24 research outputs found
Neutrino Physics with JUNO
The Jiangmen Underground Neutrino Observatory (JUNO), a 20 kton multi-purposeunderground liquid scintillator detector, was proposed with the determinationof the neutrino mass hierarchy as a primary physics goal. It is also capable ofobserving neutrinos from terrestrial and extra-terrestrial sources, includingsupernova burst neutrinos, diffuse supernova neutrino background, geoneutrinos,atmospheric neutrinos, solar neutrinos, as well as exotic searches such asnucleon decays, dark matter, sterile neutrinos, etc. We present the physicsmotivations and the anticipated performance of the JUNO detector for variousproposed measurements. By detecting reactor antineutrinos from two power plantsat 53-km distance, JUNO will determine the neutrino mass hierarchy at a 3-4sigma significance with six years of running. The measurement of antineutrinospectrum will also lead to the precise determination of three out of the sixoscillation parameters to an accuracy of better than 1\%. Neutrino burst from atypical core-collapse supernova at 10 kpc would lead to ~5000inverse-beta-decay events and ~2000 all-flavor neutrino-proton elasticscattering events in JUNO. Detection of DSNB would provide valuable informationon the cosmic star-formation rate and the average core-collapsed neutrinoenergy spectrum. Geo-neutrinos can be detected in JUNO with a rate of ~400events per year, significantly improving the statistics of existing geoneutrinosamples. The JUNO detector is sensitive to several exotic searches, e.g. protondecay via the decay channel. The JUNO detector will providea unique facility to address many outstanding crucial questions in particle andastrophysics. It holds the great potential for further advancing our quest tounderstanding the fundamental properties of neutrinos, one of the buildingblocks of our Universe
The Molecular Mechanism Depicting Caveolin-1-elicited Regulation of γ-Secretase
越來越多證據指出,amyloid-precursor protein (APP)的β以及γ的水解可能是發生在細胞膜上一種稱為lipid rafts 的特殊構造中,而caveolin-1 是此種構造上一個重要的結構蛋白。此外,在阿茲海默氏症病患腦部的海馬迴區域,caveolin-1的表現有上升的現象。這些結果都顯示caveolin-1 有可能參與在調節APP 的分解以及阿茲海默氏症的致病過程中。因此,我們嘗試去證明caveolin-1 在γ-secretase主導的蛋白質水解上扮演關鍵的角色。在本實驗中,我們觀察到在HEK293 細胞裡,大量表現的caveolin-1 會促進raft membrane 的形成以及刺激γ-secretase 對於APP 的分解。然而,在γ-secretase 的另外一個作用對象Notch 身上,偵測不到此種情況。這些結果顯示caveolin-1 可以調控γ-secretase 對於受質的選擇性。接著,利用MβCD 這種抽除膽固醇的藥物去破壞raft membrane,可以完全抑制caveolin-1 所增加的γ-secretase 活性,由此更進一步證明了細胞膜形成特殊結構這個現象。另外,我們還發現APP,nicastrin,和ERK 會與caveolin-1 共同出現在raft membrane 裡。同時,caveolin-1 被報導可以抑制ERK 的活化,而我們確實也發現caveolin-1 藉此間接地促進γ-secretase 對於APP 的水解作用。這正好可以呼應我們之前的研究結果:降低ERK 的活化可以增加γ-secretase 的活性。接下來,我們抑制dynamin-2 的表現來阻斷細胞內噬作用,觀察到caveolin-1 對於γ-secretase 的刺激作用有顯著的降低。最後,我們發現大量表現的caveolin-1 也
會增加Aβ的產生,而抑制細胞內噬作用,同樣可以阻止caveolin-1 造成的影響。總結來說,透過以上的研究,我們相信caveolin-1 在調節γ-secretase 分解APP 的活性以及阿茲海默氏症的致病過程上確實扮演了重要的角色。Accumulated evidence has suggested that β- and γ-cleavage of amyloid precursor protein (APP) may take place in specialized membrane microdomains called lipid
rafts. Caveolin-1 is an important structure protein of this membrane domain, and its expression in the hippocampus region of Alzheimer’s disease patients’ brains is upregulated. These data all imply that caveoiln-1 could play a role in modulating the proteolytic processing of APP and the pathogenesis of Alzheimer’s disease. We thus seek to determine whether caveolin-1 can actively involve in the regulation of γ-secretase-mediated proteolysis. In this study, we observe that overexpression of caveolin-1 promotes the formation of raft-membranes in HEK293 cells and concomitantly stimulates γ-secretase-mediated cleavage of APP. However, the γ-secretase-mediated S3 cleavage of Notch, another substrate of γ-secretase, is not affected by the overexpression of caveolin-1, suggesting that substrate selectivity of
γ-secretase can be modulated by caveolin-1. Consistently, the caveolin-1-enhanced γ-secretase activity can be completely abolished by a cholesterol-depleting drug called
MβCD that also destabilizes the raft membranes. This strengthen the notion that caveolin-1 can promote the formation of raft membrane. We further confirm that APP,
nicastrin, and ERK are co-localized with caveolin-1 in raft membranes. Caveolin-1 is also known to be a scaffold protein that can suppress ERK activation. In caveolin-1-overexpressed cells, the evidence that the suppression of ERK activation is prominent in accordance to our previous findings that down-regulation of ERK can promote γ-secretase activity. And then, the down-regulation of dynamin-2, a critical component of endocytosis, results in a significant decrease in caveolin-1-enhanced γ-secretase activity. Finally, we discover that overexpression of caveolin-1 also can increases Aβ production, and inhibition of endocytosis blocks this effect. Together,
