您好 這是我要報的paper.請查收. 祝 研安 ehsing 敬上
____________________
2014 Mo Cell
Mutant p53 Reprograms TNF Signaling in Cancer Cells through Interaction with the Tumor Suppressor DAB2IP
Nov 27, 2014
Nov 22, 2014
Fusion gene detection
由 sufang 在 六, 11/22/2014 - 09:01 發表 Cholangiocarcinoma cholangiocarcinoma FFPE Gene Fusion
Nov 6 2014
Nov 5 2014
Daw-yang Hwang 黃道揚 to LTCHEN, yngmiin
Dear Prof Lin, Dr. Chiang, and SF:
I designed primers for ALK, ROS-1 FGFR2, FGFR3, and I can add primers for RET.
In my research part (bladder and upper urinary tract cancer), I am collaborating with urologists in KMUH. Hopefully I can get cDNA samples from then late this month. I can run the above panel for cholangiocarcinoma, oral cancer, or any cancer samples you are interested. I am currently testing some FFPE samples.
Brief, the protocol:
1. Samples (fresh or FFPE)
2. Extract RNA
3. RT-PCR for single-starnded cDNA
4. second cDNA systhesis (double-stranded cDNA)
5. Nextera XT kit
6. Specific PCR amplification
7. Barcode PCR (up to 288 different samples in one MiSeq run)
8. Purification
8. MiSeq run
In my research part (bladder and upper urinary tract cancer), I am collaborating with urologists in KMUH. Hopefully I can get cDNA samples from then late this month. I can run the above panel for cholangiocarcinoma, oral cancer, or any cancer samples you are interested. I am currently testing some FFPE samples.
Brief, the protocol:
1. Samples (fresh or FFPE)
2. Extract RNA
3. RT-PCR for single-starnded cDNA
4. second cDNA systhesis (double-stranded cDNA)
5. Nextera XT kit
6. Specific PCR amplification
7. Barcode PCR (up to 288 different samples in one MiSeq run)
8. Purification
8. MiSeq run
Maybe we can meet sometimes to have a discussion, for:
A) where to run MiSeq
B) discuss whether we want to include potential fusion on both ends or run known fusion-direction.
A) where to run MiSeq
B) discuss whether we want to include potential fusion on both ends or run known fusion-direction.
Kindly regards,
Daw-yang
Daw-yang
Nov 5 2014
Su-Fang Lin to 陳所長
Su-Fang Lin
Dear 所長:
那我就向黃秀芬醫師申請TLCN的cholangioncarc
1) FGFR2 IHC 染色 (我們剛剛買了一些commercial的tissue array, 在測試抗體條件)
2) cDNA for 道揚's RT-PCR test.
相請教您的是，用您的名字申請? 還是用我的?
Best,
Su-Fang
Nov 6 2014
Su-Fang Lin to Daw-yang Hwang., LTCHEN
Dear Daw-Yang:
Congratulations to your 104 NHRI grant!!! 我先押寶FGFR3 gene fusions in your bladder and upper uinary tract unrothelial cancer! (by the way, we have just completed FGFR3 IHC staining for some commercial tissue arrays, in case you need it someday, please let me know :)
Su-Fang 敬賀
Nov 6 2014
Daw-yang Hwang 黃道揚 to me
Dear SF,
Thank you for your great help, I plan to run several tyrosine kinase fusion genes together in the bladder cancer samples. Which organ origins are those commercial samples?
Let me know if you want to test your oral cancer (or other cancers) samples. I will run in-house designed oncogene panel for my bladder cancer, using Fluidigm-MiSeq plateform.
Let me know if you want to test your oral cancer (or other cancers) samples. I will run in-house designed oncogene panel for my bladder cancer, using Fluidigm-MiSeq plateform.
Talk to you soon,
Daw-yang
Daw-yang
Nov 6 2014
Su-Fang Lin to Daw-yang Hwang
I remember we did include some bladder cancer tissues , please check this post
http://eln.nhri.org.tw/lims/?
http://eln.nhri.org.tw/lims/?
I will call you to discuss more about FGFR3 gene fusions in oral cancer. 你今天何時比較有空? 11點OK嗎? 或是下午、或是明天你可以的時間! 謝謝。 (對了，請再給我一次你office電話)
Nov 6 2014
Daw-yang Hwang 黃道揚 to me
I will check my user name in the NHRI system.
