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Yue Wang,1 Jie Chen,1 Han Dong,2 Rui-Lin Ma,1 Ying Zou,3 Wei Wang,4 Qingmei Zheng,5 Ying Feng,6 Zhangyun Tan,7 Xiaoqin Zeng,8 Yinqing Zhao,7 Yan Deng,1 Yanfang Wang,1 Bei Gu,9 Aijun Sun1,10 1National Clinical Research Center for Obstetric & Gynecologic Diseases Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, People’s Republic of China; 2Department of Obstetrics and Gynecology, Women and Children’s Hospital of Jinzhou, Jinzhou, Liaoning, 121000, People’s Republic of China; 3Department of Obstetrics and Gynecology, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, 410008, People’s Republic of China; 4Department of Reproductive Medicine, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, People’s Republic of China; 5Department of Gynecology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266500, People’s Republic of China; 6Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, People’s Republic of China; 7Department of Obstetrics and Gynecology, Xinhui Maternity and Children’s Hospital, Nanning, Guangxi, 529100, People’s Republic of China; 8Department of Gynecology, Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 510000, People’s Republic of China; 9Department of Obstetrics and Gynecology, Beijing Shijitan Hospital, Capital Medical University, Peking University Ninth School of Clinical Medicine, Beijing, 100038, People’s Republic of China; 10Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Beijing, 100010, People’s Republic of ChinaCorrespondence: Aijun Sun, Email saj@pumch.cnBackground: The basic medical education stage is not enough to support physicians to fully diagnose and evaluate polycystic ovary syndrome (PCOS). The study aims to discover the difference in treatment choice between participants with different annual consultation number of PCOS, to promote lifelong learning, and drive balanced development within healthcare.Methods: This is a multicenter cross-sectional survey. Participants’ basic information, knowledge of PCOS and treatment options were collected online. According to the annual consultation number of patients with PCOS, physicians were divided into three groups: 0– 50 people/yr, 50– 200 people/yr, and > 200 people/yr, and the results were derived from χ2 test, Fisher exact test, and multivariate logistic regression analysis.Results: The study analyzed 1689 questionnaires, and 1206 physicians (71.4%) received less than 50 women per year, 388 physicians (30.0%) with an annual number of 50– 200 women, and 95 physicians (5.6%) with patient turnover for more than 200 people. Reproductive endocrinologists generally have higher access to the clinic. As the number of visits increases, more and more physicians would perceive patients as more likely to have abnormal blood glucose and heavy weight. Physicians with large numbers of consultations are more likely to use Asian or Chinese standards to assess obesity. The multivariate analysis involved variables such as age, hospital level, specialty, and patient turnover annually, and more young doctors actively assessed lipid profile (odds ratio (OR) 1.56, 95% confidence interval (CI) (1.16, 2.16)), and primary hospitals (OR 0.65 CI (0.44, 0.89)) chose OGTT for blood glucose assessment less than tertiary hospitals. Physicians in secondary hospitals are more aggressive in evaluating androgens.Conclusion: Our survey found differences in endocrine assessment, metabolic screening, and treatment in PCOS women in terms of the number of obstetrician-gynecologists who received different patient consultation numbers. The importance of continuing education for physicians is emphasized, to promote lifelong learning.Keywords: polycystic ovary syndrome, consultation number, lifelong learning, insulin resistance, obesity, hyperandrogenemia, metformin

Siyun Cheng,1,2,* Zhijie Tu,2,* Shuai Zheng,3 Adeel Khan,4 Ping Yang,1 Han Shen,1 Bing Gu2,3 1Department of Clinical Laboratory, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, People’s Republic of China; 2Medical Technology School of Xuzhou Medical University, Xuzhou, People’s Republic of China; 3Department of Clinical Laboratory Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, People’s Republic of China; 4Department of Biotechnology, University of Science and Technology, Bannu, KP, Pakistan*These authors contributed equally to this workCorrespondence: Han Shen, Department of Clinical Laboratory, Nanjing Drum Tower Hospital, Nanjing, People’s Republic of China, Tel +8602583304616, Fax +02568183912, Email shenhan@njglyy.com Bing Gu, Department of Clinical Laboratory Medicine, Guangdong Provincial People’s Hospital, Guangzhou, People’s Republic of China, Tel +8602083827812, Fax +02083875881, Email gubing@gdph.org.cnIntroduction: Ultrasensitive bacterial detection methods are crucial to ensuring accurate diagnosis and effective clinical monitoring, given the significant threat bacterial infections pose to human health. The aim of this study is to develop a biosensor with capabilities for broad-spectrum bacterial detection, rapid processing, and cost-effectiveness.Methods: A magnetically-assisted SERS biosensor was designed, employing wheat germ agglutinin (WGA) for broad-spectrum recognition and antibodies for specific capture. Gold nanostars (AuNSs) were sequentially modified with the Raman reporter molecules and WGA, creating a versatile SERS tag with high affinity for a diverse range of bacteria. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) antibody-modified Fe3O4 magnetic gold nanoparticles (MGNPs) served as the capture probes. Target bacteria were captured by MGNPs and combined with SERS tags, forming a “sandwich” composite structure for bacterial detection.Results: AuNSs, with a core size of 65 nm, exhibited excellent storage stability (RSD=5.6%) and demonstrated superior SERS enhancement compared to colloidal gold nanoparticles. Efficient binding of S. aureus and P. aeruginosa to MGNPs resulted in capture efficiencies of 89.13% and 85.31%, respectively. Under optimized conditions, the developed assay achieved a limit of detection (LOD) of 7 CFU/mL for S. aureus and 5 CFU/mL for P. aeruginosa. The bacterial concentration (10– 106 CFU/mL) showed a strong linear correlation with the SERS intensity at 1331 cm− 1. Additionally, high recoveries (84.8% - 118.0%) and low RSD (6.21% - 11.42%) were observed in spiked human urine samples.Conclusion: This study introduces a simple and innovative magnetically-assisted SERS biosensor for the sensitive and quantitative detection of S. aureus or P. aeruginosa, utilizing WGA and antibodies. The developed biosensor enhances the capabilities of the “sandwich” type SERS biosensor, offering a novel and effective platform for accurate and timely clinical diagnosis of bacterial infections. Keywords: SERS, WGA, pathogenic bacteria, bacterial detection, dual-recognition

