text:
- Research
- Open Access
microRNA response elements-regulated TRAIL expression shows specific survival-suppressing activity on bladder cancer
- Youguang Zhao†1Email author,
- Ying Li†2,
- Liang Wang1,
- Hang Yang1,
- Qingtang Wang1,
- Haiyan Qi3,
- Shadan Li1,
- Peng Zhou1,
- Ping Liang1,
- Qiwu Wang1 and
- Xiaowei Li1
© Zhao et al.; licensee BioMed Central Ltd. 2013
- Received: 26 October 2012
- Accepted: 20 February 2013
- Published: 26 February 2013
Bladder transitional cell carcinoma greatly threatens human health all over the world. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows a strong apoptosis-inducing effect on a variety of cancer cells including bladder cancer. However, adenovirus-mediated TRAIL expression still showed cytotoxicity to normal cells mainly due to lack of tumor specificity.
To solve the problem, we applied miRNA response elements (MREs) of miR-1, miR-133 and miR-218 to confer TRAIL expression with specificity to bladder cancer cells.
Expression of miR-1, miR-133 and miR-218 was greatly decreased in bladder cancer than normal bladder tissue. Luciferase assay showed that application of the 3 MREs was able to restrain exogenous gene expression to within bladder cancer cells. Subsequently, we constructed a recombinant adenovirus with TRAIL expression regulated by MREs of miR-1, miR-133 and miR-218, namely Ad-TRAIL-MRE-1-133-218. qPCR, immunoblotting and ELISA assays demonstrated that Ad-TRAIL-MRE-1-133-218 expressed in bladder cancer cells, rather than normal bladder cells. The differential TRAIL expression also led to selective apoptosis-inducing and growth-inhibiting effect of Ad-TRAIL-MRE-1-133-218 on bladder cancers. Finally, bladder cancer xenograft in mouse models further confirmed that Ad-TRAIL-MRE-1-133-218 effectively suppressed the growth of bladder cancers.
Collectively, we demonstrated that MREs-based TRAIL delivery into bladder cancer cells was feasible and efficient for cancer gene therapy.
- Bladder cancer
- Adenovirus
- miRNA
- Specificity
Among the most common malignant cancers, bladder transitional cell carcinoma severely risks health of the people on the earth [1]. Downregulation of certain tumor suppressor genes was documented to largely contribute to initiation, progression, invasion and metastasis of bladder cancer [2]. Therefore, gene therapy is a reasonable strategy for bladder cancer treatment and many reports have confirmed its feasibility and effectiveness [3, 4].
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted much attention due to its specific induction of apoptosis in various types of cancer cells by binding death receptors and activating mitochondria-independent signal transduction pathway [5, 6]. Like many other cancer types, adenovirus-mediated TRAIL therapy was well demonstrated to inhibit the survival of bladder cancer cells [7–12]. More intriguingly, extensive DR4 and DR5 expressions of bladder cancer in patients ensure its responsiveness to TRAIL in future clinical treatment [13].
Cytotoxicity to normal cells, however, seriously hurdles the clinical application of adenoviral vector for cancer gene therapy, since adenoviral vector lacks the ability to discriminate cancer and normal cells. To confer adenovirus with bladder cancer specificity, researchers developed many strategies including employing cancer-specific promoter. Although UP II promoter has been used to specifically drive TRAIL expression in bladder cancer cells, more novel strategies are needed to prevent the cytotoxicity of adenovirus-based gene therapy to normal cells [14–16].
Differential expression profile of miRNAs has been widely reported between bladder cancer and normal cells [17]. Decreased expression level of certain miRNAs allows the introduced genes specifically expressed in bladder cancer cells by inserting their miRNA response elements (MREs) following the opening reading frames. So far, no groups have tested the feasibility and effectiveness of this MREs-based strategy for bladder cancer-specific gene therapy.
Here, we intended to identify suitable MREs for bladder cancer specific adenovirus-mediated TRAIL expression from the miRNAs with downregulated expression in bladder cancer, including miR-1 [18–21], miR-99a [22], miR-100 [23], miR-101 [24, 25], miR-125b [23, 26, 27], miR-133a [18, 20, 21, 23, 28–30], miR-143 [22, 23, 31–33], miR-145 [21, 23, 29–31, 34], miR-195-5p [35], miR-199a-3p [36], miR-200 [37, 38], miR-203 [39, 40], miR-205 [37], miR-218 [21, 41], miR-490-5p [42], miR-493 [43], miR-517a [44], miR-574-3p [45], miR-1826 [46] and let-7c [42].
Primary culture
We employed primary cultures derived from bladder transitional carcinoma and normal bladder mucosal cells (BMC) in this study. For the culturing of bladder cancer, the samples were obtained with written informed consent from all patients according to protocols approved by Ethical Review Board in General Hospital of Chengdu Military Area Command of Chinese PLA (Chengdu, China). All patients underwent surgical resection of bladder carcinoma at Department of Urology, General Hospital of Chengdu Military Area Command of Chinese PLA (Chengdu, China). Bladder cancer samples were sheared into small pieces, followed by mechanical manipulation to obtain single cell suspension. The primary cultures were maintained in DMEM supplemented with 15% FBS.
For primary BMC culture, the samples were obtained from 8 patients that underwent cystoscopic examination of asymptomatic haematuria (The biopsies were not malignant revealed by histopathological results). The previously described procedures that have been approved by Ethical Review Board in General Hospital of Chengdu Military Area Command of Chinese PLA (Chengdu, China) was followed to establish the primary BMC culture [47]. The BMCs were immortalized using adenoviral vector, Adeno-SV40 (Applied Biological Materials Inc., Canada), according to the manufacturer's instructions. All the patients approved the application of their samples for this study.
Construction of adenoviral vectors
Ad-EGFP and Ad-TRAIL were preserved in our laboratory. We constructed Ad-TRAIL-MRE-1-133-218 as follows. A DNA fragment was synthesized (5′-ACAAACACCACATTCCA ACAAACACCACATTCC.
