Mar 3, 2010
Feb 22, 2010
Feb 10, 2010
挖係EBV,衝衝衝!
告別牛年,意味者也告別了KSHV。不過愛KV者也別太感傷,這是暫時的。 精確點說,本人只是想把KR部份做個結束,有些東西拖越久就越難收尾,現在應是把phosphorylation及O-GlcNAcylation兩篇推出去的最佳時機,咱們要把握機會。這裡要特別感謝中研院Khoo lab的鼎力相助、Ingrid及Maruko君的鍥而不舍,俗語說“戲棚子下站久了就是你的”,我想我們會成功的。
實驗室這幾年來建立的EV/KV lytic replication system在小嬿鴨子划水的努力下,第一份manuscript也出爐了,請台大幾位老師幫忙看一下,大抵反應還不差。我的構想是這篇文章著重在兩個細胞株的分析。最難但也最重要的部份就是:細胞都爛成這樣子了,到底有多少病毒產出?這部份set-up之後往下我們就會有一個概念→何謂permissive lytic replication。其實文獻上很難找得到像我們現在手上有這麼高比例, 這麼爛的誘導系統。真是破碎得好極了,我喜歡。
小夏一年來幫忙大家做許多“臨門一腳”的工作,這也要好好提出來感恩一下。大家在傳球上籃時如果個個都想當那最後一個投籃者,那麼這個隊伍的分數一定不高。希望大家能享受團隊合作、一起得分的感覺,也希望這個精神能在我們實驗室裡永遠流傳下去。不過,三月間的poster要好好做,趁這個機會理出ER和老化/G1滯留可以做的方向. i.e. 你該對ER大喊一聲: oh, baby, I am back!
現在來談談tiger year的展望。這年裡我們將以EBV為主(70%)口腔癌為輔(30%),先講較具新鮮感的OSCC (oral squamous cancer cell)。目前我們有五株本土OSCC細胞株, cDNAs皆已置備好。我們往下需要profile (by using Q-RT-PCR) 這些細胞株中PTK的表現情形。另外,由Ingrid及小夏製備好的一些蛋白質則想用來偵測各個細胞表現integrin及differentiation狀況。最後就是B藥對這些細胞株毒殺程度及其機制。在EBV方面則可分【夢幻型計畫】及【穩紮穩打型計畫】兩類。前者包括NP460-EREVp2089的建立; Rta在EBV感染primary epithelial cell後6~48小時內表現情形、對細胞有無明顯effect (如誘導老化); Rta遙控viral genome再活化之直接機制等等。後者則包括 TW01-EREVp2089, HONE1-EREVp2089, HeLa-EZEVp2089之建立; microarray結果中cell growth arrest與cytoskeleton reorganization兩大pathway結果之驗證;以及再分析幾種細胞中的transcriptomes表現情形等等。
Tiger年裡會有一個讓大家賺外快的【一百元專區】→凡是為大家報一篇該區中的paper,立即可得新台幣一百元。目前【一百元專區】的貨源就是在本Blog右上角那些不時更新的好文 (啥米,你到現在才注意到…昏)。若都不符合您的胃口,日後我會在nas上另開一個folder,把2010前一些值得大家討論的paper放進去。或者,可email給我你懷有極大熱情的paper,我會以最寬鬆的審核制度讓你放入該區。要讓寫作技巧進步、國際會議上和你的偶像可以交談的祕訣之一就是不斷地讀paper。學習人家怎麼賣他的想法、怎樣organize他的結果。福虎年裡就讓我們初生之犢不畏虎,大家好好加油!財源廣進!
最後, as usual, 財務方面、實驗室整潔、各項運作要特別謝謝大家一年來的幫忙。各位的分憂解勞讓我覺得當老闆還真是幸福啊！祈願Tiger Year大家身體健康、智慧增長、安和豐富、阿彌陀佛 ！(最後四字=無量光、無量壽之吉祥意)
實驗室這幾年來建立的EV/KV lytic replication system在小嬿鴨子划水的努力下,第一份manuscript也出爐了,請台大幾位老師幫忙看一下,大抵反應還不差。我的構想是這篇文章著重在兩個細胞株的分析。最難但也最重要的部份就是:細胞都爛成這樣子了,到底有多少病毒產出?這部份set-up之後往下我們就會有一個概念→何謂permissive lytic replication。其實文獻上很難找得到像我們現在手上有這麼高比例, 這麼爛的誘導系統。真是破碎得好極了,我喜歡。
小夏一年來幫忙大家做許多“臨門一腳”的工作,這也要好好提出來感恩一下。大家在傳球上籃時如果個個都想當那最後一個投籃者,那麼這個隊伍的分數一定不高。希望大家能享受團隊合作、一起得分的感覺,也希望這個精神能在我們實驗室裡永遠流傳下去。不過,三月間的poster要好好做,趁這個機會理出ER和老化/G1滯留可以做的方向. i.e. 你該對ER大喊一聲: oh, baby, I am back!