Yong Ling,1,* Xuejiao Hu,1,* Guansheng Zheng,1 Weitao Ye,2 Kaixuan Yuan,1 Long Ye,1 Weiye Huang,3 Benshun Tian,1 Bing Gu1 1Department of Clinical Laboratory Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, 510000, People’s Republic of China; 2Department of Radiology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, 510000, People’s Republic of China; 3Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, 510000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Benshun Tian; Bing Gu, Department of Laboratory Medicine, Guangdong Provincial People’s Hospital, 106 Zhongshan 2nd Road, Yuexiu District, Guangzhou, Guangdong, 510000, People’s Republic of China, Email tbs20161017@163.com; gb20031129@163.comAbstract: Scrub typhus is a vector-borne infectious disease caused by Orientia tsutsugamushi. Accurate and timely diagnosis at the early infection stage could save the patients’ lives. Traditional technologies were limited to rapidly and successfully detecting Orientia tsutsugamushi due to poor specificity, especially in the condition of atypical symptoms. The technology of Metagenomic next-generation sequencing (mNGS) is amenable to finding the real pathogen because it holds potential as a diagnostic platform for unbiased pathogen identification and precision medicine. Herein, we reported two clinical case reports relative to the Orientia tsutsugamushi infection diagnosed by mNGS. We hope these two cases will improve clinical diagnosis.Keywords: scrub typhus, metagenomic next-generation sequencing, Orientia tsutsugamushi

Min Zhu,1 Yuyu Sun,1 Huawei Xue,1 Gang Wu,1 Zhen Wang,1 Junfeng Shi,1 Jiye Ma,1 Baorong Gu,1 Xiaoling Yan2 1Department of Spine Surgery, Nantong Third People’s Hospital, Affiliated Nantong Hospital 3 of Nantong University, Nantong, People’s Republic of China; 2Department of Chemotherapy, Affiliated Hospital of Nantong University, Nantong, People’s Republic of ChinaCorrespondence: Xiaoling Yan, Department of Chemotherapy, Affiliated Hospital of Nantong University, Nantong, 226001, People’s Republic of China, Email 5200007@ntu.edu.cnBackground: Osteosarcoma is a malignant tumor originating from the skeletal system. There is no effective treatment other than surgery and chemotherapy, which seriously endangers the health of children and adolescents. NEK6 is a novel discovered Serine/Threonine protein kinase that can regulate cell cycle and activate several oncogenic pathways.Methods: NEK6 expression in pan-cancer including sarcoma was evaluated using analysis tools of TIMER, UALCNA and GEPIA with TCGA database, and its association with overall survival in patients with sarcoma was also analyzed. TargetScan, tarbase, microT-CDS and Starbase online software were used to predict NEK6-targeted miRNAs, including miR-26a-5p. Tumor tissues from patients with osteosarcoma were collected for NEK6 and miRNA detection using RT-qPCR. NEK6 down-regulated by siRNAs or miR-26a-5p in osteosarcoma cells was detected by RT-qPCR, Western blot and Immunofluorescence staining assays. Effects of NEK6 knockdown on proliferation, migration, invasion and apoptosis of osteosarcoma cells were detected by CCK-8, wound healing, transwell and flow cytometry, respectively. The expressions of STAT3, metastasis and apoptosis-related genes were detected by Western blot.Results: High expression of NEK6 and low expression of miR-26a-5p were lowly expressed in osteosarcoma and they were negative correlation. NEK6 has been confirmed as a direct target for miR-26a-5p. In addition, NEK6 down-regulated by siRNAs or miR-26a-5p led to inhibition of cell proliferation, migration and invasion while promoting cell apoptosis. The levels of phosphorylated STAT3 and metastasis genes (MMP-2, MMP-9) were inhibited, while apoptotic gene Bax was promoted and Bcl2 was inhibited by miR-26a-5p upregulation.Conclusion: NEK6 can promote osteosarcoma progression via activating STAT3 signaling pathway, which is inhibited by miR-26a-5p, suggesting that NEK6 is a potential oncogene and miR-26a-5p is a suppressor of osteosarcoma. The strategy of inhibiting of NEK6 by miR-26a-5p may be an effective approach for osteosarcoma therapy.Keywords: osteosarcoma, NEK6, miR-26a-5p, STAT3 signaling pathway

We investigated rates and controls on greenhouse gas (CO2 and CH4) production in two contrasting water-saturated tundra soils within a permafrost-affected watershed near Nome, Alaska, United States. Three years of field sample analysis have shown that soil from a fen-like area in the toeslope of the watershed had higher pH and higher porewater ion concentrations than soil collected from a bog-like peat plateau at the top of the hillslope. The influence of these contrasting geochemical and topographic environments on CO2 and CH4 production was tested in soil microcosms by incubating both the organic- and mineral-layer soils anaerobically for 55 days. Nitrogen (as NH4Cl) was added to half of the microcosms to test potential effects of N limitation on microbial greenhouse gas production. We found that the organic toeslope soils produced more CO2 and CH4, fueled by higher pH and higher concentrations of water-extractable organic C (WEOC). Our results also indicate N limitation on CO2 production in the peat plateau soils but not the toeslope soils. Together these results suggest that the weathering and leaching of ions and nutrients from tundra hillslopes can increase the rate of anaerobic soil organic matter decomposition in downslope soils by (1) increasing the pH of soil porewater; (2) providing bioavailable WEOC and fermentation products such as acetate; and (3) relieving microbial N limitation through nutrient runoff. We conclude that the soil geochemistry as mediated by landscape position is an important factor influencing the rate and magnitude of greenhouse gas production in tundra soils.

