Persistence of Japanese Encephalitis Virus Is Associated with Abnormal Expression of the Nonstructural Protein NS1 in Host Cells

AbstractPersistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (103to 104PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1′ were produced in C2-2 cells. Both truncated NS1 and NS1′ contain deletions at their N-termini; however, the analyses by RT–PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus–cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed

A Novel RFID Sensing System Using Enhanced Surface Wave Technology for Battery Exchange Stations

This paper presents a novel radio-frequency identification (RFID) sensing system using enhanced surface wave technology for battery exchange stations (BESs) of electric motorcycles. Ultrahigh-frequency (UHF) RFID technology is utilized to automatically track and manage battery and user information without manual operation. The system includes readers, enhanced surface wave leaky cable antennas (ESWLCAs), coupling cable lines (CCLs), and small radiation patches (SRPs). The RFID sensing system overcomes the electromagnetic interference in the metallic environment of a BES cabinet. The developed RFID sensing system can effectively increase the efficiency of BES operation and promote the development of electric vehicles which solve the problem of air pollution as well as protect the environment of the Earth

The genome sequence of the orchid Phalaenopsis equestris

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers. (Résumé d'auteur

C. elegans EIF-3.K Promotes Programmed Cell Death through CED-3 Caspase

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Health-care herb screening of inhibiting lung cancer cell proliferation and metastasis: Utilizing HLJ1 and IL-8 promoter reporter gene assay