Cell line cultures
Human bladder transitional cell carcinoma cell line T24 and RT-4 were both purchased from the American Type Culture Collection (Manassas, VA) and were grown in McCoy's 5a Medium Modified (Life Technologies, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Rockville, MD). Human endothelial cells HUV-EC-C and normal liver cells L-02 were obtained from Shanghai Cell Collection (Shanghai, China). HUV-EC-C and L-02 cells were cultured using DMEM media supplemented with 10% (v/v) fetal bovine serum. All media was supplemented with 4 mM glutamine, 100 units/mL penicillin and 100 μg/ml streptomycin. All cells in this experiment were cultured under a 5% CO2 and humidified atmosphere at 37°C.
Quantitative PCR (qPCR)
Total RNA was extracted from 14 bladder cancer samples with Trizol solution (Sigma-Aldrich, MO) and pooled as one group for subsequent experiments. Another pool of RNA was also obtained from 8 normal bladder mucosal tissues according to the same protocol. Also, T24, RT-4, HUV-EC-C and L-02 cells were processed for extracting RNA with Trizol solution. Reverse transcription reaction was subsequently performed with TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. qPCR was finally performed with TaqMan® 2 × Universal PCR Master Mix (Applied Biosystems) on CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, CA) supplied with analytical software.
4 × 104 cells were cultured in each well of 6-well plates. TRAIL mRNA abundance was determined in Ad-TRAIL-MRE-1-133-218-infected cells after treated with 10 MOI of adenoviruses. After 48h, cells were lysed for RNA extraction and then inversely transcribed into cDNAs with Rever Tra Ace qPCR RT Kit (Toyobo, Japan) according to the manufacturer's instructions. qPCR was performed with SYBR premix Ex Taq (TaKaRa) on CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, CA) supplied with analytical software.
Immunoblotting assay
Protein in adenovirus-infected cells was quantified with immunoblotting assay. 3.5 × 105 cells were cultured in each well of 6-well plates. 10 MOI of adenoviruses were added to cell cultures. Proteins were lyzed with M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, IL) after 48 h, separated using polyacrylamide gel electrophoresis and transferred onto 0.45 μm nitrocellulose membranes. 5% fat-free dry milk was used for blocking. The membrane was then incubated with specific primary antibodies for 6h. The membrane was incubated with corresponding secondary antibody and then with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, IL).
TRAIL determination by ELISA assay
We performed ELISA assay to evaluate the secreted TRAIL protein in media. Briefly, 3.5 × 105 cells were cultured in each well of 6-well plates. 10 MOI of adenoviruses were added to cell media. After 48h, two-antibody sandwich ELISA was applied to determine human TRAIL expression level in the supernatant of cells. The involved antibodies are monoclonal mouse anti-human TRAIL antibody (R&D Systems), peroxidase-conjugated rabbit anti-goat IgG (H&L) and goat anti-human TRAIL antibody (R&D Systems). The absorbance was assessed at a 450 nm wavelength.
miRNA mimics treatment
miR-1, miR-133, miR-218 and control mimics were synthesized by GenePharma (Shanghai, China). T24 and RT-4 cells were transfected with 300 nM control mimic or the mixture of 100 nM miR-1, 100 nM miR-133 and 100 nM miR-218.
FACS analysis on apoptotic rates
3.5 × 105 cells were cultured in each well of 6-well plates. After 24h, the cells were infected with adenoviruses of 10 MOI. After 48h, the cells were stained with Annexin V-PE Apoptosis Detection Kit (Biovision, CA) based on the manufacturer's instructions. The percentages of apoptotic cells were examined with FACS analysis.
Luciferase assay
MiRNA response elements (MREs) for bladder cancer-specific downregulated miRNAs
primer sequences
Forward: 5′-TCGAGACAAACACCACATTCCA ACAAACACCACATTCCA ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTTGGAATGT GGTGTTTGTTGGAATGT GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCTACGGGT ACAAACACCTACGGGT ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTACCCGTA GGTGTTTGTACCCGTA GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCGTACTGT ACAAACACCGTACTGT ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTACAGTAC GGTGTTTGTACAGTAC GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCGGACCAAA ACAAACACCGGACCAAA ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTTTTGGTCC GGTGTTTGTTTTGGTCC GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCAAGCACAA ACAAACACCAAGCACAA ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTTTGTGCTT GGTGTTTGTTTGTGCTT GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCATCCATG ACAAACACCATCCATG ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTCATGGAT GGTGTTTGTCATGGAT GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCACCTTCA ACAAACACCACCTTCA ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTTGAAGGT GGTGTTTGTTGAAGGT GGTGTTTGTC-3′
Forward: 5′-TCGAGACAAACACCTGCACGA ACAAACACCTGCACGA ACAAACACCGC-3′
Reverse: 5′-GGCCGCGGTGTTTGTTCGTGCA GGTGTTTGTTCGTGCA GGTGTTTGTC-3′
4 × 104 cells were cultured in each well of 24-well plates. After transfecting T24, RT-4 and BMCs with the above plasmids, cells were processed with lysis buffer, and subsequently, luciferase activities were assessed with the Dual-Luciferase reporter system (Promega, WI) according to the manufacturers' instructions.
Cell viability assay
1 × 104 T24 and RT-4 cells, 1.5 × 104 primary bladder cancer cells or 2 × 104 BMCs were cultured in each well of 96-well plates. Adenoviruses of indicated MOIs were added to cell cultures. After 6d, 50 μl of MTT (1 mg/ml) was added, and 4 h later, MTT-containing media was replaced with 150 μl of DMSO. The spectrophotometric absorbance was assessed on a model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA) at 570 nm with a reference wavelength of 655 nm. Cell viability = absorbance value of infected cells / absorbance value of uninfected control cells.
Animal experiments
Procedures for animal experiments were all approved by the Committee on the Use and Care on Animals in Qingdao Municipal Hospital (Qingdao, China).
2×106 T24 cells were inoculated at the left flanks of 5-week-old female BALB/c nude mice (Institute of Animal Center, Chinese Academy of Sciences, Shanghai, China). When tumors reached 7–9 mm in diameter, 24 mice were equally assigned into 4 groups (n=6). 100 μL of PBS with or without 2×108 pfu of Ad-EGFP, Ad-TRAIL and Ad-TRAIL-MRE-1-133-218 was directly administrated into tumors by injection, respectively. The administrations were performed every other day for five times with a total dosage of 1×109 pfu of adenoviruses.