Spatial heterogeneities in soil hydrology have been confirmed as a key control on CO2 and CH4 fluxes in the Arctic tundra ecosystem. In this study, we applied a mechanistic ecosystem model, CLM-Microbe, to examine the microtopographic impacts on CO2 and CH4 fluxes across seven landscape types in Utqiaġvik, Alaska: trough, low-centered polygon (LCP) center, LCP transition, LCP rim, high-centered polygon (HCP) center, HCP transition, and HCP rim. We first validated the CLM-Microbe model against static-chamber measured CO2 and CH4 fluxes in 2013 for three landscape types: trough, LCP center, and LCP rim. Model application showed that low-elevation and thus wetter landscape types (i.e., trough, transitions, and LCP center) had larger CH4 emissions rates with greater seasonal variations than high-elevation and drier landscape types (rims and HCP center). Sensitivity analysis indicated that substrate availability for methanogenesis (acetate, CO2 + H2) is the most important factor determining CH4 emission, and vegetation physiological properties largely affect the net ecosystem carbon exchange and ecosystem respiration in Arctic tundra ecosystems. Modeled CH4 emissions for different microtopographic features were upscaled to the eddy covariance (EC) domain with an area-weighted approach before validation against EC-measured CH4 fluxes. The model underestimated the EC-measured CH4 flux by 20% and 25% at daily and hourly time steps, suggesting the importance of the time step in reporting CH4 flux. The strong microtopographic impacts on CO2 and CH4 fluxes call for a model-data integration framework for better understanding and predicting carbon flux in the highly heterogeneous Arctic landscape.

Bei Gu,1 Shiyang Zhu,2 Xuesong Ding,2 Yan Deng,2 Xiao Ma,2 Jingwen Gan,2 Yanfang Wang,2 Aijun Sun2 1Department of Obstetrics and Gynecology, Beijing Shijitan Hospital, Capital Medical University, the Ninth Clinical Medical College of Peking University, Beijing, People’s Republic of China; 2National Clinical Research Center for Obstetric & Gynecologic Diseases, Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of ChinaCorrespondence: Aijun Sun, Tel +86 18600045466, Email saj@pumch.cnBackground: Primary dysmenorrhea (PD) is one of the most common complaints in women of childbearing age. Therefore, this trial aimed to assess the efficacy and safety of low-power visible-light-activated photodynamic therapy (PDT) in the treatment of primary dysmenorrhea (PD), and to further investigate their possible mechanisms of action.Methods: This study was conducted by using a multicenter, randomized, open, parallel control design. Qualified subjects are randomly assigned to two groups: Group A (low-power visible-light-activated PDT group), Group B (placebo group) and are treated with corresponding protocols for three consecutive menstrual cycles. Baseline data are collected during the trial period. Changes in the scores of VAS scales and the fluctuation of pain factors (PGE2, PGF2α) are recorded before and after the treatment for each group. A comparison of effectiveness in pain control and symptom control is made among the two groups.Results: After treatment, for the PDT group, the scores of VAS scales decline compared with the scores before treatment. The level of pain factors including PGE2 and PGF2α also drops significantly (P < 0.05). There are no serious adverse events during the study.Conclusion: Low-power visible-light-activated PDT is a new type of treatment for primary dysmenorrhea which is safe, effective and does not affect normal pregnancy preparation. It may exert its therapeutic effect by adjusting downward the level of PGE2, PGF2α in the body. These factors can be used not only to study the treatment mechanism for primary dysmenorrhea, but also to serve as quantitative indicators for objective assessment of whether dysmenorrhea is relieved.Keywords: primary dysmenorrhea, low-power visible-light-activated photodynamic therapeutic device, prostaglandins E2, prostaglandins F2alpha

Feng Gu,1,2,&ast; Shan Hu,3,&ast; Yunjian Wu,4,&ast; Changyu Wu,4 Ying Yang,5 Bing Gu,6 Hong Du1 1Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, People’s Republic of China; 2Department of Laboratory Medicine, Xuzhou Central Hospital, Xuzhou, 221000, People’s Republic of China; 3Department of Laboratory Medicine, Xuzhou Tumor Hospital, Xuzhou, 221005, People’s Republic of China; 4School of Medical Imaging, Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China; 5Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Diseases, Beijing, 100850, People’s Republic of China; 6Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Hong Du; Ying Yang, Email hong_du@126.com; y_ying_77@163.comPurpose: Candida infection has a high mortality rate, and the increasing prevalence of non-Candida albicans drug resistance in recent years poses a potential threat to human health. Non-Candida albicans has long culture cycles, and its firm cell walls making it difficult to isolate DNA for sequencing.Materials and Methods: Fe3O4@PEI (PEI, polyvinyl imine) was mixed with clinical samples to form Fe3O4@PEI@non-Candida albicans and enriched them with magnets. Triangular silver nanoplates enhanced the surface-enhanced Raman scattering (SERS) signal. SERS was used to detect the fingerprint spectrum of non-Candida albicans. Then, orthogonal partial least squares discriminant analysis (OPLS-DA) was used to analyze the drug resistance of non-Candida albicans.Results: SERS combined with OPLS-DA could well analyze the drug resistance of non-Candida albicans. Through 10-fold-cross validation, the accuracy of training and test data is greater than 99%, indicating that the model has good classification ability. We used SERS for the first time to detect the drug resistance of non-Candida albicans directly.Conclusion: This approach can be utilized without causing damage to the cell wall and can be accomplished in as little as 90 minutes. It can provide timely guidance for the treatment of patients with good clinical application potential.Keywords: non-Candida albicans, SERS, drug resistance, OPLS-DA

