Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection reagent for siRNAs. Our results indicate that the PEI-NH-SWNTs and PEI-NH-MWNTs produced in this study are capable of delivering siRNAs into HeLa-S3 cells to suppress gene expression and may therefore be considered as novel nonviral gene delivery reagents

Identification and analysis of ice plant (Mesembryanthemum crystallinum) conserved and novel microRNAs by high-throughput sequencing and their association with salt stress response

Ice plant (Mesembryanthemum crystallinum L.) is a facultative halophyte that high degree of salt tolerance can be induced at a specific developmental stage. Several levels of regulation, including genetic/epigenetic, transcriptional/post-transcriptional, and translational/post-translational, are involved in the complex process of stress induction. High-throughput sequencing was used to identify miRNA profiles in ice plant. Sequences of poly (A)+ RNA and small RNA (sRNA) from 3-day-old seedlings (control) and 200 mM NaCl-treated 6 h (salt) were obtained and analyzed. Total 3,089,326 and 3,157,610 redundant clean reads of 18- to 25-nt sRNA, representing 1,106,388 and 1,118,741 unique sRNA sequences were generated from control and salt libraries, respectively. The 24-nt sRNAs were the most abundant sRNA and exhibited the highest sequence diversity, consistent with the profiles obtained by other plant sRNA. miRBase annotation of ice plant sRNA revealed 143 conserved miRNAs belonging to 38 families. Among them, miR166 was the most abundant family and miRNA156/157 consisted of the largest numbers. The hairpin precursor structures of total 19 conserved mcr-miRNAs belonging to 12 miRNA families and 24 novel mcr-miRNAs were identified according to strict criteria. Most of them paired with the star sequences, with two exceptions. Minimal folding free energy index of most mcr-miRNA candidates were greater than 0.85, a threshold value to distinguish miRNA precursor and other structural RNAs. Three conserved mcr-miR156 candidates were salt-induced and two of them were highly abundant in both control and salt libraries, indicating the conserved miR156/157 family was highly expressed at the early stage of development and was responsive to salt stress. The further investigation for the expression of mcr-miRNA and potential target gene would help understanding of miRNA regulation in response to salt stress in ice plant. Two strategies, poly(A)-tailing and stem-loop RT-PCR, were used to evaluate the expression profiles of conserved and novel mcr-miRNA in control and 200 mM NaCl-treated seedlings. Poly(A)-tailing RT-PCR was able to detect the expression profile of mcr-miRNA families. Five miRNA families, namely miR159, miR162, miR168, miR171, and miR396,were stress-responsive and their potential target gene families exhibited reciprocal expression patterns under salt stress. Furthermore, stem-loop RT-PCR was performed to detect individual miRNA expressions using optimalized miRNA-specific forward primers. Nine pairs of salt-responsive individual mcr-miRNAs/target genes including mcr-miR156-1/SPL6, mcr-miR159-1/MYB33, mcr-miR162/DCL1, mcr-miR164/NAC100, mcr-miR168/AGO1, mcr-miR168/CYP76C4, mcr-miR169-2/NFYA1, mcr-miR396/FBP, and mcr-miR403/AGO2 showed reciprocal correlation patterns. Degradome