4 was intravenously administered to mice with acute myocardial ischemia–reperfusion injury. CNP–Tβ4 revealed a stronger fibrin-targeting ability than the NP–Tβ4 group and accumulated mainly in the infarcted area and colocalized with fibrin. Subsequently, treatment with CNP–Tβ4 resulted in a better therapeutic effect. Conclusion: CRKEA modification favored Tβ4 accumulation and retention in the infarcted region, leading to augmented functional benefits. Fibrin-targeting delivery system represents a generalizable platform technology for regenerative medicine. Keywords: thymosin beta 4, fibrin, CREKA, targeting delivery, ischemia reperfusion, cardiovascular

Fei Lu,1,2 Zhiyong Pang,2,3 Jingjing Zhao,2 Kai Jin,4 Haichun Li,2 Qiang Pang,2 Long Zhang,2 Zhiqing Pang2 1Department of Pharmacy, Xianju People’s Hospital, Xianju, Zhejiang, 2Department of Pharmaceutics, Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, School of Pharmacy, Fudan University, Shanghai, 3Chongyang Center for Disease Control and Prevention, Xianning, Hubei, 4School of Life Science, Fudan University, Shanghai, People’s Republic of China Abstract: The blood–brain barrier is a formidable obstacle for glioma chemotherapy due to its compact structure and drug efflux ability. In this study, a dual-targeting drug delivery system involving Angiopep-2-conjugated biodegradable polymersomes loaded with doxorubicin (Ang-PS-DOX) was developed to exploit transport by the low-density lipoprotein receptor-related protein 1 (LRP1), which is overexpressed in both blood–brain barrier and glioma cells. The polymersomes (PS) were prepared using a thin-film hydration method. The PS were loaded with doxorubicin using the pH gradient method (Ang-PS-DOX). The resulting PS were uniformly spherical, with diameters of ~135 nm and with ~159.9 Angiopep-2 molecules on the surface of each PS. The drug-loading capacity and the encapsulation efficiency for doxorubicin were 7.94%±0.17% and 95.0%±1.6%, respectively. Permeability tests demonstrated that the proton diffusion coefficient across the PS membrane was far slower than that across the liposome membrane, and the common logarithm value was linearly dependent on the dioxane content in the external phase. Compared with PS-DOX, Ang-PS-DOX demonstrated significantly higher cellular uptake and stronger cytotoxicity in C6 cells. In vivo pharmacokinetics and brain distribution experiments revealed that Ang-PS-DOX achieved a more extensive distribution and more abundant accumulation in glioma cells than PS-DOX. Moreover, the survival time of glioma-bearing rats treated with Ang-PS-DOX was significantly prolonged compared with those treated with PS-DOX or a solution of free doxorubicin. These results suggested that Ang-PS-DOX can target glioma cells and enhance chemotherapeutic efficacy. Keywords: Angiopep-2, dual targeting, biodegradable polymersomes, doxorubicin, glioma treatment

Alcohol use disorders (AUD) are commonly occurring, heritable and polygenic disorders with etiological origins in the brain and the environment. To outline the causes and consequences of alcohol-related milestones, including AUD, and their related psychiatric comorbidities, the Collaborative Study on the Genetics of Alcoholism (COGA) was launched in 1989 with a gene-brain-behavior framework. COGA is a family based, diverse (~25% self-identified African American, ~52% female) sample, including data on 17,878 individuals, ages 7-97 years, in 2246 families of which a proportion are densely affected for AUD. All participants responded to questionnaires (e.g., personality) and the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) which gathers information on psychiatric diagnoses, conditions and related behaviors (e.g., parental monitoring). In addition, 9871 individuals have brain function data from electroencephalogram (EEG) recordings while 12,009 individuals have been genotyped on genome-wide association study (GWAS) arrays. A series of functional genomics studies examine the specific cellular and molecular mechanisms underlying AUD. This overview provides the framework for the development of COGA as a scientific resource in the past three decades, with individual reviews providing in-depth descriptions of data on and discoveries from behavioral and clinical, brain function, genetic and functional genomics data. The value of COGA also resides in its data sharing policies, its efforts to communicate scientific findings to the broader community via a project website and its potential to nurture early career investigators and to generate independent research that has broadened the impact of gene-brain-behavior research into AUD.