Jingwen Lyu,1,2,&ast; Qianyun Deng,2,&ast; Rongqing Li,3 Benshun Tian,1,2 Yunhu Zhao,2 Xuejiao Hu,2 Maohua Zhou,2 Bing Gu2 1Xuzhou Key Laboratory of Laboratory Diagnostics, School of Medical Technology, Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China; 2Laboratory Medicine, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510000, People’s Republic of China; 3Jiangsu Province Key Laboratory of Immunity and Metabolism, Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Maohua Zhou; Bing Gu, Laboratory Medicine, Guangdong Provincial People’s Hospital, 106 Zhongshan 2nd Road, Yuexiu District, Guangzhou, Guangdong, 510000, People’s Republic of China, Tel +86-20-83827812-62462, Email zhoumaohua@gdph.org.cn; gb20031129@163.comBackground: Pneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed.Case Presentation: Here, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient’s guardian. Hence, the child was discharged from the hospital and eventually died.Conclusion: This case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.Keywords: metagenomic next-generation sequencing, Pneumocystis jirovecii, cytomegalovirus, pneumonitis, HIV-negative

Yang Yu,1,* Wenfeng Wang,2,* Zuquan Xiong,3 Zhenyu Yang,1 Jun Li,1 Yifan Shen,1 Bin Gu1 1Urology Department, Shanghai Pudong New Area People’s Hospital, Shanghai, People’s Republic of China; 2Cancer Institute, Fudan University Shanghai Cancer Center, Fudan University, Shanghai, People’s Republic of China; 3Urology Department, Shanghai Huashan Hospital, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Bin GuUrology Department, Shanghai Pudong New Area People’s Hospital, 490 Chuanhuan South Road, Shanghai, 201200, People’s Republic of ChinaTel/Fax +86-2120509000Email ibippan3998@163.comPurpose: This study aimed to compare perioperative outcomes of open partial nephrectomy (OPN) and laparoscopic partial nephrectomy (LPN) from a retrospective single-center dataset.Patients and Methods: A retrospective single-center analysis of 210 patients who underwent open (n=91) or laparoscopic (n =119) partial nephrectomy for RCC was conducted between 2012 and 2015. All patients were grouped into low complexity, moderate complexity, and high complexity according to the R.E.N.A.L. Nephrometry Score, respectively. The rates of intraoperative and postoperative complications estimated blood loss, warm ischemia time, operative time, conversion of laparoscopic procedure to open surgery, and postoperative length of stay were assessed for both procedures.Results: In low complexity group (n=93), patients undergoing LPN (n=52) under ischemia conditions had significantly longer renal artery clamp time (p < 0.001) and operative time (p = 0.001) compared with OPN (n=41). However, patients undergoing LPN had a significantly less postoperative length of stay (p=0.005) and estimated blood loss (p < 0.001) compared with OPN. There was no statistically significant difference in the rates of complications between LPN and OPN. In the moderate complexity group (n=114), 67 and 47 patients underwent LPN and OPN, respectively. LPN had notably longer warm ischemia time (p < 0.001) and operative time (p < 0.001) compared with OPN. There were no statistically significant differences in the rates of complications, estimated blood loss, and postoperative length of stay between LPN and OPN. In the high complexity group (n=3), all patients underwent OPN.Conclusion: OPN and LPN procedures performed in patients with low and moderate complexity tumors based on the RENAL Nephrometry score offer acceptable and comparable results. When applied to low complexity tumors, our data suggest that laparoscopic NSS is an effective, minimally invasive therapeutic approach with the advantages of less blood loss, earlier hospital discharge, and more rapid convalescence.Keywords: renal cell carcinoma, open partial nephrectomy, laparoscopic partial nephrectomy, R.E.N.A.L. Nephrometry Score