T-24 cancer xenograft was established by incubating 1.5×106 cells at the right flanks of 5-week-old female BALB/c nude mice. 24 mice were equally divided into 4 groups (n=6). The doses of used adenoviruses and injection procedures were the same as those on T24 tumor xenograft.
We periodically measured tumor diameter using calipers. Tumor volume (mm3) = maximal length (mm) × perpendicular width (mm) 2 / 2.
Liver function evaluation
To evaluate the hepatoxicity induced by adenovirus treatment, BALB/c mice (n=5) were intravenously injected with 1×109 pfu of indicated adenoviruses every other day for five times. On day 11, their blood (600 mL/mice) was harvested by cardiac puncture, followed by being incubated with 12 U of heparin. Alanine aminotransferase (ALT) levels in blood were detected at the Clinical Laboratory, Qingdao Manucipal Hospital (Qingdao, China).
Histological staining
On day 7 after adenovirus injection, one mouse was sacrificed from each group and its tumor, brain and liver were collected and fixed according to the routine procedures. Histological staining was then performed on formalin-fixed, paraffin-embedded tumor, brain and liver tissue sections using the streptavidinbiotin peroxidase complex method. Anti-TRAIL antibody (Santa Cruz Biotechnology, CA) was used to specifically recognize TRAIL protein. The sections were finally counterstained with hematoxylin.
Statistical analysis
The statistical tests in this manuscript were two-tailed student's t-test. Differences were considered as statistically significant (*) when P < 0.05 and statistically very significant (**) when P < 0.01.
The expression levels of 8 miRNAs were greatly reduced in bladder cancer cells
Application of MREs of miR-1, miR-133 and miR-218 restrained exogenous gene expression within bladder cancer cells
To assess if MREs of miR-1, miR-99a, miR-101, miR-133a, miR-218, miR-490-5p, miR-493 and miR-517a could be used for bladder cancer-specific delivery of exogenous genes, we constructed a series of reporter plasmids containing luciferase regulated by their MREs. The data revealed that luciferase expression was only slightly affected in bladder cancer cells transfected with the reporter plasmids that were regulated by MREs of miR-1, miR-101, miR-133a, miR-218 and miR-490-5p (Figure 2b). Furthermore, inhibitory effect on luciferase expression was greater than 80% in bladder mucosal cells (BMCs) when MREs of miR-1, miR-133a and miR-218 were used (P<0.01) (Figure 2b). Furthermore, HUV-EC-C and normal liver cells L-02 have been shown to have much higher expression level of miR-1, miR-133a and miR-218 than bladder cancer samples (Additional file 2: Figure S2).
Bladder cancer-specific expression of TRAIL genes was achieved by employing MREs of miR-1, miR-133 and miR-218
To confirm if combined application of MREs of miR-1, miR-133 and miR-218 conferred TRAIL expression with bladder cancer specificity, we simultaneously inserted the 3 MREs immediately following TRAIL-encoding open reading frame on adenoviral vectors (Figure 1a). qPCR assay showed that the modified adenovirus, Ad-TRAIL-MRE-1-133-218, had a similar level of TRAIL gene to that of Ad-TRAIL in bladder cancer while TRAIL expression was greatly suppressed in Ad-TRAIL-MRE-1-133-218-infected BMC (Figure 1b). Immunoblotting and ELISA assays also confirmed that Ad-TRAIL-MRE-1-133-218 infection resulted in TRAIL expression with a comparative level with Ad-TRAIL, but almost no TRAIL expression was detected in normal bladder mucosal cells infected with Ad-TRAIL-MRE-1-133-218 (Figure 1c and d).
To confirm MRE-regulated TRAIL expression was dependant on the level of corresponding miRNAs, Ad-TRAIL-MRE-1-133-218-infected T24 cells were treated with mixed mimics of miR-1, miR-133 and miR-218. Elevated expression level of these miRNAs led to a great reduction in TRAIL expression in bladder cancer cells (Figure 1e).
The above results verified that simultaneous application of MREs of miR-1, miR-133 and miR-218 conferred adenovirus-mediated TRAIL expression with bladder cancer specificity.
MREs-regulated adenovirus-mediated TRAIL expression specifically activated extrinsic apoptotic pathway in bladder cancer cells
We subsequently examined the activation of extrinsic apoptosis pathway in T24, RT-4 and BMC cells by immunoblotting assay. The data showed that caspase-8 was cleaved in Ad-TRAIL and Ad-TRAIL-MRE-1-133-218-infected bladder cancer cells as well as Ad-TRAIL-infected BMCs. However, this cleavage did not take place in Ad5-TRAIL-MRE-1-133-218-treated normal bladder mucosal cells (Figure 3b). Similarly, cleavages of caspase-3 and PARP proteins were also observed in the same patterns as caspase-8, suggesting extrinsic apoptotic pathway was selectively activated in bladder cancer cells when Ad5-TRAIL-MRE-1-133-218 was used (Figure 3b).
Ad-TRAIL-MRE-1-133-218 decreased the survival of bladder cancer cells rather than normal bladder mucosal cells
We next investigated the viability of bladder cancer cells and BMCs with MTT assay, when Ad-EGFP, Ad-TRAIL and Ad-TRAIL-MRE-1-133-218 were added to the indicated cell cultures. The data revealed that Ad-TRAIL-MRE-1-133-218 had a comparative tumor-suppressing capacity on T24 and RT-4 bladder cancer cells as well as primary bladder carcinoma cells with Ad-TRAIL (Figure 3c). However, Ad-TRAIL had cytotoxicity to both cancerous and normal bladder cells. In contrast, administration of Ad-TRAIL-MRE-1-133-218 did not affect the survival of BMCs.
Collectively, we proved that Ad-TRAIL-MRE-1-133-218 inhibited the viability of bladder cancer cells without significant cytotoxicity to normal cells.