analysis validated mcr-miR164, mcr-miR166-1, mcr-miR169-1, mcr-miR169-2, and mcr-miR403 specifically cleaved the transcripts of target genes. Target predictions using ice plant transcriptome revealed mcr-miRNAs function in transcriptional regulation, chromosome remodeling, ion homeostasis, photosynthesis, protein degradation, signal transduction, hormone-related, and redox reaction. High-throughput sequencing is powerful technique to identify miRNA profile and stem-loop real-time PCR is highly specific method for miRNA quantification. Characterization of mcr-miRNAs using these two strategies is useful in exploring the unique mechanism of salt tolerance in ice plant.冰花(Mesembryanthemum crystallinum L.)為一種耐鹽模式植物，其耐鹽能力會隨著發育時期的轉變與鹽逆境的誘導而提高，目前已鑑定出特定機制在基因、轉錄與後轉錄、以及轉譯與後轉譯層次參與調控冰花耐鹽機制的誘導。在本論文中利用高通量定序法鑑定並分析冰花的基因表現圖譜，以播種後三天大的小苗為對照組以及200 mM鹽處理6小時的小苗為實驗組，進行mRNA與small RNA (sRNA)的高通量定序，定序結果在長度介於18到25個核苷酸的sRNA中，對照組與實驗組分別獲得3,089,326與3,157,610條重複序列，以及去除重複序列後得到的 1,106,388 與1,118,741條單一序列。分析核苷酸長度的分布，發現長度24個核苷酸的sRNA佔有最大的比例，並且具有最高的序列歧異度，此現象與分析其他植物的sRNA的結果相同。利用miRBase進行sRNA的身份鑑定得到143個高度保留性的miRNA，分別屬於38種miRNA家族，其中miR166家族具有最高的表現量而miR156家族則具有較多的成員。接著以嚴格的設定條件進行冰花miRNA的鑑定，共有19個高度保留性的miRNA與24個在其他物種中尚未被鑑定的miRNA，在冰花的transcriptome中可以找到具有hairpin結構的pre-miRNA，除了兩個例外，其餘均可鑑定到miRNA*的序列。在這些高度保留性以及嶄新的miRNA中，大多數的minimal folding free energy index大於0.85，此數值為區別miRNA與其他sRNA的閥值，以上參數皆可提高經由預測所得到的冰花特有miRNA的真實性。三個冰花miRNA成員在鹽逆境下具有明顯的表現量差異，其中兩個在對照組與實驗組當中都具有極高的表現量，顯示miRNA156家族在冰花發育的早期大量表現並參與鹽誘導相關基因的調控。進一步分析冰花miRNA與目標基因的表現情形，以期瞭解冰花中miRNA所調控的耐鹽機制。利用poly(A)-tailing與stem-loop RT- PCR兩種策略在對照組及鹽處理的實驗組中對冰花保留性與嶄新的miRNA進行偵測並定量分析，其中poly(A)-tailing RT- PCR可有效的偵測miRNA基因家族的表現，鑑定出五個對鹽有反應的miRNA基因家族miR159、miR162、miR168、miR171與miR396，且在鹽逆境下皆與預測之目標基因家族表現呈現相互消長的趨勢。進一步利用stem-loop RT- PCR搭配2-3核苷酸互補的最佳化miRNA專一性引子，可專一性並零敏的偵測個別miRNA的表現，一共鑑定出九個對鹽處理有反應並具有相互消長表現的個別miRNA與目標基因的組合，分別是mcr-miR156-1/SPL6、mcr-miR159-1/MYB33、mcr-miR162/DCL1、 mcr-miR164/NAC100、mcr-miR168/AGO1、mcr-miR168/CYP76C4、mcr-miR169-2/NFYA1、mcr-miR396/FBP與mcr-miR403/AGO2。由植物降解組的分析中證實mcr-miR164、mcr-miR166-1、mcr-miR169-1、mcr-miR169-2與mcr-miR403專一性的切割並調控目標基因在鹽逆境下的表現。預測冰花中受到miRNA調控的目標基因群，發現其功能參與轉錄的調控、染色體的重組、細胞內離子的平衡、光合作用、蛋白的降解、訊息傳遞、氧化還原反應與賀爾蒙相關基因的調控等生理反應。高通量定序法與stem-loop RT- PCR可大量且專一性的鑑定並分析miRNA的表現，兩種策略的搭配可有效的進一步探究後轉譯修飾參與調節冰花的耐鹽性。Contents 摘要…………………………………………………………….……………………....I Abstract………………………………………………………………………..……..III Contents…………………………………………………….…………………………V Contents of Tables……………………………………………..……………………VII Contents of Figures…..…………………………………………………………….VIII Chapter One………..………………………………………………..…………………1 Abstract…………………………………………………………….…………….…2 Introduction…………………………………………………………..……………..4 Research objectives and strategies…………..……………………...……………..13 Materials and methods.............................................................................................15 Result and Discussion..............................................................................................21 Conclusion…...……………………………………………………….……………34 Tables………………..………….…………………………………………............35 Figures......................................................................................................................65 References…………………………………………………….…………………...79 Appendix……………………………………………………….………………….94 Chapter Two…...…………………………………………..…………………………95 Abstract……………………..…………………………….…………………….