Junzhu Wu,1,2,* Jingjing Zhao,1,3,* Bo Zhang,1 Yong Qian,1 Huile Gao,1 Yuan Yu,1 Yan Wei,1 Zhi Yang,1 Xinguo Jiang,1 Zhiqing Pang1 1Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, 2School of Pharmacy, Dali University, Xiaguan, 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: For a nanoparticulate drug-delivery system, crucial challenges in brain-glioblastoma therapy are its poor penetration and retention in the glioblastoma parenchyma. As a prevailing component in the extracellular matrix of many solid tumors, fibrin plays a critical role in the maintenance of glioblastoma morphology and glioblastoma cell differentiation and proliferation. We developed a new drug-delivery system by conjugating polyethylene glycol–polylactic acid nanoparticles (NPs) with cysteine–arginine–glutamic acid–lysine–alanine (CREKA; TNPs), a peptide with special affinity for fibrin, to mediate glioblastoma-homing and prolong NP retention at the tumor site. In vitro binding tests indicated that CREKA significantly enhanced specific binding of NPs with fibrin. In vivo fluorescence imaging of glioblastoma-bearing nude mice, ex vivo brain imaging, and glioblastoma distribution demonstrated that TNPs had higher accumulation and longer retention in the glioblastoma site over unmodified NPs. Furthermore, pharmacodynamic results showed that paclitaxel-loaded TNPs significantly prolonged the median survival time of intracranial U87 glioblastoma-bearing nude mice compared with controls, Taxol, and NPs. These findings suggested that TNPs were able to target the glioblastoma and enhance retention, which is a valuable strategy for tumor therapy. Keywords: CREKA peptide, nanoparticles, retention effect, paclitaxel, glioblastoma

Zhiqing Pang,1,2 Huile Gao,1,2 Jun Chen,1,2 Shun Shen,1,2 Bo Zhang,3 Jinfeng Ren,1,2 Liangran Guo,1,2 Yong Qian,1,2 Xinguo Jiang,1,2 Heng Mei31Department of Pharmaceutics, School of Pharmacy, 2Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, Fudan University, Shanghai, 3Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of ChinaBackground: A novel brain drug delivery system using cationic bovine serum albumin (CBSA)-conjugated biodegradable polymersomes (CBSA-PO) was prepared, and its intracellular delivery mechanism and brain delivery kinetics were evaluated.Methods and results: Biodegradable poly(ethylene glycol)-poly(ε -caprolactone) (PEG-PCL) was used to prepare the polymersomes, and thiolated CBSA was conjugated with the surface of the polymersome. Transmission electron microscopy and dynamic light scattering showed that the CBSA-PO had a round and vesicle-like shape, with a mean diameter of around 100 nm. Coupling of CBSA with polymersomes was confirmed by X-ray photoelectron spectroscopy. Uptake of CBSA-PO by bEnd.3 cells was significantly higher than that of unconjugated polymersomes, but was inhibited by low temperature, free CBSA, and poly-L-lysine, indicating that endocytosis was energy-driven and absorptive-mediated. Cell viability assays confirmed the good safety profile of biodegradable CBSA-PO. Pharmacokinetic results demonstrated that the polymersomes had long circulation times, and CBSA conjugation on the polymersomes significantly increased the blood–brain barrier permeability surface area product by 3.6-fold and the percentage of injected dose per gram brain (% ID/g brain) by 2.1-fold. Capillary depletion experiments showed that CBSA-PO was distributed into the brain parenchyma in a time-dependent manner, with few polymersomes detected, indicating that conjugation of polymersomes with CBSA significantly improved their transcytosis across the brain–blood barrier.Conclusion: These results suggest that CBSA-PO is a promising drug brain delivery carrier with low toxicity.Keywords: brain delivery kinetics, biodegradable polymersomes, cationic albumin, brain–blood barrier