You can call me at 11AM, 07-3121101 x 2266 (this is the lab, I will be there at 11AM)
(Cellular phone is 0932351464 or 0975356113 if case you need them)
(Cellular phone is 0932351464 or 0975356113 if case you need them)
Talk to you soon,
Daw-yang
Daw-yang
Nov 6 2014
Su-Fang Lin to Daw-yang Hwang
OK, I will call you at 11AM (lab phone 我比較會講話)
剛剛那個post 連結請用 DYH (user name), DYH123 (password) 登入。與NHRI account 無關喔。
剛剛那個post 連結請用 DYH (user name), DYH123 (password) 登入。與NHRI account 無關喔。
Best,
Su-Fang
Nov 6 2014
Daw-yang Hwang 黃道揚 to me
Dear SF,
Below is the estimated cost of my fusion gene analysis:
Nov 6 2014
Below is the estimated cost of my fusion gene analysis:
A. The cost for known FGFR2 fusion gene detection is minimal:
1. primer (some cost, < NTD 5000)
2. RNA extraction (no cost in NHRI)
3. RT-PCR (some cost)
4. Run gel for positive band
5. Sanger sequencing band in 4. (some cost)
Total cost should be less than NTD 10000
1. primer (some cost, < NTD 5000)
2. RNA extraction (no cost in NHRI)
3. RT-PCR (some cost)
4. Run gel for positive band
5. Sanger sequencing band in 4. (some cost)
Total cost should be less than NTD 10000
For unknwon TK fusion (including FGFR2-fusion, and all other TK candidates) (48 samples)
1. primer (no cost, since I am going to order these primers anyway)
2. RNA extraction (no cost in NHRI)
3. RT-double stranded cDNA (second stranded cDNA kit 10000)
4. Nextera XT library kit (~20000)
5. fusion specific PCR (Fluidigm plate 20 000)
6. Barcode PCR (free from my previous lab)
7. MiSeq (NTD 60 000)
Total cost for run 48 samples in all possible fusion genes is NTD 110,000, 96 samples will be NTD 160,000 (if still use one MiSeq kit)
1. primer (no cost, since I am going to order these primers anyway)
2. RNA extraction (no cost in NHRI)
3. RT-double stranded cDNA (second stranded cDNA kit 10000)
4. Nextera XT library kit (~20000)
5. fusion specific PCR (Fluidigm plate 20 000)
6. Barcode PCR (free from my previous lab)
7. MiSeq (NTD 60 000)
Total cost for run 48 samples in all possible fusion genes is NTD 110,000, 96 samples will be NTD 160,000 (if still use one MiSeq kit)
For your references, the cost for RNA-Seq (rRNA-depletion, minimal 10M read; we get 15M reads) is ~NTD 28000 (威健 OR 基米 has similar prices).
Regards,
Daw-yang
Daw-yang
Nov 6 2014
Su-Fang Lin to Daw-yang Hwang.
Thanks a lot for the valuable info! And sorry I had been in a meeting whole afternoon, in case you had called me.
Best,
Su-Fang
Su-Fang
11/05/2014
Nov 20, 2014
CTCF and HOXs
由 sufang 在 四, 11/20/2014 - 12:53 發表 EBV and KSHV CTCF
Mol Cell Biol. 2014 Oct;34(20):3867-79. doi: 10.1128/MCB.00567-14. Epub 2014 Aug 18.
CTCF controls HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure in NT2/D1 cells.
Xu M1, Zhao GN1, Lv X1, Liu G1, Wang LY2, Hao DL1, Wang J3, Liu DP4, Liang CC1.
Author information
Abstract
HOX cluster genes are activated sequentially in their positional order along the chromosome during vertebrate development. This phenomenon, known as temporal colinearity, depends on transcriptional silencing of 5' HOX genes. Chromatin looping was recently identified as a conserved feature of silent HOX clusters, with CCCTC-binding factor (CTCF) binding sites located at the loop bases. However, the potential contribution of CTCF to HOX cluster silencing and the underlying mechanism have not been established. Here, we demonstrate that the HOXA locus is organized by CTCF into chromatin loops and that CTCF depletion causes significantly enhanced activation of HOXA3 to -A7, -A9 to -A11, and -A13 in response to retinoic acid, with the highest effect observed for HOXA9. Our subsequent analyses revealed that CTCF facilitates the stabilization of Polycomb repressive complex 2 (PRC2) and trimethylated lysine 27 of histone H3 (H3K27me3) at the human HOXA locus. Our results reveal that CTCF functions as a controller of HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Mol Cell Biol. 2014 Oct;34(20):3867-79. doi: 10.1128/MCB.00567-14. Epub 2014 Aug 18.