…96 Introduction…………………………………………..……………………………98 Materials and methods...........................................................................................108 Result and Discussion.............................................................................................113 Conclusion……...……………………………………….…..……………………124 Future perspective..................................................................................................125 Tables………………..……...……………………………………………............127 Figures....................................................................................................................140 References……………………………………………….……………………….15

Analysis of the Sliding Contact Behavior of a Centrifugal Clutch

The analysis of the sliding contact behavior for friction lining is an important issue in design of centrifugal clutches. In this study, a numerical model based on the moment equilibrium and contact mechanics has been developed to analyze the dynamics of a commercial centrifugal clutch. The proposed dynamic model can estimate the rotational speed of initial contact, output torque, and contact pressure for friction lining during the clutch operation. The comparison of the numerical and experimental results for the output torque of the analyzed centrifugal clutch shows the simulation agrees well with the experimental data. The difference for the maximum output torque is around 1.9%, which shows the effectiveness of the proposed method. The proposed dynamic model can reduce unnecessary physical prototypes and experimental cost, and is expected to be used to improve the design of the centrifugal clutches.</jats:p

Analyses of ATPase activity and tissue-specific expression of a salt-induced ice plant protein mcSKD1

mcSKD1為耐鹽植物冰花(Mesembryanthemum crystallinum L.)之鹽誘導基因，本論文主要是探討mcSKD1蛋白在冰花耐鹽機制中所扮演的角色。mcSKD1蛋白胺基酸序列與老鼠 SKD1 (suppressor of K+ transport growth defect) 及酵母菌 VPS4 (vacuolar protein sorting) 蛋白有很高的相似性，三者皆具有一段高度保留的 ATPase (or AAA) domain，屬於 AAA-type (ATPase associated with a variety of cellular activities) ATPase 蛋白家族。由酵母菌互補測試中發現，老鼠 SKD1 與冰花 mcSKD1 蛋白皆具有幫助鉀離子吸收的功能，此外 mcSKD1 蛋白還與鈉離子的區隔有關。而 酵母菌VPS4 蛋白已被證實具有 ATPase 酵素活性，並參與酵母菌液泡蛋白的運輸，在 endosomal transport 系統中扮演重要的角色。為了探討 mcSKD1 蛋白於冰花細胞內的功能與耐鹽機制的關係，本論文利用大腸桿菌大量表現重組 mcSKD1 蛋白，並以親合性管柱純化，分析其 ATPase 酵素活性及蛋白特性，測得酵素活性 Km = 2.73 mM、Kcat = 60.32mol/min/g，經由酵素親合性分析與 nucleotide binding assay，發現在高濃度 ATP 下，ADP 會與 mcSKD1 蛋白緊密結合，導致 ATPase 酵素活性的降低。此外利用 K177A、E231Q、K177A/E231Q 等定點突變探討高度保留性序列 Walker A、B motifs 對 mcSKD1 酵素活性的影響，發現突變株與野生株的相對酵素活性分別為 K177A：41 %、E231Q：14 %、K177A/E231Q：160 %，顯示 Walker A、B motifs 在水解 ATP 的反應中扮演重要的角色。 以專一性抗體偵測 mcSKD1 蛋白於冰花各器官的累積情形，在加鹽及未加鹽處理的葉、根，花與種子中皆可偵測到 mcSKD1 蛋白的累積，顯示 mcSKD1 為持續表現之蛋白。利用免疫螢光法偵測 mcSKD1 蛋白的組織表現專一性，發現 mcSKD1 蛋白在根的皮層及韌皮部、葉的腎型細胞、花粉囊與發育中的種皮內累積，推測與冰花生殖細胞生長發育時的營養吸收及鹽逆境下鈉離子的區隔相關。此外由蔗糖梯度離心與免疫金標定法發現，mcSKD1 蛋白分佈於密度很輕的microsome fractions，且聚集形成small vesicles，顯示 mcSKD1 可能與 VPS4 蛋白具有類似的功能，參與蛋白經由內膜系統運輸之過程(endomembrane protein trafficking)。根據以上結果推測，mcSKD1 蛋白在鹽逆境下，利用其 ATPase 酵素活性，提高液泡蛋白運輸之效率，在腎型細胞中參與鈉離子的區隔，協助根部鉀離子的吸收，以調節滲透潛勢，降低鹽逆境所造成的傷害。