the present study strongly suggests that caveolin-1 plays an important role in the regulation of γ-secretase-catalyzed proteolysis of APP and may exacerbate the
pathogenesis of Alzheimer’s disease.Table of contents I
List of Figures III
摘要 1
Abstract 3
Introduction 5
Alzheimer’s disease 5
Aβ generation and γ-secretase 6
Lipid rafts and secretase-mediated proteolysis of APP 9
Caveolin and its relationship with AD 10
Objectives and hypothesis 12
Materials and methods 14
Reagents 14
Cell culture 15
Generation of DNA Constructs 15
Transient transfection of mammalian cells 16
Cell-based γ-secretase assays 16
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis 18
Transfection of small interfering RNAs (siRNAs) targeting dynamin-2 19
Aβ ELISA 20
Membrane fractionation 20
Results 23
The expression of exogenous caveolin-1 facilitates the formation of raft-membranes 23
Overxepression of caveolin-1 increases γ-secretase-mediated cleavage of APP, while Notch processing seems unchanged. 24
Caveolin-1-induced formation of raft microdomains is a structurally critical for the enhancement of γ-secretase activity and the recruitment of this protease and APP 25
Endocytosis plays a crucial role in the caveolin-1-elicited stimulation of γ -secretase activity 27
Discussion 30
Differential processing between APP and Notch 30
Caveolin-1-mediated endocytosis and APP proteolysis 32
Aβ production 33
Summary 34
Reference 35
List of Figures
Figure 1. Tetracycline-inducible expression of caveolin-1 and the quantitative measurement of γ-secretase activity. 43
Figure 2. The tetracycline-inducible expression of caveolin-1 is functional in the formation of raft-membranes. 44
Figure 3. Overxepression of caveolin-1 increases γ-secretase-mediated cleavage of APP. 45
Figure 4. Overexpression of caveolin-1 dose not affect Notch processing. 47
Figure 5. Cholesterol depletion completely abolishes caveolin-1-induced γ-secretase activity. 48
Figure 6. Overexpression of caveolin-1 recruits APP, mature nicastrin, and ERK into raft-fraction. 49
Figure 7. Overexpression of caveolin-1 suppresses ERK activation. 50
Figure 8. Inhibition of endocytosis decreases caveolin-1-stimulated γ-secretase activity. 52
Figure 9. Overexprssion of caveolin-1 increases extracellular Aβ, which can be blocked by the inhibition of endocytosis. 53
Figure 10. Model for the molecular mechanisms depicting caveolin-1-elicited regulation of γ-secretase. 5
Patterning nanowire and micro-/nanoparticle array on micropillar-structured surface: Experiment and modeling
Object Investigation of Industrial Heritage: The Forging and Metallurgy Shop in Taipei Railway Workshop
As a special plant for train maintenance in northern Taiwan, the Taipei Railway Workshop was founded in 1885 and moved in 2011, reflecting the changes in Taiwan’s history, transportation, and industrial technology. Now, it is planned to change the maintenance plant into a railway museum in the form of an in situ site. This study briefly introduces the historical background and present situation of the Taipei Railway Workshop and takes its forging workshop as the object for investigation and exhibition planning. According to the preservation and maintenance methods of the cultural heritage of the museum, the investigation process proposed includes four steps: Site exploration, object registration, object research, and exhibition planning. The work area in the plant is divided into shaping and forging areas, as based on the categories of the machines on the site of the forging workshop. In this study, a total of 85 industrial relics in the forging workshop are registered for systematic research. The working conditions, including machine parts for train maintenance, manufacturing processes of parts, and the relationship between in-line on-site machines and tools, of the forging workshop before closing are restored, as based on the principles of machine manufacturing, literature, and retired workers’ oral histories. Finally, an in situ exhibition plan of the forging workshop is put forward based on the results of the object research
MPT0B098, a Microtubule Inhibitor, Suppresses JAK2/STAT3 Signaling Pathway through Modulation of SOCS3 Stability in Oral Squamous Cell Carcinoma.
Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC
Zebrafish Model-Based Assessment of Indoxyl Sulfate-Induced Oxidative Stress and Its Impact on Renal and Cardiac Development
Kidney disease patients may have concurrent chronic kidney disease-associated mineral bone disorder and hypertension. Cardiovascular disease (CVD) and neuropathy occur due to kidney failure-induced accumulation of uremic toxins in the body. Indoxyl sulfate (IS), a product of indole metabolism in the liver, is produced from tryptophan by the intestinal flora and is ultimately excreted through the kidneys. Hemodialysis helps renal failure patients eliminate many nephrotoxins, except for IS, which leads to a poor prognosis. Although the impacts of IS on cardiac and renal development have been well documented using mouse and rat models, other model organisms, such as zebrafish, have rarely been studied. The zebrafish genome shares at least 70% similarity with the human genome; therefore, zebrafish are ideal model organisms for studying vertebrate development, including renal development. In this study, we aimed to investigate the impact of IS on the development of zebrafish embryos, especially cardiac and renal development. At 24 h postfertilization (hpf), zebrafish were exposed to IS at concentrations ranging from 2.5 to 10 mM. IS reduced survival and the hatching rate, caused cardiac edema, increased mortality, and shortened the body length of zebrafish embryos. In addition, IS decreased heart rates and renal function. IS affected zebrafish development via the ROS and MAPK pathways, which subsequently led to inflammation in the embryos. The results suggest that IS interferes with cardiac and renal development in zebrafish embryos, providing new evidence about the toxicity of IS to aquatic organisms and new insights for the assessment of human health risks. Accordingly, we suggest that zebrafish studies can ideally complement mouse model studies to allow the simultaneous and comprehensive investigation of the physiological impacts of uremic endotheliotoxins, such as IS, on cardiac and renal development
Combination treatment significantly induces apoptosis in OSCC cells.
<p>(<b>A</b>) OEC-M1 cells were treated with MPT0B098 (0.25 μM), cisplatin (5 μM), 5-fluorouracil (5-FU, 5 μM), cisplatin (5 μM)+5-FU (5 μM), 098 (0.25 μM)+ 5-FU (5 μM) and 098 (0.25 μM)+ cisplatin (5 μM) for the indicated times. The cell viability was assessed by MTT assay. (<b>B</b>) OEC-M1 cells were treated with MPT0B098 (0.25 μM), cisplatin (5 μM), 5-fluorouracil (5-FU, 5 μM), cisplatin (5 μM)+5-FU (5 μM), 098 (0.25 μM)+ 5-FU (5 μM) and 098 (0.25 μM)+ cisplatin (5 μM) for the indicated times and the caspase-3 activity was assessed. (<b>C</b>) OEC-M1 and HSC-3 cells were stimulated with IL-6 (10 ng/ml) in the presence or absence of MPT0B098 (0.25 μM) for the indicated times. Whole cell lysates were immunoblotted with antibodies to phosphorylated proteins (p-JAK2 and p-STAT3) and total level of proteins (JAK2 and STAT3). GAPDH was used as protein loading control. (<b>D</b>) OEC-M1 and HSC-3 cells were stimulated with IL-6 (10 ng/ml) in the presence or absence of MPT0B098 (0.25 μM) for 24 hrs and the caspase-3 activity was assessed. All data are presents as mean ± SE relative to DMSO vehicle control from three replicate experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p
MPT0B098 induces the cell cycle arrest and apoptosis.