CTCF controls HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure in NT2/D1 cells.
Xu M1, Zhao GN1, Lv X1, Liu G1, Wang LY2, Hao DL1, Wang J3, Liu DP4, Liang CC1.
Author information
Abstract
HOX cluster genes are activated sequentially in their positional order along the chromosome during vertebrate development. This phenomenon, known as temporal colinearity, depends on transcriptional silencing of 5' HOX genes. Chromatin looping was recently identified as a conserved feature of silent HOX clusters, with CCCTC-binding factor (CTCF) binding sites located at the loop bases. However, the potential contribution of CTCF to HOX cluster silencing and the underlying mechanism have not been established. Here, we demonstrate that the HOXA locus is organized by CTCF into chromatin loops and that CTCF depletion causes significantly enhanced activation of HOXA3 to -A7, -A9 to -A11, and -A13 in response to retinoic acid, with the highest effect observed for HOXA9. Our subsequent analyses revealed that CTCF facilitates the stabilization of Polycomb repressive complex 2 (PRC2) and trimethylated lysine 27 of histone H3 (H3K27me3) at the human HOXA locus. Our results reveal that CTCF functions as a controller of HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Remarkable Role of Indoleamine 2,3-Dioxygenase and Tryptophan Metabolites
由 sufang 在 四, 11/20/2014 - 13:20 發表 Pre-published IDO1 KYN
Remarkable Role of Indoleamine 2,3-Dioxygenase and Tryptophan Metabolites in Infectious Diseases: Potential Role in Macrophage-Mediated Inflammatory Diseases (PubMed) (pdf 4235)
Figure 1. Schematic overview of the kynurenine pathway. It is estimated that only 1% of dietary tryptophan (TRP) can be converted into serotonin (5-HT). The remaining 99% of TRP is metabolized via the kynurenine (KYN) pathway. Tryptophan hydroxylase (TH), 5-hydroxy TRP (5-HTP), N-acetylserotonin (NAS), indoleamine 2,3-dioxygenase 1 and 2 (IDO1/2), tryptophan 2,3-dioxygenase (TDO), kynurenine 3-monooxygenase (KMO), kynurenine aminotransferase (KAT I, II, III), kynurenic acid (KA), anthranilic acid (AA), 3-hydroxykynurenine (3-HK), xanthurenic acid (XA), 3-hydroxyanthranilic acid (3-HAA), 3-hydroxyanthranilic acid oxidase (3-HAO), quinolinic acid (QUIN), quinolinic-acid phosphoribosyl transferase (QPRT), nicotinic acid mononucleotide (NaMN), nicotinamide mononucleotide adenylyltransferase (NMNAT), nicotinamide adenine dinucleotide (NAD).
Figure 2: T-cell immune regulation by IDO1. (a) IDO1 is induced by IFN-γ-dependent and/or -independent signal pathways, depending on the variety of immune stimuli by macrophages and dendritic cells (DCs) [28, 83]. IDO1 activity is suppressed by the formation of NO or the competitive enzyme inhibitor, 1MT. Marked increases in IDO1 suppress immune responses by locally depleting L-TRP and preventing T-cell proliferation [44]. Expression of IDO1 has been observed in certain types of activated macrophages and DCs. IDO1-expressing cells deplete TRP from the extracellular milieu and secrete TRP metabolites, including KYN, 3-HK, 3-HAA, and QUIN, which induce T-cell apoptosis and suppress immune responses in vitro. (b) CD19+ plasmacytoid DCs (pDCs) express high levels of IDO1, which can activate mature regulatory T (Treg) cells via activation of the protein kinase general control nonderepressing-2 (GCN2) pathway of protein synthesis inhibition [84]. pDC-produced IDO1 and activated Treg can convert naïve T cells into new Treg. IDO1 acts in an autocrine manner to suppress pDC production of IL-6, which prevents the conversion of Treg into IL-17-producing Th17 proinflammatory cells [79]. IDO1 also downregulates type I IFN (IFN-α) production by pDC [80].