Xiaoxiao Wu,1,&ast; Yongyan Chen,1,&ast; Yangheng Zhang,2,&ast; Yunjie Shan,1 Zhiyue Peng,1 Bing Gu,1,3 Huan Yang1 1Xuzhou Key Laboratory of Laboratory Diagnostics, School of Medical Technology, Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China; 2Department of Periodontology, Nanjing Stomatological Hospital of Nanjing University School of Medicine, Nanjing, 210008, People’s Republic of China; 3Laboratory Medicine, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, 510000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Huan Yang; Bing Gu Email yanghuan2015@tmu.edu.cn; gb20031129@163.comBackground: Shigella infection has always been a global burden, and it particularly threatens children between the ages of 1 and 5 years. Economically underdeveloped countries are dominated by Shigella flexneri infection. The most effective method to treat Shigella is antibiotics, but with the abuse of antibiotics and the prevalence of multidrug resistance, we urgently need a relatively safe non-antibiotic treatment to replace it. Ultrasmall Au nanoclusters (NCs) have special physical and chemical properties and can better interact with and be internalized by bacteria to disrupt the metabolic balance. The purpose of this study was to explore whether Au NCs may be a substitute for antibiotics to treat Shigella infections.Methods: Au NCs and Shigella Sf301, R2448, and RII-1 were cocultured in vitro to evaluate the bactericidal ability of Au NCs. The degree of damage and mode of action of Au NCs in Shigella were clearly observed in images of scanning electron microscopy (SEM). In vivo experiments were conducted to observe the changes in body weight, clinical disease activity index (DAI) and colon (including length and histopathological sections) of mice treated with Au NCs. The effect of Au NCs was analysed by measuring the content of lipocalin-2 (LCN2) and Shigella in faeces. Next, the changes in Shigella biofilm activity, the release of reactive oxygen species (ROS), the changes in metabolism-related and membrane-related genes, and the effect of Au NCs on the body weight of mice were determined to further analyse the mechanism of action and effect.Results: Au NCs (100 μM) interfered with oxidative metabolism genes, induced a substantial increase in ROS levels, interacted with the cell membrane to destroy it, significantly killed Shigella, and effectively alleviated the intestinal damage caused by Shigella in mice. The activity of the biofilm formed by Shigella was reduced.Conclusion: The effective antibacterial effect and good safety suggest that Au NCs represent a good potential alternative to antibiotics to treat Shigella infections.Keywords: Shigella, Au nanoclusters, antibacterial effect, reactive oxygen species, oxidative stress

Shan Hu,1– 3,* Haiquan Kang,4,* Feng Gu,2,* Chongwen Wang,1,5 Siyun Cheng,3 Wenjing Gong,1 Liping Wang,1 Bing Gu,3,4 Ying Yang1 1Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Diseases, Beijing, 100850, People’s Republic of China; 2Department of Laboratory Medicine, Xuzhou Tumor Hospital, Xuzhou, 221005, People’s Republic of China; 3Xuzhou Key Laboratory of Laboratory Diagnostics, Medical Technology School of Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China; 4Department of Laboratory Medicine, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221006, People’s Republic of China; 5College of Life Sciences, Anhui Agricultural University, Hefei, 230036, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ying Yang; Bing Gu Email y_ying_77@163.com; gb20031129@163.comPurpose: Candidemia infection is common in the clinic and has a high mortality rate. Candida albicans, Candida tropicalis, and Candida krusei are very important and common pathogenic species. Candida is difficult to isolate from clinical samples and culture, and immunological detection cannot distinguish these related strains. Furthermore, Candida has a complex cell wall, which causes difficulties in the extraction of DNA for nucleic acid detection. The purpose of this study was to establish a protocol for the direct identification of Candida from serum.Materials and Methods: We synthesized Fe3O4@PEI (where PEI stands for polyethylenimine) magnetic nanoparticles to capture Candida and prepared positively charged silver nanoparticles (AgNPs+) as the substrate for surface-enhanced Raman scattering (SERS). Candida was directly identified from serum by SERS detection.Results: Orthogonal partial least squares discriminant analysis (OPLS-DA) was used as the multivariate analysis tool. Principal component analysis confirmed that this method can clearly distinguish common Candida. After 10-fold cross-validation, the accuracy of training data in this model was 100% and the accuracy of test data was 99.8%, indicating that the model has good classification ability.Conclusion: The detection could be completed within 40 minutes using Fe3O4@PEI and AgNPs+ prepared in advance. This is the first time that Fe3O4@PEI was used in the detection of Candida by SERS. We report the first rapid method to identify fungi directly from serum without breaking the cell wall to extract DNA from the fungi.Keywords: capture, surface-enhanced Raman scattering, positively charged silver nanoparticles, orthogonal partial least squares discriminant analysis, 10-fold cross-validation

Ziyan Kong,1,2,* Xuemei Liu,3,* Chenxi Li,1,* Siyun Cheng,1 Fei Xu,3 Bing Gu1,4 1Medical Technology School, Xuzhou Medical University, Xuzhou, Jiangsu Province, People’s Republic of China; 2Department of Laboratory Medicine, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu Province, People’s Republic of China; 3Department of Laboratory Medicine, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, People’s Republic of China; 4Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fei XuDepartment of Laboratory Medicine, Children’s Hospital of Nanjing Medical University, Nanjing 210009, People’s Republic of ChinaEmail 350826448@qq.comBing GuDepartment of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou 221006, People’s Republic of ChinaEmail gb20031129@163.comPurpose: The continuous emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious public health problem globally, especially for children, but data on CRKP infection in pediatric patients are limited. This study aimed to identify epidemiological and molecular patterns of CRKP among pediatric patients in Jiangsu province, China.Patients and Methods: CRKP were consecutively collected from the Children’s Hospital of Nanjing Medical University in China from July 2018 to May 2019. Then, CRKP strains were performed for further study: antimicrobial susceptibility testing, drug-resistance determinants screening and homology analysis.Results: We collected 94 CRKP from 94 children. Overall, blaKPC-2 (79.8%) was the predominant carbapenemase gene, followed by blaNDM-1(14.9%), blaIMP-4 (5.3%) and blaNDM-5(4.3%). Notably, two isolates coharbored blaKPC-2 and blaIMP-4, and two isolates coharbored blaKPC-2 and blaNDM-5. MLST analysis revealed that 14 distinct sequence types (STs) were identified, of which ST11 was the most common sequence type identified. Moreover, two novel STs, ST4854 and ST4855, were detected in this study. PFGE revealed that a predominant cluster consisting of KPC-2-producing CRKP ST11 clone isolates was identified and was distributed mainly in the pediatric intensive care unit (PICU) and cardiac intensive care unit (CCU). Moreover, this is the first report to identify the dissemination of ST716 CRKP coproducing KPC-2 and IMP-4 clones.Conclusion: Clonal dissemination of KPC-2-producing CRKP ST11 was observed in multiple departments. Moreover, two novel STs (ST4854 and ST4855) were identified, which indicates an increased diversity of CRKP strains. To our knowledge, this is the first report that identified the dissemination of Klebsiella pneumoniae coproducing KPC-2 and IMP-4 clones among children, which represents a significant health risk to pediatric patients. Active surveillance and effective control measures are urgently needed to prevent further transmission of these strains among children.Keywords: Klebsiella pneumoniae, carbapenemase, KPC-2, ST11, clonal dissemination, children