Ad-TRAIL-MRE-1-133-218 suppressed the growth of bladder cancer xenograft in mouse models
To test the side effect induced by these adenoviruses, we injected Ad-EGFP, Ad-TRAIL and Ad-TRAIL-MRE-1-133-218 into BALB/c mice. On day 11, their blood was collected and assayed for ALT level in serum. Ad-TRAIL treatment was found to cause an elevated level of serum ALT in mice. In contrast, Ad-TRAIL-MRE-1-133-218 did not significantly change the ALT level in the blood of mice, showing no cytotoxicity to liver cells (Figure 4c).
Also, TRAIL expression was evaluated in the tumor and liver sections from the T24 tumor-bearing mice that received the injection of Ad-EGFP, Ad-TRAIL and Ad-TRAIL-MRE-1-133-218. The histological staining showed that Ad-TRAIL-MRE-1-133-218 treatment resulted in high expression of TRAIL in tumors as Ad-TRAIL infection (Figure 4d). Importantly, TRAIL expression was not detected in liver section from Ad-TRAIL-MRE-1-133-218-treated group, whereas Ad-TRAIL-infected mice had an extensive TRAIL expression in their livers (Figure 4d).
In this study, we experimentally confirmed expression profiles of 20 miRNAs in bladder cancer and corresponding noncancerous bladder tissues. qPCR assay showed that all of them had lower abundance in bladder cancer in comparison with normal bladder tissue. Our results were in accordance with previous reports from other research groups. The differential expression level of these miRNAs made it feasible that their MREs can be utilized to control TRAIL expression specifically in bladder cancer cells.
Luciferase reporter assays showed that miR-1, miR-99a, miR-101, miR-133a, miR-218, miR-490-5p, miR-493 and miR-517a only had limited suppressive effect on luciferase expression in bladder cancer cells when their MREs were applied. Further investigation indicated that MREs of miR-1, miR-133a and miR-218 inhibited luciferase expression in normal bladder cells. Therefore, MREs of miR-1, miR-133a and miR-218 were believed to prevent exogenous gene expression from normal bladder mucosal cells without affecting its expression in bladder cancer cells.
UPII promoter has been utilized for specific TRAIL expression in bladder cancer cells. However, gene expression controlled by this promoter is not strictly bladder cancer-specific, due to the remaining activity of UPII promoter in normal bladder mucosal cells [49]. Therefore, other strategies should be developed for preventing TRAIL expression from normal bladder cells. We employed multidisciplinary approaches to prove that TRAIL expression was greatly inhibited in Ad-TRAIL-MRE-1-133-218-infected normal bladder epithelial cells. These data demonstrated this recombinant adenovirus as a vehicle for TRAIL expression with a high bladder cancer-specificity.
As expected, Ad-TRAIL-MRE-1-133-218 induced extrinsic pathway-mediated apoptosis in bladder cancer cells, rather than normal bladder mucosal cells. Subsequent cell viability assay and animal experiments showed that Ad-TRAIL-MRE-1-133-218 greatly suppressed the growth of bladder cancer. More importantly, survival of normal bladder epithelial cells was almost not affected by Ad-TRAIL-MRE-1-133-218, suggesting biosafety of this MREs-regulated TRAIL-expressing adenoviral vector.
To further improve the biosafety of the adenoviral vector expressing TRAIL, other MREs should also be applied to suppress the undesirable exogenous gene expression in normal tissue, such as liver. miR-122 has been extensively reported to be highly expressed in normal hepatic cells and downregulated in hepatocellular carcinoma, and thus, its MRE can be utilized to prevent cytotoxicity from liver cells [50].
TRAIL has been demonstrated as a potent anti-tumor cytokine in our study. Other therapeutic cytokines also act as candidates for cancer gene therapy, especially the natural inhibitors against signaling pathway that is critical for cancer progression. For example, DKK1 has been shown to suppress the gastric cancer progression by inhibiting WNT/β-catenin pathway [51]. Our novel MRE-regulated adenoviral vector is believed to be a suitable expression vehicle for these inhibitors with high bladder cancer specificity.
We generated a bladder cancer-specific adenoviral vector that expressed TRAIL based on MREs of miRNAs whose levels were reduced in bladder cancer. The anti-tumor capacity and biosafety of this new adenoviral vector was proved by a series of experimental approaches. We proposed that the MREs-targeted adenovirus is a promising tool for gene therapy against bladder cancer.