現在來談談tiger year的展望。這年裡我們將以EBV為主(70%)口腔癌為輔(30%),先講較具新鮮感的OSCC (oral squamous cancer cell)。目前我們有五株本土OSCC細胞株, cDNAs皆已置備好。我們往下需要profile (by using Q-RT-PCR) 這些細胞株中PTK的表現情形。另外,由Ingrid及小夏製備好的一些蛋白質則想用來偵測各個細胞表現integrin及differentiation狀況。最後就是B藥對這些細胞株毒殺程度及其機制。在EBV方面則可分【夢幻型計畫】及【穩紮穩打型計畫】兩類。前者包括NP460-EREVp2089的建立; Rta在EBV感染primary epithelial cell後6~48小時內表現情形、對細胞有無明顯effect (如誘導老化); Rta遙控viral genome再活化之直接機制等等。後者則包括 TW01-EREVp2089, HONE1-EREVp2089, HeLa-EZEVp2089之建立; microarray結果中cell growth arrest與cytoskeleton reorganization兩大pathway結果之驗證;以及再分析幾種細胞中的transcriptomes表現情形等等。
Tiger年裡會有一個讓大家賺外快的【一百元專區】→凡是為大家報一篇該區中的paper,立即可得新台幣一百元。目前【一百元專區】的貨源就是在本Blog右上角那些不時更新的好文 (啥米,你到現在才注意到…昏)。若都不符合您的胃口,日後我會在nas上另開一個folder,把2010前一些值得大家討論的paper放進去。或者,可email給我你懷有極大熱情的paper,我會以最寬鬆的審核制度讓你放入該區。要讓寫作技巧進步、國際會議上和你的偶像可以交談的祕訣之一就是不斷地讀paper。學習人家怎麼賣他的想法、怎樣organize他的結果。福虎年裡就讓我們初生之犢不畏虎,大家好好加油!財源廣進!
最後, as usual, 財務方面、實驗室整潔、各項運作要特別謝謝大家一年來的幫忙。各位的分憂解勞讓我覺得當老闆還真是幸福啊！祈願Tiger Year大家身體健康、智慧增長、安和豐富、阿彌陀佛 ！(最後四字=無量光、無量壽之吉祥意)
Jan 28, 2010
小嬿Progress Report有感
Teacher蔡: (1) Fig 1C western 太髒, not qualified for publication (要改進!). (2) EAD clone 88 is a good one for IFA (3) Akata strain 比 B95-8 transforms B cell 能力弱很多. (3) Dox-treated ERKV中 knock-downKR (siRNA),用以證實ER是透過KR做事... (4) ACIF是做好EBNA1 IFA之唯一良藥. (5)淑君的Blood paper中述及primary infection可有Z, R, 一直到VCA. (6)葉醫師的differentiation系統為Sphenoidal sinus, 並未有un-differentiated cells.
Teacher陳M.-R.: (1) MA 220 is a good antibody for Dox-EREV8 late gene expression (又是Fig 1C!) (2) Mechanism of ER-mediated activation of KR promoter.
Teacher陳J.-Y.: 要做與疾病,癌症有關的為佳!
SFL: (1) 一篇會令人開悟的文章, 這裡.(2) 293的Vitronectin receptor是怪胎 (ITGAVB1). (3)NA中ER的量與EREV8中ER的量是可以比較估算的.
Teacher陳M.-R.: (1) MA 220 is a good antibody for Dox-EREV8 late gene expression (又是Fig 1C!) (2) Mechanism of ER-mediated activation of KR promoter.
Teacher陳J.-Y.: 要做與疾病,癌症有關的為佳!
SFL: (1) 一篇會令人開悟的文章, 這裡.(2) 293的Vitronectin receptor是怪胎 (ITGAVB1). (3)NA中ER的量與EREV8中ER的量是可以比較估算的.