A salt-induced gene mcSKD1 was identified from halophyte Mesembryanthemum crystallinum L. (ice plant). The main focus of this thesis is to examine the roles of mcSKD1 protein participate in the salt-tolerant mechanism of ice plant. The amino acid sequence of mcSKD1 was highly homologous to mouse SKD1 (suppressor of K+ transport growth defect) and yeast VPS4 (vacuolar protein sorting). All of them have a highly conserved ATPase (or AAA) domain, belong to AAA-type (ATPase associated with a variety of cellular activities) ATPase protein family. Yeast complementation assay showed mcSKD1 and mouse SKD1 were involved in K+ uptake and mcSKD1 was responsible for the compartmentation of Na+. VPS4 was confirmed with vacuolar protein sorting of endosomal transport system through the ATPase activity. To examine the cellular function of mcSKD1 and roles in salt-tolerant mechanism in ice plant, recombinant mcSKD1 was over-expressed in E. coli and purified by affinity column to analyze ATPase activity and enzymatic properties of protein. The enzyme kinetics showed mcSKD1 has ability to hydrolyze ATP with a Km of 2.73 mM and a Kcat of 60.32mol/min/g. Use the affinity- and nucleotide-binding assay, we found the ATPase activity was greatly reduced by ADP tightly bound with mcSKD1 in high ATP concentrations. The structure of ATPase domain was further examined by point mutation of highly conserved Walker A and B motifs. Three mutants were constructed, namely K177A, E231Q and K177/E231Q. The wild-type and mutants' relative ATPase activity were K177A: 41%, E231Q: 14 % and K177A/E231Q: 160 %. The results indicated that the Walker A and B motifs were important to ATP hydrolysis ability of mcSKD1. Accumulation of mcSKD1 at different organs was detected by anti-mcSKD1 antibody. We found mcSKD1 accumulated in leaves, roots, flowers and seeds of unstressed and stressed plants. The results indicated mcSKD1 was constitutively expressed protein. The immune-fluorescence was used to detect the tissue-specific expression of mcSKD1. The mcSKD1 was specifically accumulated in cortex and phloem of root, bladder cells of leaves, pollen sac and developing seed coat. The results suggested mcSKD1 plays a role in nutrient uptake of developing reproductive cells and responsible for the compartmentation of excess Na+ under salinity stress. Furthermore, the accumulation of mcSKD1 was found to localize at low-density small vesicles by gradient ultra-centrifugation and immuno-gold labeling. The results indicated mcSKD1 had similar function with VPS4, which was involved in cellular protein sorting. In conclusion, the ATPase activity of mcSKD1 drives the energy obtained by ATP hydrolysis and uses in compartmentation of excess Na+ in bladder cells and K+ uptake in roots, and as the result, the ion homeostasis and osmotic potential was maintained.目錄： 中文摘要…………………………………………………………………...I 英文摘要………………………………………………………………… .II 壹、前人研究……………………………………………………………...1 一、耐鹽機制的模式植物冰花…………………………………………….2 二、冰花耐鹽相關 mcSKD1 基因與 AAA-type ATPase………………….3 三、酵母菌液泡蛋白運輸之相關蛋白 VPS……………………………...4 貳、材料與方法…………………………………………………………...9 一、實驗材料……………………………………………………………….9 (一)、冰花的培養………………………………………………………….9 (二)、癒傷組織的培養…………………………………………………….9 二、實驗方法………………………………………………………………10 (一)、mcSKD1 基因構築於表現載體 pET32a(+)……………………… 10 (二)、mcSKD1-(His)6 蛋白的表現與分析……………………………..12 (三)、冰花組織石臘切片…………………………………………………16 (四)、冰花蛋白的萃取……………………………………………………17 (五)、蔗糖梯度離心………………………………………………………18 (六)、冰花組織塑膠切片…………………………………………………18 (七)、免疫金標定…………………………………………………………19 參、結果………………………………………………………………....21 肆、討論………………………………………………………………....29 