Authors' Affiliations
- Jacobs BL, Lee CT, Montie JE: Bladder cancer in 2010: how far have we come?. CA Cancer J Clin. 2010, 60 (4): 244-272. 10.3322/caac.20077.View ArticleGoogle Scholar
- Voutsinas GE, Stravopodis DJ: Molecular targeting and gene delivery in bladder cancer therapy. J Buon. 2009, 14 (Suppl 1): S69-S78.Google Scholar
- Malmstrom PU, Loskog AS, Lindqvist CA, Mangsbo SM, Fransson M, Wanders A, Gardmark T, Totterman TH: AdCD40L immunogene therapy for bladder carcinoma–the first phase I/IIa trial. Clin Cancer Res. 2010, 16 (12): 3279-3287. 10.1158/1078-0432.CCR-10-0385.View ArticleGoogle Scholar
- Vardouli L, Lindqvist C, Vlahou K, Loskog AS, Eliopoulos AG: Adenovirus delivery of human CD40 ligand gene confers direct therapeutic effects on carcinomas. Cancer Gene Ther. 2009, 16 (11): 848-860. 10.1038/cgt.2009.31.View ArticleGoogle Scholar
- Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH: Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med. 1999, 5 (2): 157-163. 10.1038/5517.View ArticleGoogle Scholar
- Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME, Dixit VM: FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death–inducing signaling complex. Cell. 1996, 85 (6): 817-827. 10.1016/S0092-8674(00)81266-0.View ArticleGoogle Scholar
- Zhao Y, Li Y, Wang Q, Wang L, Yang H, Li M: Increased antitumor capability of fiber-modified adenoviral vector armed with TRAIL against bladder cancers. Mol Cell Biochem. 2011, 353 (1–2): 93-99.View ArticleGoogle Scholar
- Metwalli AR, Khanbolooki S, Jinesh G, Sundi D, Shah JB, Shrader M, Choi W, Lashinger LM, Chunduru S, McConkey DJ, McKinlay M, Kamat AM: Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells. Cancer Biol Ther. 2010, 10 (9): 885-892. 10.4161/cbt.10.9.13237.View ArticleGoogle Scholar
- White-Gilbertson SJ, Kasman L, McKillop J, Tirodkar T, Lu P, Voelkel-Johnson C: Oxidative stress sensitizes bladder cancer cells to TRAIL mediated apoptosis by down-regulating anti-apoptotic proteins. J Urol. 2009, 182 (3): 1178-1185. 10.1016/j.juro.2009.05.005.View ArticleGoogle Scholar
- Sun B, Moibi JA, Mak A, Xiao Z, Roa W, Moore RB: Response of bladder carcinoma cells to TRAIL and antisense oligonucleotide, Bcl-2 or clusterin treatments. J Urol. 2009, 181 (3): 1361-1371. 10.1016/j.juro.2008.10.148.View ArticleGoogle Scholar
- Szliszka E, Mazur B, Zydowicz G, Czuba ZP, Krol W: TRAIL-induced apoptosis and expression of death receptor TRAIL-R1 and TRAIL-R2 in bladder cancer cells. Folia Histochem Cytobiol. 2009, 47 (4): 579-585.Google Scholar
- Shrader M, Pino MS, Lashinger L, Bar-Eli M, Adam L, Dinney CP, McConkey DJ: Gefitinib reverses TRAIL resistance in human bladder cancer cell lines via inhibition of AKT-mediated X-linked inhibitor of apoptosis protein expression. Cancer Res. 2007, 67 (4): 1430-1435. 10.1158/0008-5472.CAN-06-1224.View ArticleGoogle Scholar
- Li Y, Jin X, Li J, Jin X, Yu J, Sun X, Chu Y, Xu C, Li X, Wang X, Kakehi Y, Wu X: Expression of TRAIL, DR4, and DR5 in bladder cancer: correlation with response to adjuvant therapy and implications of prognosis. Urology. 2012, 79 (4): 968 e967-968 e915.View ArticleGoogle Scholar
- Zhai Z, Wang Z, Fu S, Lu J, Wang F, Li R, Zhang H, Li S, Hou Z, Wang H, Rodriguez R: Antitumor effects of bladder cancer-specific adenovirus carrying E1A-androgen receptor in bladder cancer. Gene Ther. 2012, 19 (11): 1065-1074. 10.1038/gt.2011.180.View ArticleGoogle Scholar
- Wang D, Wang Z, Tian J, He X, Chowdhury WH, Zhang X, Li S, Rodriguez R: Prostate stem cell antigen enhancer and uroplakin II promoter based bladder cancer targeted tissue-specific vector. Urol Oncol. 2010, 28 (2): 164-169. 10.1016/j.urolonc.2008.02.002.View ArticleGoogle Scholar
- Zhu H, Zhang ZA, Xu C, Huang G, Zeng X, Wei S, Zhang Z, Guo Y: Targeting gene expression of the mouse uroplakin II promoter to human bladder cells. Urol Res. 2003, 31 (1): 17-21.Google Scholar
- Catto JW, Alcaraz A, Bjartell AS, De Vere WR, Evans CP, Fussel S, Hamdy FC, Kallioniemi O, Mengual L, Schlomm T, Visakorpi T: MicroRNA in prostate, bladder, and kidney cancer: a systematic review. Eur Urol. 2011, 59 (5): 671-681. 10.1016/j.eururo.2011.01.044.View ArticleGoogle Scholar
- Yamasaki T, Yoshino H, Enokida H, Hidaka H, Chiyomaru T, Nohata N, Kinoshita T, Fuse M, Seki N, Nakagawa M: Novel molecular targets regulated by tumor suppressors microRNA-1 and microRNA-133a in bladder cancer. Int J Oncol. 2012, 40 (6): 1821-1830.Google Scholar
- Yoshino H, Enokida H, Chiyomaru T, Tatarano S, Hidaka H, Yamasaki T, Gotannda T, Tachiwada T, Nohata N, Yamane T, Seki N, Nakagawa M: Tumor suppressive microRNA-1 mediated novel apoptosis pathways through direct inhibition of splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) in bladder cancer. Biochem Biophys Res Commun. 2012, 417 (1): 588-593. 10.1016/j.bbrc.2011.12.011.View ArticleGoogle Scholar
- Yoshino H, Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Nishiyama K, Nohata N, Seki N, Nakagawa M: The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Br J Cancer. 2011, 104 (5): 808-818. 10.1038/bjc.2011.23.View ArticleGoogle Scholar
- Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Uchida Y, Kawahara K, Nishiyama K, Seki N, Nakagawa M: Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer. Urol Oncol. 30 (4): 434-443.Google Scholar
- Han Y, Chen J, Zhao X, Liang C, Wang Y, Sun L, Jiang Z, Zhang Z, Yang R, Chen J, Li Z, Tang A, Li X, Ye J, Guan Z, Gui Y, Cai Z: MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One. 2011, 6 (3): e18286-10.1371/journal.pone.0018286.View ArticleGoogle Scholar