Jan 22, 2010
B292 相關
1/21/2010 (Thu) 第一次討論會議
Speaker 1-北醫劉景平: Structures of 292, J-30, SAHA etc. One OCH3 group in 292 is enough, two OCH3 increases IC50 from 50 nM to 1400 nM; replace with OH increases IC50 from 50 nM to 500 nM. SAHA is a planer structure, quite stable. SAHA is a pan-HDACi, with uM range. B291 is also a pan-HDACi, with 100 nM range. Wishing 292 is more specific.
Speaker 2-程韻靜: Effects of 292 (1/3) inhibits tubulin polymerization in KB cell; (2/3) inhibits tube formation in SH-SY5Y; (3/3) Animal models (CAM, xenograft in H460 58.7% inhibition, orthotopic PANC-1 with Luc expression); Calcium influx was increased in Taxol or J-30 (not as good as Taxol) treated neuronal cells, but neither SAHA nor 292 had effects in calcium influx; While most cells exhibit 50~100 nM IC50, HUVEC is 42 uM and WI-38 is 12 uM (so interesting!!!)
Speaker 3-夏興國: (1) B292 and EMT seemed not have correlation in breast and lung cancer cells (2) B292 and Adhesion: CL1-0 扁平+292 (uM) =扁平. CL1-5 細長+292 (nM)=morphological changes and tubulin disperse.... (但butyrate, TSA, SAHA都能造成相同現象(FAK and paxillin increases) 耶...
Speaker 4-吳仲峻: TWO1 (72 nM), NA (35 nM with EAD 5~10%), HONE-1 (66 nM), HA (42 nM).
------------------------------------------------------------------------------
11/1/09 SFL wrote:
與PrCA/AR有關:Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation.
與Tau有關:To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation.
有趣, 有趣..
有空要瞭解一下HSP90,就這麼查吧. "hsp90 review AND cell cycle [jour]"
SAHA 相關 Chiang's recent paper [Here].
Speaker 1-北醫劉景平: Structures of 292, J-30, SAHA etc. One OCH3 group in 292 is enough, two OCH3 increases IC50 from 50 nM to 1400 nM; replace with OH increases IC50 from 50 nM to 500 nM. SAHA is a planer structure, quite stable. SAHA is a pan-HDACi, with uM range. B291 is also a pan-HDACi, with 100 nM range. Wishing 292 is more specific.
Speaker 2-程韻靜: Effects of 292 (1/3) inhibits tubulin polymerization in KB cell; (2/3) inhibits tube formation in SH-SY5Y; (3/3) Animal models (CAM, xenograft in H460 58.7% inhibition, orthotopic PANC-1 with Luc expression); Calcium influx was increased in Taxol or J-30 (not as good as Taxol) treated neuronal cells, but neither SAHA nor 292 had effects in calcium influx; While most cells exhibit 50~100 nM IC50, HUVEC is 42 uM and WI-38 is 12 uM (so interesting!!!)
Speaker 3-夏興國: (1) B292 and EMT seemed not have correlation in breast and lung cancer cells (2) B292 and Adhesion: CL1-0 扁平+292 (uM) =扁平. CL1-5 細長+292 (nM)=morphological changes and tubulin disperse.... (但butyrate, TSA, SAHA都能造成相同現象(FAK and paxillin increases) 耶...
Speaker 4-吳仲峻: TWO1 (72 nM), NA (35 nM with EAD 5~10%), HONE-1 (66 nM), HA (42 nM).
------------------------------------------------------------------------------
11/1/09 SFL wrote:
與PrCA/AR有關:Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation.
與Tau有關:To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation.
有趣, 有趣..
有空要瞭解一下HSP90,就這麼查吧. "hsp90 review AND cell cycle [jour]"
SAHA 相關 Chiang's recent paper [Here].
Jan 16, 2010
Jan 7, 2010
Dec 21, 2009
Dec 18, 2009
新的挑戰—for YCK (更新)
真是不好意思,我又改變順序了
1. 回溶HeLaTet (#3、#4)及HONE1Tet
2. 測試Bsd在TW03、CGHNK2及Zeo對HeLaTet、HONE1Tet的耐受性
3. 293FT用來包pLentiTR6, pLentiFlag-gZ, -cZ及GFP (每種包3個well, 就是半盤6-well plate之意).
同時,可否問一下進階業務,"LONZA"有款叫KGM的medium,專門用來養keratinocyte,是否有進一部資料可供參考! 謝謝.