Bo Yang,* Baoyu Gu,* Jing Zhang, Long Xu, Yong Sun Department of Ophthalmology, Shenzhen Hospital of Integrated Chinese and Western Medicine, Shenzhen 518101, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yong SunDepartment of Ophthalmology, Shenzhen Hospital of Integrated Chinese and Western Medicine, 3 Shajing Street, Bao’an District, Shenzhen 518101, Guangdong Province, People’s Republic of ChinaTel +86 755-2772-2241Email wk9710@163.comBackground: CASC8 lncRNA has been proven to be oncogenic in a variety of cancers, but its role in other types of cancer remains unclear. This study was to investigate the role of CASC8 in retinoblastoma (Rb).Methods: RT-qPCR was performed to determine the expression of CASC8 and miR34a in paired Rb and nontumor tissue. Overexpression of CASC8 and miR34a in Rb cells was achieved to evaluate the interaction between them. Methylation-specific PCR was used to analyze the effect of CASC8 overexpression on MIR34A gene methylation. CCK8 assays were used to analyze cell proliferation.Results: The results showed that CASC8 expression was upregulated and miR34a expression downregulated in Rb tissue. Moreover, miR34a expression was negatively correlated with the of CASC8 expression in Rb tissue. Overexpression of CASC8 decreased expression of miR34a and increased methylation of MIR 34A in Rb cells. In addition, overexpression of CASC8 reduced the inhibitory effects of miR34a on Rb-cell proliferation.Conclusion: CASC8 may promote Rb cell proliferation by downregulating miR34a methylation.Keywords: retinoblastoma, CASC8, miR34a, methylation, proliferation

Huan Yang,1,* Rui Cai,1,* Yangheng Zhang,2,* Yongyan Chen,1 Bing Gu1,3 1Xuzhou Key Laboratory of Laboratory Diagnostics, School of Medical Technology, Xuzhou Medical University, Xuzhou 221004, People’s Republic of China; 2Department of Periodontology, Nanjing Stomatological Hospital of Nanjing University School of Medicine, Nanjing 210008, People’s Republic of China; 3Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou 221006, People’s Republic of China*These authors contributed equally to this workCorrespondence: Bing GuDepartment of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou 221006, People’s Republic of ChinaEmail binggu2015@xzhmu.edu.cnBackground: Clostridium difficile infection (CDI) has become one of the most important factors threatening human health, and about 20– 30% antibiotic-related diarrhea cases and almost all pseudomembranous enteritis cases are related to CDI. The high recurrence of Clostridium difficile (C. difficile) and the emergence of drug resistance make clinical treatment of CDI difficult. Therefore, there is an urgent need to develop a non-antibiotic-alternative therapy against CDI. Gold nanoclusters (AuNCs) can better interact with bacteria due to its ultrasmall size. The aim of the present study was to explore whether AuNCs could be used as an antibacterial agent to kill C. difficile.Methods: AuNCs and C. difficile were co-cultivated in an anaerobic atmosphere to evaluate the bactericidal effect of AuNCs. The bacterial growth rate was estimated by using two concentrations of AuNCs (50 and 100 μM). The damage of AuNCs to C. difficile is detected by SYTOX Green staining methods and SEM image analysis. The mechanism of AuNCs on C. difficile was explored by reactive oxygen species (ROS) detection. The toxic effect of AuNCs on human cells was evaluated by MTT method.Results: AuNCs (100 μM) killed C. difficile drastically. AuNCs increased the release of ROS by about 5 fold and destroyed the membrane integrity of C. difficile cells without causing significant toxic effect on human cells.Conclusion: AuNCs showed great potential as an alternative to traditional antibiotics in killing C. difficile and may prove to be an alternative to treat CDI.Keywords: Clostridium difficile, antibacterial effect, gold nanoclusters, reactive oxygen species