- Song T, Xia W, Shao N, Zhang X, Wang C, Wu Y, Dong J, Cai W, Li H: Differential miRNA expression profiles in bladder urothelial carcinomas. Asian Pac J Cancer Prev. 2010, 11 (4): 905-911.Google Scholar
- Kottakis F, Polytarchou C, Foltopoulou P, Sanidas I, Kampranis SC, Tsichlis PN: FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. Mol Cell. 2011, 43 (2): 285-298. 10.1016/j.molcel.2011.06.020.View ArticleGoogle Scholar
- Friedman JM, Liang G, Liu CC, Wolff EM, Tsai YC, Ye W, Zhou X, Jones PA: The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2. Cancer Res. 2009, 69 (6): 2623-2629. 10.1158/0008-5472.CAN-08-3114.View ArticleGoogle Scholar
- Baffa R, Fassan M, Volinia S, O'Hara B, Liu CG, Palazzo JP, Gardiman M, Rugge M, Gomella LG, Croce CM, Rosenberg A: MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets. J Pathol. 2009, 219 (2): 214-221. 10.1002/path.2586.View ArticleGoogle Scholar
- Huang L, Luo J, Cai Q, Pan Q, Zeng H, Guo Z, Dong W, Huang J, Lin T: MicroRNA-125b suppresses the development of bladder cancer by targeting E2F3. Int J Cancer. 2011, 128 (8): 1758-1769. 10.1002/ijc.25509.View ArticleGoogle Scholar
- Uchida Y, Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Kawahara K, Nishiyama K, Seki N, Nakagawa M: MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines. Urol Oncol. 2011, 31 (1): 115-123.View ArticleGoogle Scholar
- Chiyomaru T, Enokida H, Tatarano S, Kawahara K, Uchida Y, Nishiyama K, Fujimura L, Kikkawa N, Seki N, Nakagawa M: miR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer. Br J Cancer. 2010, 102 (5): 883-891. 10.1038/sj.bjc.6605570.View ArticleGoogle Scholar
- Ichimi T, Enokida H, Okuno Y, Kunimoto R, Chiyomaru T, Kawamoto K, Kawahara K, Toki K, Kawakami K, Nishiyama K, Tsujimoto G, Nakagawa M, Seki N: Identification of novel microRNA targets based on microRNA signatures in bladder cancer. Int J Cancer. 2009, 125 (2): 345-352. 10.1002/ijc.24390.View ArticleGoogle Scholar
- Villadsen SB, Bramsen JB, Ostenfeld MS, Wiklund ED, Fristrup N, Gao S, Hansen TB, Jensen TI, Borre M, Orntoft TF, Dyrskjot L, Kjems J: The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer. Br J Cancer. 2012, 106 (2): 366-374. 10.1038/bjc.2011.520.View ArticleGoogle Scholar
- Song T, Zhang X, Wang C, Wu Y, Dong J, Gao J, Cai W, Hong B: Expression of miR-143 reduces growth and migration of human bladder carcinoma cells by targeting cyclooxygenase-2. Asian Pac J Cancer Prev. 2011, 12 (4): 929-933.Google Scholar
- Noguchi S, Mori T, Hoshino Y, Maruo K, Yamada N, Kitade Y, Naoe T, Akao Y: MicroRNA-143 functions as a tumor suppressor in human bladder cancer T24 cells. Cancer Lett. 2011, 307 (2): 211-220. 10.1016/j.canlet.2011.04.005.View ArticleGoogle Scholar
- Ostenfeld MS, Bramsen JB, Lamy P, Villadsen SB, Fristrup N, Sorensen KD, Ulhoi B, Borre M, Kjems J, Dyrskjot L, Orntoft TF: miR-145 induces caspase-dependent and -independent cell death in urothelial cancer cell lines with targeting of an expression signature present in Ta bladder tumors. Oncogene. 2010, 29 (7): 1073-1084. 10.1038/onc.2009.395.View ArticleGoogle Scholar
- Fei X, Qi M, Wu B, Song Y, Wang Y, Li T: MicroRNA-195-5p suppresses glucose uptake and proliferation of human bladder cancer T24 cells by regulating GLUT3 expression. FEBS Lett. 2012, 586 (4): 392-397. 10.1016/j.febslet.2012.01.006.View ArticleGoogle Scholar
- Shatseva T, Lee DY, Deng Z, Yang BB: MicroRNA miR-199a-3p regulates cell proliferation and survival by targeting caveolin-2. J Cell Sci. 2011, 124 (Pt 16): 2826-2836.View ArticleGoogle Scholar
- Wiklund ED, Bramsen JB, Hulf T, Dyrskjot L, Ramanathan R, Hansen TB, Villadsen SB, Gao S, Ostenfeld MS, Borre M, Peter ME, Orntoft TF, Kjems J, Clark SJ: Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. Int J Cancer. 2011, 128 (6): 1327-1334. 10.1002/ijc.25461.View ArticleGoogle Scholar
- Adam L, Zhong M, Choi W, Qi W, Nicoloso M, Arora A, Calin G, Wang H, Siefker-Radtke A, McConkey D, Bar-Eli M, Dinney C: miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy. Clin Cancer Res. 2009, 15 (16): 5060-5072. 10.1158/1078-0432.CCR-08-2245.View ArticleGoogle Scholar
- Bo J, Yang G, Huo K, Jiang H, Zhang L, Liu D, Huang Y: microRNA-203 suppresses bladder cancer development by repressing bcl-w expression. Febs J. 2011, 278 (5): 786-792. 10.1111/j.1742-4658.2010.07997.x.View ArticleGoogle Scholar
- Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan M, Bassi P, Sevignani C, Byrne D, Negrini M, Pagano F, Gomella LG, Croce CM, Baffa R: Micro-RNA profiling in kidney and bladder cancers. Urol Oncol. 2007, 25 (5): 387-392. 10.1016/j.urolonc.2007.01.019.View ArticleGoogle Scholar
- Tatarano S, Chiyomaru T, Kawakami K, Enokida H, Yoshino H, Hidaka H, Yamasaki T, Kawahara K, Nishiyama K, Seki N, Nakagawa M: miR-218 on the genomic loss region of chromosome 4p15.31 functions as a tumor suppressor in bladder cancer. Int J Oncol. 2011, 39 (1): 13-21.Google Scholar
- Han Y, Chen J, Zhao X, Liang C, Wang Y, Sun L, Jiang Z, Zhang Z, Yang R, Chen J, Li Z, Tang A, Li X, Ye J, Guan Z, Gui Y, Cai Z: MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One. 6 (3): e18286-Google Scholar
- Ueno K, Hirata H, Majid S, Yamamura S, Shahryari V, Tabatabai ZL, Hinoda Y, Dahiya R: Tumor suppressor microRNA-493 decreases cell motility and migration ability in human bladder cancer cells by downregulating RhoC and FZD4. Mol Cancer Ther. 2012, 11 (1): 244-253. 10.1158/1535-7163.MCT-11-0592.View ArticleGoogle Scholar
- Yoshitomi T, Kawakami K, Enokida H, Chiyomaru T, Kagara I, Tatarano S, Yoshino H, Arimura H, Nishiyama K, Seki N, Nakagawa M: Restoration of miR-517a expression induces cell apoptosis in bladder cancer cell lines. Oncol Rep. 2011, 25 (6): 1661-1668.Google Scholar