----------------------------------------------------------
on 12/2/2009 Crazy SFL wrote:
哈哈哈, 新年新氣象,這是給YCK和YLC的小禮物。先把手邊的東西忙完後,請著手下面有趣的實驗:
1. 回溶HeLaTet (#3、#4)及HONE1Tet
2. 測試Bsd在TW03、CGHNK2及Zeo對HeLaTet、HONE1Tet的耐受性
3. 293FT用來包pLentiTR6, pLentiFlag-gZ, -cZ及GFP (每種包3個well, 就是半盤6-well plate之意).
同時,可否問一下進階業務,"LONZA"有款叫KGM的medium,專門用來養keratinocyte,是否有進一部資料可供參考! 謝謝.
----------------------------------------------------------
on 12/2/2009 Crazy SFL wrote:
哈哈哈, 新年新氣象,這是給YCK和YLC的小禮物。先把手邊的東西忙完後,請著手下面有趣的實驗:
- Try to establish TW03-TetR, CGHNK2-TetR, CGHNC8-TetR and TW2.6-TetR。
- Try to establish Luc, ER and gZ (B95-8 strain) in HONE1Tet, NP460Tet, TW03Tet, CGHNK2Tet, CGHNC8Tet and TW2.6Tet。這裡有個重點就是請再重讀一下原instruction manual, 把induction量弄到和293TetER相可匹配的程度。我們需要步步為營。
Dec 1, 2009
量化西方墨漬法
以後實驗室quantitate protein band請依以下步驟進行:
- Plasmid 最好是一起抽的一批, 因實驗室LB/BHI已過使用期限,希望大家每次抽質體時盡量使用新鮮media及LB plate (1-cm厚度最佳),超過一個月以上的media及plate請自行定時清理(請不要當福壽螺到處蔓延留種,謝謝)。
- 若質體中有SV40-ori, 請以293T為host (DLR/expression etc),其餘它種質體則用293。
- Transfection (24~48 h)每次請做p-100/1 ml-lysate的份量(6x p-35/1.2-ml), protein lysate量一次到位,不要老是重複在養細胞/TF效率好像有問題/plasmid不夠...(姑且稱之為小鼻子小眼睛的實驗)
- 檢測各質體間表現量:(i) 請以wild-type為標準 (所以wild-type可能要多transfect一份),293T為host的取5-ug及10-ug, 293為host的取25-ug及50-ug protein extracts run數片小膠,確定所使用的西方墨漬條件可分出一倍和兩倍間的差異。若是過強,那就請在砍primary/ secondary antibody濃度、incubation時間、ECL強度上三件事情上做調整。(ii)依前步驟所訂出的條件run一片小膠檢查各檢體間是否差異很大,如有二倍以上差異而各步驟又沒有太大疑慮,那表示可能有protein stability/proteosome involved,值得設計其他實驗來分析之.兩倍以下差異者微調input量可繼續往下的實驗。(Plasmid, 細胞用畢的請定時清理,不要當福壽螺到處蔓延留種,謝謝)。
- 附註定量software有三款,請在Lab Protocol中找到小抄。
Novartis 石明石博士 Michael Shi 演講
- TKI 258 was a FGFR1 inhibitor, but the use of TKI 258 causes FGF23/bFGF increased.
- Melanoma trial found FGF mediated escape form anti-angiogenesis (sorxxxtinb/...) Early phase: VEGFR2 blockadge.
- Phase 3 trials: (a) FGFR3 expression/t94,14) trans-location in melanoma (b) FGFR3 mutations in bladder cancer (c) FGFR1 expression in breast cancer [這裡若有蒲教授的data就好了!!]
- AUY 922 is an inhibitor of HSP90 for GI tract tumor.
- BGT226 is is on clinical trial for NPC (與中文大學馬教授合作,明年AACR發表) 另一位Mike補充陳所長有拿藥用在oral cavity (張光裕醫師 was in charge, he found this drug induced autophagic death in a panel of oral cancer cells)
- BKM120 is a PI3K -specific inhibitor; BGT226 and BE2 232 are dual kinase inhibitors (mTOR and PI3K) 這部份還是沒有搞清楚.
- ASA 404 in NSCLC.
- LED 225 is for Smoothened/Hedgehog pathway.
- MGM 黃秀芬醫師介紹 Tissue bank/LCM slides for Chlorangiocarcinoma...
Nov 21, 2009
知識分享 - immunopreciptitation
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