Ziyan Kong,1,* Rui Cai,1,* Chen Cheng,1 Chuanling Zhang,2 Haiquan Kang,3 Ping Ma,3 Bing Gu1,3 1Medical Technology School, Xuzhou Medical University, Xuzhou, Jiangsu Province, People’s Republic of China; 2Department of Laboratory Medicine, XuZhou Children’s Hospital, Xuzhou, Jiangsu Province, People’s Republic of China; 3Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Bing GuDepartment of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou 221006, People’s Republic of ChinaTel +8615862163560Email gb20031129@163.comPurpose: Carbapenem-resistant Klebsiella pneumoniae (CRKP) have emerged worldwide and also being a major threat to children and neonate. In this study, we describe a nosocomial outbreak of NDM-5-producing Klebsiella pneumoniae in neonatal unit of a teaching hospital in China from September 2015 to September 2016.Patients and methods: We collected 12 carbapenem-resistant K. pneumoniae outbreak strains from 12 newborns and characterized these isolates for their antimicrobial susceptibility, clone relationships, and multi-locus sequence types using vitek-2 compact system, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Resistant genes were detected by using PCR and sequencing. Plasmid conjugation experiment was carried out to determine the transferability of carbapenem resistance. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and southern blotting were conducted for plasmid profiling.Results: All 12 K. pneumoniae isolates were resistant to carbapenems and carried blaNDM-5, blaTEM-1 and blaSHV-11. Furthermore, PFGE analysis showed that NDM-5-producing K. pneumoniae were clonally related and MLST assigned them to sequence type 337. Conjugative assays showed that plasmids harboring blaNDM-5 gene were self-transmissible. Plasmid analysis suggested that all blaNDM-5 gene located on a ∼45 kb IncX3 type plasmid.Conclusion: To the best of our knowledge, this is the first report of a clone outbreak of blaNDM-5-carrying K. pneumoniae isolates from neonates. There is an urgent need for effective infection control measures to prevent blaNDM-5 variants from becoming epidemic in the neonates in the future.Keywords: Klebsiella pneumoniae, carbapenemases, blaNDM-5, ST337, neonate, IncX3

Meixian Chen,1,* Lisheng Zhou,1,* Zhaoxia Liao,1 Xijiu Ye,1 Xujun Xuan,2 Beibei Gu,1 Fuding Lu1 1Department of Anesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China; 2Department of Andrology, The Seventh Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fuding LuDepartment of Anesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of ChinaTel +86-13560371081Email lfd19870901@126.comBackground: Osteosarcoma is one of the most common primary bone cancers with predominant occurrence in children and adolescents. This study aimed to determine the effects of sevoflurane treatment on the osteosarcoma progression and to explore the underlying molecular mechanisms.Materials and methods: The mRNA and protein expression levels were determined by qPCR and Western blot, respectively. Osteosarcoma cell proliferation, apoptosis and invasion were determined by MTT, caspase-3 activity, colony formation and Transwell invasion assays, respectively. The interaction between miR-203 and WNT2B 3ʹ untranslated region was confirmed by luciferase reporter assay.Results: Sevoflurane treatment for 6 hrs concentration-dependently suppressed cell viability, increased caspase-3 activity and up-regulated miR-203 expression in both U2OS and MG63 cells. MiR-203 overexpression suppressed cell viability, increased caspase-3 activity and suppressed cell growth and invasion of osteosarcoma cells. In addition, miR-203 knockdown attenuated the tumor-suppressive effects of sevoflurane treatment on osteosarcoma cells. Mechanistic studies showed that miR-203 repressed the expression of WNT2B in U2OS cells, and inhibition of miR-203 attenuated the suppressive effects of sevoflurane on WNT2B expression. More importantly, WNT2B overexpression attenuated the effects of sevoflurane treatment on cell viability, caspase-3 activity, cell growth and invasion of U2OS cells. MiR-203 overexpression suppressed Wnt/β-catenin signalling. Similarly, sevoflurane suppressed the activity of Wnt/β-catenin signalling, which was partially reversed by miR-203 knockdown and WTN2B overexpression.Conclusion: Our data showed the tumor-suppressive effects of sevoflurane on osteosarcoma cells, and mechanistic studies revealed that sevoflurane inhibited osteosarcoma cell proliferation and invasion partly via targeting the miR-203/WNT2B/Wnt/β-catenin axis.Keywords: osteosarcoma, proliferation, invasion, sevoflurane, miR-203, WNT2B, Wnt/β-catenin

Yizhao Xie,1,2,* Bingxin Gu,2–5,* Xichun Hu,1,2 Yingjian Zhang,2–5 Jian Zhang,1,2 Zhonghua Wang,1,2 Yannan Zhao,1,2 Chengcheng Gong,1,2 Yi Li,1,2 Zhongyi Yang,2–5 Biyun Wang,1,21Department of Medical Oncology, Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of China; 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China; 3Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of China; 4Center for Biomedical Imaging, Fudan University, Shanghai, People’s Republic of China; 5Shanghai Engineering Research Center of Molecular Imaging Probes, Shanghai, People’s Republic of China*These authors contributed equally to this workBackground: Platinum-based chemotherapy is widely used as first-line therapy for metastatic triple-negative breast cancer (mTNBC). Intratumor heterogeneity derived from fluorine-18 fluorodeoxyglucose (18,F-FDG) positron emission tomography/computed tomography (PET/CT) is a potential predictor of treatment outcomes and the prognosis of breast cancer. However, the presence of multiple lesions and complex calculation methods leads to difficulties in the clinical use of this parameter for metastatic breast cancer. The aim of this study is to provide a convenient and effective measurement of intratumor heterogeneity to predict treatment outcomes for mTNBC patients with lung metastasis.Patients and methods: We enrolled mTNBC patients with lung metastasis who underwent 18,F-FDG PET/CT scans before first‐line therapy from three clinical trials (NCT01287624, NCT02341911 and NCT02546934). Apart from the regular FDG parameters, including standard uptake value (SUV), total lesion glycolysis (TLG) and metabolic tumor volume (MTV), we defined the lung index as the SUVmean divided by the difference between the SUVmax and SUVmean for the targeted lung lesion. The MTV was automatically exported from the manual delineation using software based on an adaptive threshold of SUV intensity >2.5 within the contouring margin. The TLG was calculated using the following formula: TLG=SUVmean×MTV. Progression‐free survival (PFS) and overall survival were estimated by the Kaplan–Meier method, and univariate and multivariate analyses were performed using the Cox proportional hazard model.Results: The data from 31 patients were available for analysis. The median PFS of low-lung index (LI) patients was 8.1 months, which was significantly longer than that of high-LI patients (HR=3.3, 95% CI 1.5–7.3, p=0.003). Patients with low TLG had a significantly better PFS than those with high TLG (HR=2.6, 95% CI 1.2–5.8, p=0.014). Patients with low TLG had significantly longer overall survival than those with high TLG (31.2 months vs 13.9 months, HR=3.1, 95% CI 1.2–8.6, p=0.029). Multivariate analysis confirmed the predictive value of LI and TLG.Conclusions: This study proposed a new “PET biopsy” method to evaluate the intratumor heterogeneity of mTNBC on pretreatment 18,F-FDG PET/CT scans and indicated the predictive value of LI and TLG for first-line platinum-based treatment outcomes and overall survival. These findings could help clinicians recognize patients who are likely to not only have a favorable response to platinum-based therapy but also a good prognosis.Keywords: triple-negative breast cancer, chemotherapy, PET, tumor heterogeneity