- Tatarano S, Chiyomaru T, Kawakami K, Enokida H, Yoshino H, Hidaka H, Nohata N, Yamasaki T, Gotanda T, Tachiwada T, Seki N, Nakagawa M: Novel oncogenic function of mesoderm development candidate 1 and its regulation by MiR-574-3p in bladder cancer cell lines. Int J Oncol. 2012, 40 (4): 951-959.Google Scholar
- Hirata H, Hinoda Y, Ueno K, Shahryari V, Tabatabai ZL, Dahiya R: MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer. Carcinogenesis. 2012, 33 (1): 41-48. 10.1093/carcin/bgr239.View ArticleGoogle Scholar
- Woodman JR, Mansfield KJ, Lazzaro VA, Lynch W, Burcher E, Moore KH: Immunocytochemical characterisation of cultures of human bladder mucosal cells. BMC Urol. 2011, 11: 5-10.1186/1471-2490-11-5.View ArticleGoogle Scholar
- He X, Liu J, Yang C, Su C, Zhou C, Zhang Q, Li L, Wu H, Liu X, Wu M, Qian Q: 5/35 fiber-modified conditionally replicative adenovirus armed with p53 shows increased tumor-suppressing capacity to breast cancer cells. Hum Gene Ther. 2011, 22 (3): 283-292. 10.1089/hum.2010.058.View ArticleGoogle Scholar
- Lin JH, Wu XR, Kreibich G, Sun TT: Precursor sequence, processing, and urothelium-specific expression of a major 15-kDa protein subunit of asymmetric unit membrane. J Biol Chem. 1994, 269 (3): 1775-1784.Google Scholar
- Ma L, Liu J, Shen J, Liu L, Wu J, Li W, Luo J, Chen Q, Qian C: Expression of miR-122 mediated by adenoviral vector induces apoptosis and cell cycle arrest of cancer cells. Cancer Biol Ther. 2010, 9 (7): 554-561. 10.4161/cbt.9.7.11267.View ArticleGoogle Scholar
- Wang B, Liu J, Ma LN, Xiao HL, Wang YZ, Li Y, Wang Z, Fan L, Lan C, Yang M, Hu L, Wei Y, Bian XW, Chen D, Wang J: Chimeric 5/35 adenovirus-mediated Dickkopf-1 overexpression suppressed tumorigenicity of CD44(+) gastric cancer cells via attenuating Wnt signaling. J Gastroenterol. 2012, Epub ahead of print,Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Prologue: Dad.
I remember the moment you changed my life forever. You may have wrecked my life or maybe you saved it; could be both. You were repairing the plumbing of a large pipe organ in a dark gothic style church in Powelton Village. I was maybe four, probably not long before we were whisked away to California. I have no idea how you came to be hired to fix a pipe organ, you weren't specifically qualified, but I remember you saying "plumbing is plumbing." a trade you taught yourself from reading the Time-Life books on home repair and improvement. The fact that this event occurred in a church is vastly ironic considering my feelings about religion. You were on your back, half submerged in a crawl space, I was handing you the tools that you asked for, and retrieving the ones you didn't need. At some point I anticipated the tool you were going to need and had it ready before you even asked. You were impressed with this and said: "Wow Josh, you're a really good helper!" I was elated! It was as if the "Powelton Village Gothic Church Angels" (good name for a band) were blasting trumpets in heaven in celebration of my astonishing feat! I, Josh Wright, had just demonstrated a usefulness in this world at the age four, and it was witnessed and acknowledged by my father! This was the birthdate of my greatest asset and my greatest curse, my work ethic. My goal in life from that moment forward was to show the whole world what a good helper I was. I'm not sure if giving birth to a work ethic in a church is blasphemy, but I'm pretty sure it was a Protestant church, so it should be ok.
I'm pretty sure this was the church, and I think it sits on the corner of 36th and Baring St. Pearl St is right behind the church, were I apprenticed with the junk art sculptor Leo Sewell. He's still at it to this day.
I don't know what the church is called but I call it "The Holy Church of the Self-Esteemer."
3321 Spring Garden St
We would go to dad's on weekends and have a blast. We'd play music, go on walks to the art museum, around the parks, nothing but freedom and fun. We revelled in the lack of rules. When I ran away from mom I went straight to dads, imagining that life from then on would be great. He was ill prepared for a thirteen year old to show up out of the blue and say: "Hi, you need to be my dad now". Even after all those years of forced separation when he wanted nothing more than to have me and Ben full time. I think he had given up on that dream and moved on. I finally had enough of mom and her abuse, I couldn't take any more. So, dreaming of the unending freedom and happiness I'd tasted on those weekend visits, I hopped on the train and went to dad's. He got me into a school, set me up in a room on the second floor (called: "second floor middle"), and my new life began. It wasn't quite what I'd hoped for. It sucked balls.
I had to learn the commute to a new school where I was one of two white kids, and involved more walking than I cared for; my dad ate nothing but stir-fry-tofu-veggie-hippie- grain-crap that tasted like sawdust soaked in soy sauce; my room was cold and dark, and I had no friends. It was far from what I expected. Real life replaced my colorful fantasy with a cold, grey, mildewy harshness. Eventually things started to improve. I managed to find lights for my room, got used to being called "cracker" and "white bread" at school, and convinced dad that teenagers required junk food to properly develop.
Teachers, administrators, parents, education policymakers, idealists...none of them can dictate or control, even with the best of intentions, what a child experiences in school. When you throw a large group of children together in a building and control what they do, when they eat, when they come and go, the vast majority of what they're subjected to is each other.