近年來，由於人類遭逢最嚴重的疾病威脅 (癌症)，導致每年死亡人數不斷攀升，且越來越糟。許多的藥理學家便嘗試著想從傳統的保健草藥中尋求解答，然而，面對數以千萬計的保健藥材和未知的抗癌機轉，並沒有一個具專一性、而又高效率的篩選藥物方法。根據先前以基因晶片分析肺癌的相關研究中，我們已經發現抑癌基因 (HLJ1) 在肺癌病人有不正常的表現，且與其預後存活率相關。在本研究中，我們將建立利用抑癌基因 (HLJ1) 和血管新生因子 (IL-8) 的啟動子之報導基因分析 (reporter gene assay) 模式，來篩選抑制肺癌細胞生長、轉移、及調控血管新生之保健植物的平台。首先，使用CL1-0細胞和MTT分析來決定九十種中草藥的細胞毒性；隨後分別轉殖HLJ1和IL-8啟動子載體至CL1-0細胞，然後處理使細胞死亡20%的藥物濃度 (IC20)；24小時後，使用報導基因分析偵測螢光酵素活性。接著以西方墨點法 (western blotting) 分析CL1-0和CL1-5細胞中HLJ1蛋白質的表現量，來確認報導基因分析的結果。為了更進一步研究大量表現HLJ1的影響，使用細胞移動和侵入分析 (wound-healing and invasion assay) 來看能否抑制癌細胞的移動。實驗結果顯示有36種中草藥可以在CL1-0細胞中增加HLJ1啟動子的活性，有25種中草藥可以在CL1-5細胞中增加HLJ1啟動子的活性。而有37種中草藥可以在CL1-0和CL1-5細胞中誘導HLJ1蛋白質的表現。從這37種藥中挑選HLJ1表現量最高的五種中草藥，進行細胞移動和侵入分析；結果發現這五種中草藥可以抑制癌細胞的移動，而且這些中草藥可能是經由影響HLJ1啟動子上的YY-1結合區域 (binding domain) 或AP-1結合區域，進而使得HLJ1的表現上升。在IL-8的部份，有15種中草藥可以在CL1-5細胞中抑制IL-8啟動子的活性；從這15種藥中挑選IL-8表現最低的六種中草藥，進行電泳移動分析 (electromobility shift assay, EMSA)，結果發現有3種藥可能是經由影響IL-8啟動子上的轉錄因子NF-κB和AP-1，進而減少IL-8的表現。本論文中，我們建立一個以HLJ1和IL-8作為目標的藥物篩選平台，可以大量地篩選中草藥。由結果顯示，有數種測試之中草藥可以調節HLJ1或IL-8啟動子的活性，推測這些中草藥的萃取物可能含有重要的成分可以直接或間接地促進HLJ1或抑制IL-8的表現，進而抑制癌細胞的移動與侵入。Recently, the most severe disease, cancer, has attacked human and cause lots of people dead in every years and getting worse. A lot of pharmacologists try to find the answers from the traditional Chinese herb medicines. However, there is no specific and high-throughput way to screen thousands of these “health-care” herbs to unknown anti-cancer pathway. According to our previous microarray's studies, we have identified an oncosuppressor gene (HLJ1) which was reduced expression in highly metastatic lung cancer cells and might relate to patients' survival rate. In this study, the selected herbs were screened for their role in regulating the promoter activity of the HLJ1 and IL-8. First, we used MTT assay to determine cytotoxicity of ninety traditional Chinese medicines in CL1-0 cells. Subsequently, we transfected HLJ1 and IL-8 promoter construct to CL1-0 cells and then treated with the IC20 concentration of the herb medicines. After 24 hours, we measured the luciferase activity by reporter gene assay. To confirm the results of reporter assay, we carried out the western blotting of HLJ1 in CL1-0 and CL1-5 cells. Then we further performed wound-healing assay (migration assay) and invasion assay to investigate which traditional Chinese medicines can suppress cancer cell metastasis. We have identified thirty-six traditional Chinese herb medicines which can enhance HLJ1 promoter activity in CL1-0 and twenty-five traditional Chinese medicines which can enhance HLJ1 promoter activity in CL1-5. Furthermore, thirty-seven herb medicines could induce HLJ1 protein expression in CL1-0 or CL1-5. We also found that there are five of the thirty-seven Chinese herb medicines can suppress cancer cell migration and invasion. These Chinese herb medicines may induce HLJ1 promoter activity through affecting YY-1 and AP-1 protein binding affinity. In addition, our results also revealed that there are fifteen Chinese herb medicines that can suppress IL-8 promoter activity. Electromobility shift assay further confirmed that there are three Chinese herb medicines may suppress IL-8 promoter activity through inhibiting NF-κB and AP-1 binding affinity. In this study, we have set up a HLJ1 and IL-8 targeting drug-screening platform respectingly, which could be used to screen thousands of traditional Chinese herb (heath-care) medicines. Our results showed that there are several herb medicines that can up-regulate HLJ1 and down-regulate IL-8 promoter activity and suggested that these herb medicines may have the important elements that can indirectly or directly induce HLJ1 or repress IL-8 expression to suppress cancer cell migration and invasion.中文摘要..................................................i 英文摘要.................................................ii 目錄.....................................................iv 圖表目錄.................................................vi 縮寫字對照表...........................................viii 緒論 一、肺癌............................................1 二、癌轉移..........................................2 三、中草藥治療癌症..................................3 四、篩選平台........................................3 五、報導基因分析....................................4 六、熱休克蛋白......................................5 七、發炎反應與癌症..................................7 八、目的與策略......................................9 實驗材料與方法 一、 細胞株的選用...................................10 二、 細胞培養與分盤.................................10 三、 選擇中草藥.......................................11 四、 藥劑的準備.......................................12 五、 細胞增生分析...................................12 六、 質體的製備.......................................12 七、 細胞轉染作用.....................................15 八、 冷光報導基因分析.................................16 九、 細胞total RNA萃取................................16 十、 cDNA的合成.......................................17 十一、 蛋白質樣品製備及定量分析.....................18 十二、 西方墨點法...................................18 十三、 細胞遷移分析.................................19 十四、 細胞基質侵襲力分析...........................20 十五、 核內核外蛋白質分離萃取.......................20 十六、電泳移動分析..................................20 實驗結果 一、 測定中草藥對CL1-0細胞的毒性......................23 二、 利用磷酸鈣方法可以成功地轉殖質體至CL1-0細胞中....23 三、 報導基因分析平台可以大量地篩選對標的基因有影響的中草藥.....23 四、 利用報導基因分析平台發現有36種中草藥在CL1-0細胞中可以促進HLJ1 啟動子冷光酵素活性增加.......................24 五、 利用報導基因分析平台發現有25種中草藥在CL1-5細胞中可以促進HLJ1 啟動子冷光酵素活性增加.......................24 六、 36種中草藥對肺腺癌細胞CL1-0中HLJ1的蛋白質表現有影響........24 七、 37種中草藥對肺腺癌細胞CL1-5中HLJ1的蛋白質表現有影響........24 八、 確認CL1-5細胞中，CHM-10、CHM-37、CHM-45、CHM-76和CHM-82在HLJ1蛋白質層次上的表現..............................25 九、 中草藥增加HLJ1的表現進而影響肺腺癌細胞CL1-5遷移的能力.......25 十、 中草藥增加HLJ1的表現進而影響肺腺癌細胞CL1-5侵入的能力.......25 十一、 中草藥CHM-76會增加HLJ1 啟動子上AP-1結合區域的轉錄活性..26 十二、 中草藥會增加HLJ1 啟動子上YY-1結合區域的轉錄活性.........................................26 十三、 混合的中草藥會促進肺腺癌細胞中HLJ1 啟動子冷光酵素活性的表現增加............................26 十四、 混合的中草藥會促進肺腺癌細胞中HLJ1蛋白質的表現上升........................................27 十五、 15種中草藥對肺腺癌細胞CL1-5中IL-8 啟動子的活性有影響........................................27 十六、 中草藥可以經由中草藥影響轉錄因子與啟動子序列的結合進而抑制IL-8 啟動子的轉錄活性..........................27 討論....................................................29 結論....................................................35 參考文獻................................................36 實驗結果表圖............................................46 附表....................................................72 附圖....................................................90 附錄....................................................96 圖表目錄 圖表一、 MTT分析結果....................................46 圖二、 測試磷酸鈣方法(Calcium phosphate)轉殖質體至CL1-0細胞中的結果....48 圖三、 利用報導基因分析平台和肺腺癌細胞CL1-0大量地篩選中草藥對標的基因影響的結果...................................50 圖四、 利用報導基因分析平台和肺腺癌細胞CL1-5大量地篩選對標的基因有影響中草藥的結...................................51 圖五、 利用報導基因分析平台發現有36種中草藥在CL1-0細胞中可以促使HLJ1啟動子冷光酵素活性增加.........................52 圖六、 利用報導基因分析平台發現有25種中草藥在CL1-5細胞中可以促使HLJ1啟動子冷光酵素活性增加.........................53 圖七、 36種中草藥對肺腺癌細胞CL1-0中HLJ1的蛋白質表現影響的結果....................54 圖八、 37種中草藥對肺腺癌細胞CL1-5中HLJ1的蛋白質表現影響的結果....................55 圖九、 確認CL1-5細胞中，CHM-10、CHM-37、CHM-45、CHM-76和CHM-8蛋白質層次上的表現.................................56 圖十、 中草藥增加HLJ1的表現進而影響肺腺癌細胞CL1-5遷移的能力........................57 圖十一、 加入中草藥對肺腺癌細胞CL1-5侵犯能力的影響......60 圖十二、 中草藥影響HLJ1啟動子上AP-1結合區域的冷光酵素活性結果........................61 圖十三、 中草藥影響HLJ1啟動子上YY-1結合區域的冷光酵素活性結果........................62 圖十四、 混合中草藥促使肺腺癌細胞CL1-0中HLJ1啟動子冷光酵素活性的結果...............63 圖十五、 混合中草藥促使肺腺癌細胞CL1-5中HLJ1啟動子冷光酵素活性的結果...............64 圖十六、 混合的中草藥會促使肺腺癌細胞中HLJ1蛋白質的表現上升...............65 圖十七、 混合的中草藥會促使肺腺癌細胞中HLJ1蛋白質的表現上升...............66 圖十八、 中草藥對肺腺癌細胞CL1-5中IL-8啟動子活性影響的結果...............68 圖十九、 使用電泳移動分析方法觀察中草藥調控IL-8轉錄因子活性的結果...69 圖二十、 使用電泳移動分析方法觀察中草藥調控IL-8轉錄因子活性的結果....70 圖二十一、 使用電泳移動分析方法觀察中草藥調控IL-8轉錄因子活性的結果..71 附表一、 選擇中草藥(單一藥方)............................72 附表二、 選擇中草藥(混合藥方)............................88 附表三、 pGL3-basic primer...............................89 附表四、 Real-time PCR primer............................89 附圖一、 報導基因分析原理................................90 附圖二、 報導基因發光原理................................91 附圖三、 HLJ1啟動子全長序列..............................92 附圖四、 IL-8 wild-type啟動子序列........................93 附圖五、 pGL3-basic vector map...........................94 附錄、 熱休克蛋白分類與功能..............................9