Figure 3: IDO1 induction and proinflammatory cytokines. IFN-γ drives intense enzymatic IDO1 activity, resulting in TRP depletion and high-level production of immunoregulatory TRP metabolites, KYNs, which may foster Tregs expansion. Induced Tregs use TGF-β to maintain an IDO1-dependent regulatory environment, with IDO1 mostly functioning as a signaling molecule. Both mechanisms are interrupted by IL-6, which drives IDO1 degradation as potent inflammatory stimuli enter the local environment [85].
Figure 4: The mechanism of IDO1 regulation on viral infectious diseases. Innate defense occurs when pathogens contact or invade host cells and elicit the production of cytokines and chemokines, which in turn induce an influx of immune cells that affect pathogen clearance. Type I IFNs are critical mediators of innate immunity and limit disease caused by many viruses [73, 86]. The enhancement of TRP breakdown by IDO1 regulates the signal pathway for IFNs production, and TRP metabolites might contribute to the function of IFNs producing cells.
Remarkable Role of Indoleamine 2,3-Dioxygenase and Tryptophan Metabolites in Infectious Diseases: Potential Role in Macrophage-Mediated Inflammatory Diseases (PubMed) (pdf 4235)
Protocols-including KDF
#1由 sufang 在 一, 05/18/2015 - 08:50 發表。
Keratinocyte culture method
說在前面:
Rheinwald Lab所寄給我們的Human Keratinocyte Culture Methods第一段便比較下面三個廠商來的media, 說請大家用K-sfm來養"low-to-moderate density"的keratinocytes (primary or immortalized)
| K-sfm | Life Technologies | grow well from low to moderate density |
| EpiLife | Life Technologies | |
| KGM (or MCDB154) | Clonetics | very short replicative lifespan |
第二段:
須牢記於心的是, K-sfm的營養成分僅適用於低密度、聚集性的keratinocyte生長, 因此用K-sfm養的細胞, 1/3滿度前就必須passage.
第三段: 碰到OSCC的話, 建議與irradiated 3T3 fibroblasts共養、養在DMEM/F12 + 10-20% calf serum + 0.4 ug/ml hydrocortisone.
以下是養細胞步驟, Adapt from 宛樺labnotes and Rheinwald Lab.
For a p100 dish (or T75) --> plaing 104 – 105 cells / dish
For a p60 dish (or T25) --> plaing 103 – 104 cells / dish
Cultures are refed every two days.
5-8 days after plating, cells are ready for the next passage.
–It is important to subculture keratinocytes no more than 8 days after they have been plated, even if they have not reached the desired density (每一繼代不超過8天).
–A healthy, proliferative cell population should contain primarily small cells which have a population doubling time of ~24-30 hours (正常健康的小細胞約24–30小時複製一代)
–It is best practice to count the cells at each subculture and replate at a defined number, avoid the common practice of many labs for serially passaging cell lines of plating 1:4 or 1:10 dilutions (維持每次繼代種入固定細胞數, 不要用1:4, 1:10這種不良繼代習慣).
Growing keratinocytes to high density (提高細胞培養密度之培養方法)
–K-sfm medium was not designed to grow cells to high density with retention of viability. To grow keratinocytes to high density a medium containing higher concentrations of nutrients is needed. We have found that keratinocytes and SCC cells that have been grown up to ~1/3 confluence in K-sfm can be switched at this time to a 1:1 mixture of K-sfm and a medium we call “DF-K” (note: K-sfm + DF-K --> KDF medium).
–K-sfm medium was not designed to grow cells to high density with retention of viability. To grow keratinocytes to high density a medium containing higher concentrations of nutrients is needed. We have found that keratinocytes and SCC cells that have been grown up to ~1/3 confluence in K-sfm can be switched at this time to a 1:1 mixture of K-sfm and a medium we call “DF-K” (note: K-sfm + DF-K --> KDF medium).
–SF notes: 請大家漸漸把immortalized keratinocytes 馴服在KDF medium中, 謝謝! These incudes
* CGHNK2
* OKF4hTERT
* NP460hTERT
* NP550hTERT
* DOK
Recipe for the above Rheinwald lab modification of KSFM:
(GIBCO K-sfm +25 µg/ml BPE +0.4 mM CaCl2 +0.2 ng/ml EGF +pen/strep)
(GIBCO K-sfm +25 µg/ml BPE +0.4 mM CaCl2 +0.2 ng/ml EGF +pen/strep)
500 ml K-sfm (Invitrogen/Life Technologies, which comes with 0.1 mM CaCl2)
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.5 ml 0.3 M CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
5 ml Penicillin/Streptomycin (100X conc. solution) (Life Technologies)
Swirl to mix and then filter-sterilize through a 0.2 μm pore-size sterilization filter. The medium remains good for at least one month at 4oC.