<p>(<b>A</b>) OEC-M1 cells were treated with 0.25 or 0.5 μM of MPT0B098 for 12 hrs. Cells were then evaluated for effects on cell cycle using PI staining and analyzed by flow cytometry. Percentages of cells in different phases were shown. The data are representative of three independent experiments. (<b>B</b>) OEC-M1 cells were treated with different concentrations of MPT0B098 for 12 hrs. Apoptosis was assessed by annexin V/PI staining and analyzed by flow cytometry. The data are represented as mean ± SE; ***, <i>p</i><0.001 versus vehicle control. (<b>C</b>) OEC-M1 cells were incubated with various concentrations of MPT0B098 for 12~24 hours and caspases-3 activity was assessed. The data are represented as mean ± SE; **, <i>p</i><0.01; ***, <i>p</i><0.001 versus vehicle control. (<b>D</b>) OEC-M1 cells were treated with different concentrations of MPT0B098 for 24 hrs. The proteolytic cleavage of caspase-3 and PARP were determined by Western blot analysis. GAPDH was used as protein loading control. (<b>E</b>) OEC-M1 cells were treated with different concentrations of MPT0B098 for 24 hrs. Effects on the expression of Pim-1, Mcl-1, Bcl-2 and survivin were determined by western blot analysis. GAPDH was used as protein loading control.</p
MPT0B098 modulates JAK2/STAT3 pathway in OSCC cells.
<p>(<b>A</b>) OEC-M1 cells were treated with MPT0B098 (0.25 μM) for the indicated times. The phosphorylated STAT3 (pSTAT3) and total level of STAT3 were determined by Western blotting. The level of STAT3 mRNA was determined by RT-PCR. GAPDH was used as loading control. (<b>B</b>) Western blot analysis of STAT3 levels after 48 hrs transfection of OEC-M1 cells with control shRNA (NS) or two shRNA constructs (shSTAT3 #1, #2) against STAT3. GAPDH was used as loading control. These shRNA transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; *, <i>p</i><0.05; **, <i>p</i><0.01 versus vehicle control. (<b>C</b>) Western blot analysis of endogenous STAT3 protein level in OSCC cells. GAPDH was used as loading control. (<b>D</b>) OSCC cells were incubated with various concentrations of MPT0B098 for 24 hrs and caspases-3 activity was assessed. Data are presents as mean ± SE relative to vehicle control from three replicate experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. (<b>E</b>) OEC-M1 and HSC-3 cells were treated with 0.25 μM of MPT0B098 for indicated times. The phosphorylated proteins (p-JAK1, p-JAK2 and p-TYK2) and total level of proteins (JAK1, JAK2 and TYK2) were determined by Western blotting. GAPDH was used as loading control.</p
MPT0B098 inhibits the proliferation and induces microtubules depolymerization in OSCC cells.
<p>(<b>A</b>) Chemical structure of MPT0B098. (<b>B</b>) OSCC cells were treated with increasing concentrations of MPT0B098 for 72 hrs and the cell viability was assessed by MTT assay. Data are presents as mean ± SE relative to DMSO vehicle control (indicated as 0 μM) from three replicate experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. (<b>C</b>) OEC-M1 and HSC-3cells were incubated at 37°C from 0~60 min in the presence of 0.25 μM of MPT0B098. Free form and polymer form of microtubule were purified and assessed by Western blot analysis. (<b>D</b>) OEC-M1 and HSC-3 cells were treated with 0.25 μM of MPT0B098 from 0~60 min. Cells were fixed and then immunostained with anti-α-tubulin (green) antibody and then stained with DAPI (blue), followed by confocal microscopy. Scale bar = 7.5 μm. (<b>E</b>) OEC-M1 and HSC-3 cells were treated with MPT0B098 for 120 min at 37°C. For recovery (Rec.) assay, cells were treated with MPT0B098 for 60 min and then the drug was washed out to allow the microtubules to repolymerize for another 60 min. Cell lysates were analyzed by western blot using the anti-α-tubulin antibody. (<b>F</b>) The recovery assay was also done in OEC-M1 cells for immunostaining. After drug treatment, OEC-M1 cells were fixed and then immunostained with anti-α-tubulin (green) antibody and then stained with DAPI (blue), followed by confocal microscopy. Scale bar = 7.5 μm.</p