One-Step Process for High-Performance, Adhesive, Flexible Transparent Conductive Films Based on p‑Type Reduced Graphene Oxides and Silver Nanowires

This work demonstrates a one-step process to synthesize uniformly dispersed hybrid nanomaterial containing silver nanowires (AgNWs) and p-type reduced graphene (p-rGO). The hybrid nanomaterial was coated onto a polyethylene terephthalate (PET) substrate for preparing high-performance flexible transparent conductive films (TCFs). The p-rGO plays the role of bridging discrete AgNWs, providing more electron holes and lowering the resistance of the contacted AgNWs; therefore, enhancing the electrical conductivity without sacrificing too much transparence of the TCFs. Additionally, the p-rGO also improves the adhesion between AgNWs and substrate by covering the AgNWs on the substrate tightly. The study shows that coating of the hybrid nanomaterials on the PET substrate demonstrates exceptional optoelectronic properties with a transmittance of 94.68% (at a wavelength of 550 nm) and a sheet resistance of 25.0 ± 0.8 Ω/sq. No significant variation in electric resistance can be detected even when the film was subjected to a bend loading with a radius of curvature of 5.0 mm or the film was loaded with a reciprocal tension or compression for 1000 cycles. Furthermore, both chemical corrosion resistance and haze effect were improved when p-rGO was introduced. The study shows that the fabricated flexible TCFs have the potential to replace indium tin oxide film in the optoelectronic industry