Bin Gu,1 Meng Li,2 Yan Zhang,3 Lingyun Li,4 Kun Yao,5 Shizhi Wang3 1Department of Neurosurgery, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China; 2Department of Neurosurgery, Suqian First Hospital, Suqian, China; 3Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, China; 4Department of Microbiology and Immunology, Nanjing Medical University, Nanjing, China; 5Department of Developmental Genetics, Nanjing Medical University, Nanjing, China Purpose: We previously identified human herpesvirus 6 (HHV-6) infection in the pathogenesis of glioma. Direct repeat (DR)7, encoded by HHV-6, has been reported to possess malignant transforming activity and involved in Hodgkin’s lymphoma carcinogenesis. Here, we aimed to determine the role of DR7 in the development and progression of glioma. Patients and methods: A total of 27 glioma and 30 normal brain tissues were collected for detection of DR7. Glioma cell proliferation, colony formation, cell cycle, migration, invasion and angiogenesis were evaluated by Cell Counting Kit-8 (CCK-8), soft agar, propidium iodide staining, wound healing, Transwell and chick embryo chorioallantoic membrane assays, respectively. The potential mRNA targets of DR7 were determined using mRNA microarray and validated via Western blot and ELISA. Results: DR7 could be detected in the 13 glioma tissues with a positive rate of 48.15%, but only the 5 normal brain tissues with a lower positive rate of 16.7%. The two strains of cells isolated from glioma tissues were also found to express DR7. CCK-8 and soft agar assays showed enhanced proliferation and colony formation in the cells expressing DR7 which might be in relation to acceleration of the G1/S phase transition by DR7. Further analyses showed that DR7 could promote glioma cell migration, invasion and angiogenesis. Expression profiles identified hundreds of differentially expressed mRNAs, among which P53, extracellular matrix (ECM) fibronectin, integrin receptor ITGβ5 and specific inhibitors of MMPs, tissue inhibitor of MMPs (TIMP)-2 and TIMP-4, were downregulated, whereas ECM-degrading proteinase MMP-3, proinflammatory cytokines IL-1β, IL-6 and IL-8, were upregulated by DR7, respectively. Conclusion: We observed existence of DR7 in the glioma tissues, and overexpression of DR7 could promote glioma cell development and progression, which might be through creating an inflammatory microenvironment and enhancing degradation of ECM. Keywords: HHV-6, DR7, development, progression, glioma

Bin Gu,1 Lingyun Li,2 Meng Li,3 Jinfeng Wang,4 Guofeng Zhang,4 Kun Yao,4 Shizhi Wang5 1Department of Neurosurgery, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China; 2Department of Developmental Genetics, Nanjing Medical University, Nanjing, China; 3Department of Neurosurgery, Suqian First Hospital, Suqian, China; 4Department of Microbiology and Immunology, Nanjing Medical University, Nanjing, China; 5Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, China Purpose: We previously found the involvement of human herpesvirus 6 (HHV-6) infection in the pathogenesis of glioma. U94/rep, encoded by HHV-6, has been identified to play a vital role in viral gene expression and latency. Recent studies have shown its inhibition of angiogenesis and tumorigenesis in endothelial cells and prostate cancer cell line PC3, respectively. Here, we aimed to investigate the role of U94/rep in the development and progression of glioma.Patients and methods: A total of 20 glioma tissues with positive HHV-6 infection were used for detection of U94/rep. MTT, soft agar, propidium iodide staining, wound healing, Transwell, and chick embryo chorioallantoic membrane assays were applied for evaluation of glioma cells’ proliferation, colony formation, cell cycle, migration, invasion, and angiogenesis, respectively.Results: U94/rep transcripts could be detected in 11 out of 20 glioma tissues with positive HHV-6 infection. Furthermore, MTT and soft agar assays revealed that overexpression of U94/rep inhibited glioma cell proliferation and colony formation, which may be attributed to the cell cycle arrest at S phase induced by U94/rep. Further analysis demonstrated that U94/rep inhibited glioma cells’ migration and invasion and ex vivo angiogenesis. Reduced expression of proangiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, and type IV collagenases, MMP-2 and MMP-9, was detected in cells overexpressing U94/rep. These decreased factors may undermine glioma cell migration, invasion, and angiogenesis.Conclusion: Our results demonstrated that U94/rep could inhibit malignant phenotypes of glioma cells, indicating that it is a potential target for therapeutic intervention. Keywords: HHV-6, U94/rep, tumorigenesis, glioma