Liangran Guo1,2,4, Li Fan1,2, Jinfeng Ren1,2, Zhiqing Pang1,2, Yulong Ren1,2, Jingwei Li1,2, Ziyi Wen1,3, Yong Qian1,2, Lin Zhang1,2, Hang Ma4, Xinguo Jiang1,2 1School of Pharmacy, Fudan University, Zhangheng Road, Shanghai, 2Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, Shanghai, 3School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People's Republic of China; 4College of Pharmacy, University of Rhode Island, RI, USAAbstract: The intractability of non-small cell lung cancer (NSCLC) to multimodality treatments plays a large part in its extremely poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for selective induction of apoptosis in cancer cells; however, many NSCLC cell lines are resistant to TRAIL-induced apoptosis. The therapeutic effect can be restored by treatments combining TRAIL with chemotherapeutic agents. Actinomycin D (ActD) can sensitize NSCLC cells to TRAIL-induced apoptosis by upregulation of death receptor 4 (DR4) or 5 (DR5). However, the use of ActD has significant drawbacks due to the side effects that result from its nonspecific biodistribution in vivo. In addition, the short half-life of TRAIL in serum also limits the antitumor effect of treatments combining TRAIL and ActD. In this study, we designed a combination treatment of long-circulating TRAIL liposomes and ActD liposomes with the aim of resolving these problems. The combination of TRAIL liposomes and ActD liposomes had a synergistic cytotoxic effect against A-549 cells. The mechanism behind this combination treatment includes both increased expression of DR5 and caspase activation. Moreover, systemic administration of the combination of TRAIL liposomes and ActD liposomes suppressed both tumor formation and growth of established subcutaneous NSCLC xenografts in nude mice, inducing apoptosis without causing significant general toxicity. These results provide preclinical proof-of-principle for a novel therapeutic strategy in which TRAIL liposomes are safely combined with ActD liposomes.Keywords: TRAIL, actinomycin D, liposomes, combination treatment, non-small cell lung cancer

We have previously described local aldosterone synthesis in mouse colon. In the renin-angiotensin-aldosterone system (RAAS), angiotensin II (Ang II) peptide is the physiological factor which stimulates aldosterone synthesis in the adrenal glands. We have recently demonstrated that Ang II stimulates aldosterone synthesis also in mouse colon. Here, we conducted a 75-min ex vivo incubation of murine colonic tissue and evaluated the effects of three other Ang peptides, Ang I (1 µM), Ang III (0.1 µM) and Ang (1-7) (0.1 µM) on aldosterone synthesis. As a possible mechanism, their effects on tissue levels of the rate-limiting enzyme, aldosterone synthase (CYP11B2) were measured by ELISA and Western blot. Ang III significantly elevated the amount of tissue CYP11B2 protein in colon. The values of released aldosterone in colon tissue incubation were increased over the control in the presence of Ang I, II or III, however, being statistically nonsignificant. In Western blot analysis, the values of tissue CYP11B2 protein content were elevated by Ang I and II. Ang (1-7) alone in colon did not influence CYP11B2 protein levels in the incubation experiment but showed higher aldosterone release without statistical significance. Ang (1-7) showed an antagonistic effect towards Ang II in release of aldosterone in adrenal gland. An overall estimation of a single peptide (three measured variables), the results were always in an increasing direction. The responses of aldosterone synthesis to high levels of glucose (44 mM) and potassium (18.8 mM) as physiological stimulators in vivo were investigated in the colon incubation. Glucose, equal to four times the concentration of the control buffer in the incubation, showed higher values of aldosterone release in colon than control without statistical significance similarly to the effect seen in adrenal glands. Increasing the concentration of potassium in the incubation buffer exerted no effect on colonic aldosterone production. Intriguingly, no correlation was found between aldosterone release and the tissue CYP11B2 protein content in colon. In summary, the response of colonic aldosterone synthesis to different Ang peptides resembles, but is not identical to, the situation in the adrenal glands. [ABSTRACT FROM AUTHOR]

Abstract Microstructured molds are essential for fabricating various components ranging from precision optics and microstructured surfaces to microfluidics. However, conventional fabrication technology such as photolithography requires expensive equipment and a large number of processing steps. Here, we report a facile method to fabricate micromolds based on a reusable photoresponsive hydrogel: Uniform micropatterns are engraved into the hydrogel surface using photo masks under UV irradiation within a few minutes. Patterns are replicated using polydimethylsiloxane with minimum feature size of 40 μm and smoothness of Rq ~ 3.4 nm. After replication, the patterns can be fully erased by light thus allowing for reuse as a new mold without notable loss in performance. Utilizing greyscale lithography, patterns with different height levels can be produced within the same exposure step. We demonstrate the versatility of this method by fabricating diffractive optical elements devices and a microlens array and microfluidic device with 100 µm wide channels.