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.5 ml 0.3 M CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
5 ml Penicillin/Streptomycin (100X conc. solution) (Life Technologies)
Swirl to mix and then filter-sterilize through a 0.2 μm pore-size sterilization filter. The medium remains good for at least one month at 4oC.
Recipe for DF-K medium:
(DMEM + F12 (1:1 v/v) +2 mM L-glutamine +25 µg/ml BPE +0.4 mM CaCl2 + 0.2ng/ml EGF )
(DMEM + F12 (1:1 v/v) +2 mM L-glutamine +25 µg/ml BPE +0.4 mM CaCl2 + 0.2ng/ml EGF )
250 ml DMEM (High Glucose, no pyruvate, no glutamine, no calcium (Life Technologies catalog #21068-028)
250 ml Ham’s F-12 medium (Life Technologies catalog #11765-054)
3.75 ml 200 mM L-Glutamine (=100x for 2 mM) (Invitrogen/Life Technologies)
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.42 ml 0.3 mM CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
250 ml Ham’s F-12 medium (Life Technologies catalog #11765-054)
3.75 ml 200 mM L-Glutamine (=100x for 2 mM) (Invitrogen/Life Technologies)
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.42 ml 0.3 mM CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
5 ml Penicillin/Streptomycin (100X conc. solution) (Life Technologies)
Once cultures have grown to ~1/3 confluence in KSFM they either should be subcultured or else subsequently fed daily with a 1:1 mixture of KSFM and DF-K until confluent. Such cultures are suitable for protein, RNA, and DNA extracts or FACS analysis, but may not subculture as well as cells grown to no more than ~1/3 confluence in K-sfm.
Nov 18, 2014
Cloning of colorful CXCL1, 2, 3, IDO1, TDO2
由 sufang 在 二, 11/18/2014 - 15:13 發表 Pre-published CXCL 素菁
| ID | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | |
| CXCL1 | 4.89 | 5.66 | 2.24 | 0.9 | 1 | 0.9 | 2.33 | 3.67 | 2.1 | 4.45 | 1.17 | 9.2 | 1.06 | 7.18 | 1.29 | 1.7 | 3.57 | 2.51 | 1.29 | 0.58 | |
| ID | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | AVE |
| CXCL1 | 0.9 | 2.46 | 1.89 | 2.44 | 3.56 | 1.8 | 2.47 | 4.94 | 1.95 | 2.33 | 0.72 | 1.2 | 6.39 | 3.09 | 1.32 | 0.48 | 4.18 | 2.59 | 2.46 | 1.07 | 2.64825 |
| ID | AC/DOK2 | ACst/DOK3 | NNK/DOK1 | NNKst/DOK3 | NIC/DOK1 | 4NQO mo | |||||||||||||||
| CXCL1 | 1.48 | 1.15 | 2.35 | 2.06 | 0.15 | 3.82 | |||||||||||||||
Entrez Gene:
CXCL1=GRO-a=GMSA (4q21)
CXCL2=GRO-b (4q21)
CXCL3=GRO-c (4q21)
CXCR2 =IL8RB (2q35)
| ID | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | |
| CXCR2 | 0.65 | 0.36 | 0.38 | 0.35 | 0.65 | 0.18 | 0.48 | 0.21 | 0.64 | 0.39 | 0.76 | 0.16 | 0.8 | 0.62 | 0.33 | 2.28 | 0.43 | 0.29 | 1.1 | 0.4 | |
| ID | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | AVE |
| CXCR2 | 0.93 | 0.24 | 0.42 | 0.25 | 1.24 | 0.54 | 0.37 | 1.26 | 0.89 | 0.35 | 0.84 | 0.2 | 1.01 | 0.57 | 1.81 | 0.52 | 0.38 | 0.74 | 0.56 | 0.45 | 0.63 |
| ID | AC/DOK2 | ACst/DOK3 | NNK/DOK1 | NNKst/DOK3 | NIC/DOK1 | 4NQO mo | |||||||||||||||
| CXCR2 | 0.77 | 1.57 | 2.03 | 0.57 | 1.47 | 1.34 | |||||||||||||||
Subscribe to:
Posts (Atom)