伍、參考文獻…………………………………………………………....36 圖表： 表一、MS 培養基配方…………………………………………………….41 表二、水耕液配方……………………………………………………....42 表三、本論文使用的引子…………………………………………………43 表四、本論文使用的大桿菌菌株…………………………………………44 表五、野生型 mcSKD1-(His)6 蛋白之純化表………………………….45 圖一、pMCSKD32 載體之構築…………………………………………….46 圖二、mcSKD1-(His)6 蛋白的基因與胺基酸序列………………………47 圖三、大量表現之mcSKD1-(His)6 與 mcSKD1K177A-(His)6 蛋白於不 同誘導時間下的蛋白圖譜………………………………………..48 圖四、大量表現之 mcSKD1E231Q-(His)6 與 mcSKD1K177A/ E231Q-(His)6 蛋白於不同誘導時間下的蛋白圖譜…………...49 圖五、利用 Anti-His 抗體偵測野生型與突變型 mcSKD1-(His)6 蛋白…………………………………………………………………50 圖六、mcSKD1-(His)6 蛋白的純化…………………………………....51 圖七、mcSKD1-(His)6K177A 蛋白的純化……………………………...52 圖八、mcSKD1E231Q-(His)6 蛋白的純化……………………………….53 圖九、mcSKD1K177A/ E231Q-(His)6 蛋白的純化…………………….54 圖十、純化後之野生型與突變型 mcSKD1-(His)6 蛋白圖譜………...55 圖十一、mcSKD1-(His)6 蛋白 N 端序列分析………………………….56 圖十二、ATPase 酵素活性測試………………………………………...58 圖十三、不同 ATP 濃度與 mcSKD1-(His)6 蛋白之 ATPase 酵素活性 關係圖…………………………………………………………………………..59 圖十四、mcSKD1-(His)6 蛋白受質親合性之分析………………………60 圖十五、mcSKD1-(His)6 蛋白nucleotide binding assay……………61 圖十六、野生型與突變型 mcSKD1-(His)6 蛋白 ATPase 酵素活性之 比較………………………………………………………………..62 圖十七、水耕培養之不同冰花器官中 mcSKD1 蛋白的累積情形………63 圖十八、土耕培養之不同冰花器官中 mcSKD1 蛋白的累積情形……..64 圖十九、mcSKD1 蛋白在冰成熟根的組織專一性表現………………...65 圖二十、mcSKD1 蛋白在冰花幼根的組織專一性表現………………...66 圖二十一、mcSKD1 蛋白在種子發育階段的組織專一性表現………...67 圖二十二、mcSKD1 蛋白在冰花花藥的組織專一性表現……………...68 圖二十三、mcSKD1蛋白在冰花葉部的組織專一性表現…………………69 圖二十四、mcSKD1蛋白在冰花癒傷組織中的分佈情形………………..70 圖二十五、蔗糖梯度離心…………………………………………………71 圖二十六、mcSKD1 蛋白在葉部細胞的累積情況……………………...72 圖二十七、mcSKD1 蛋白在根部細胞的累積情況……………………...73 圖二十八、mcSKD1 蛋白在癒傷組織的累積情況……………………...74 圖二十九、NSF 蛋白 D2 domain 3D 結構圖。………………………..7

Increased medications in the aged patients with burning mouth syndrome: A potentially overlooked risk factor

Background/purpose: Burning mouth syndrome (BMS) is a chronic oral mucosal burning and pain disorder, predominantly affecting the middle-aged and elderly women. This study aimed to explore the association between polypharmacy and BMS, with consideration of the patients’ age and sex. Materials and methods: Medical data from 115 BMS patients and 115 age- and sex-matched control subjects were collected. Medications were categorized into four groups based on the number of medications taken. The chi-square test, Student's t-test, and binary logistic regression were performed to evaluate the association between polypharmacy and BMS, after adjustment for age and sex. Results: A higher proportion of individuals with medications was observed in the BMS group than in the control group (P = 0.0015). Among BMS patients, the older group (≥ 65 years), especially the female BMS patients, tended to take more medications. Binary logistic regression analysis revealed a significant association between polypharmacy and increased odds of having BMS. After adjustment for age and sex, the patients in the minor polypharmacy group (1–4 medications) had significantly higher odds of having BMS compared to those in the non-pharmacy group (no medications), with an adjusted odds ratio of 5.015 (P < 0.001). Conclusion: These findings suggest a potential association between multiple medications and the risk of having BMS and supported the need to consider polypharmacy as a contributing factor for the older BMS patient. However, future studies should be conducted to explore the exact dose–response trend and the influence of other associated factors on BMS