"An institution is any structure or mechanism of social order and cooperation governing the behavior of a set of individuals within a given community — may it be human or a specific animal one. Institutions are identified with a social purpose, transcending individuals and intentions by mediating the rules that govern cooperative living behavior."(from Wikipedia)
Music; making weird the new normal
My father used to practice saxophone for at least eight hours a day. I'm not exaggerating. He'd start warming up with long tones at eight in the morning. By nine-thirty he'd be doing scales; major, minor, dorian, mixolydian, harmonic, etc...
By ten o'clock he'd be mixing in arpeggios; I liked hearing those...up to a point. Then he'd play a single note, strain and warp it until he was squeezing out the harmonics; this could become harsh and grating. People in the neighborhood were subjected to this cacophony on a daily basis. I would get embarrassed when he played weird or harsh sounds, worried that neighbors and people walking by wouldn't understand that he was a musical genius exploring his instrument, pushing the envelope of his musical confines. Unconventional and unselfconscious, he pioneered and proliferated a musical form that is still considered below "underground." Now he tours the world playing for crowds that sometimes exceed twenty, when back in philly in the eighties he was lucky to get five friends to show up, and even luckier if half of them- stayed after they heard the grunts, snorts, screams, and amelodic rants that spewed out of the bell of his sax. I was at once ashamed and proud. Ashamed by the derision and criticism from the mainstream, "that's not music!" Proud when I heard people say, "he's a genius, nobody can play like him!" That dichotomy is still with me.
Inevitably I started playing music. Or rather I was pressured into it. By pressured I mean to say that whenever I showed any musical inclination I was praised and encouraged. I remember a time living in the suburbs I was on the schoolbus and there was a kid sitting in front of me with a pair of drumsticks practicing rudiments on the back of the seat in front of him. I was impressed with how intricate and coordinated it was. So I started playing the drums. Music for me at the time was more a display of athletic prowess than a form of artistic expression. Later I learned that girls liked musicians. This became the overarching inspiration to practice longer and work harder to be the best drummer in Philly. I started practicing diligently four hours minimum a day sorry dad, I had school. The more I practiced, the better I got, the more other musicians wanted to play with me, and best of all: I was the focus of an increasing number of beautiful young girls. It became my "football," complete with fans and cheerleaders, and I was working hard to become the star quarterback.
Philly Punk scene, mid-eighties
Abe's was a cheesesteak joint during the day, and weekend nights was a punk club. Not really much of a club, just a room in the back of the store, a cleaned out storeroom. There was a small stage in the back which could barely fit four people let alone a full band. There was a tiny outside yard/alley which was crucial when you needed to come up for air. We'd cram fifty people in that musty dark room, if enough people were stage diving we could shoehorn in a few more. It was standing room only, no room for slamdancing, so it was pogo or no-go. It was like a tightly packed can of upright sardines with a similar smell, plus a touch of sweaty leather, beer, cigarettes, and Aqua-net (gotta keep those mohawks structurally sound.) If the crowd was jumping up and down, you were forced to do the same. If you fell or were pushed down it could mean death (no joke), but fortunately there was an unspoken rule that if anyone went down everyone around would yank them up immediately. It was a combination of carnage and release, anger and friendship, and a collective shared consciousness; the knowledge that the world was bullshit and lies, surrounded by superficial materialism. We all were groping for something real.
The first show I ever went to was at Abe's. I remember I was fourteen or fifteen and awkward. Nervous as hell, I had to walk a gauntlet of leather and spikes, all looking at me the skinny square kid in jeans and a stupid T-shirt. Wide-eyed and self conscious, I made my way to the back. As I approached the back room the music grew to a deafening roar. Terrified, I wanted to go back, get out of there, this wasn't for me, but there was a press of people behind, all pushing forward, toward the din. Soon I squeezed into the back room which was packed. I could barely see the band over the crowd which was jumping up and down in loose chorus. Before I had a chance to worm my way over to the safety of a wall I got a face full of combat boot, tumbled to the floor with the owner of the boot, then I was immediately yanked to my feet and ignored while my nose and lip dripped blood. My fear was replaced by exhilaration, as I was squeezed in with no choice but to bob up and down with the crowd, arms in the air to catch the next stage dive (not with my face this time). Eventually the music died, mohawks withered and sagged with sweat and beer, sticky punks searched leather jackets for any cigarettes that may have survived the onslaught, then piled outside into the cold, and spoke of second wind parties. "Hey kid, you got blood on your face. Wanna come to a party?"
I was hooked. I felt at home with them, they were honest and irreverent, my people.
In this crowd, a nerd could also be a badass, value in a person wasn't tied to a front, no masks. Nobody trying to be who they were not, only trying to figure out who they were. The greatest insult was if you were labeled a "poser," dressing and acting punk to be cool.
Sometimes loud unfettered music and a combat boot in the face were just what the doctor ordered.
Rock star
At fifteen I started playing in bands. The first was "Mega-Faction," a punk-metal comedy of errors. We played maybe two gigs, one at a party and the other at Abe's Steaks. That show was a disaster. We were terrible. I remember my bass drum kept sliding away from me and I had to stop playing frequently to pull it back. We cut our set short and got off that stage a fast as we could. Defeated and dejected, we never- played again, which was a relief for me because I didn't like the music.
I played in many bands during these years. Honestly I don't remember them all
clearly. Also some are not worth writing about. The music was never what I wanted to play, not until I played with my brother.
The next band was a slightly better fit: "Ghosts Before Breakfast," the name was an homage to a Fellini short by the same name. They played an odd sort of psychedelic art-rock. We played a bunch of shows and parties. One show I remember fondly was at St Mary's church on the University of Pennsylvania campus. A guy named Tim Dunn ran a soup kitchen out of the church, and funded the kitchen with shows. They were promoted as "Rock Against Hunger". Many of the punk shows around West Philly were these benefits. I think the organization was Tim and a guy named Michael McGettigen. There were many shows at St Mary's, and I always liked seeing a catholic parish hall filled with punks, it made me feel like there was hope for the world.
I don't clearly recall the timeline of the bands I played with, but some time later I got into a band called "Uh-Oh," which was a highly percussive art-funk-new-wave-no-genre- -tag-describes-what the hell we were doing. At the core of the music was the percussives of Dan Melamed and the keyboards and bass of Valerie Opaleski. This was new to me. This music was intricate, difficult, required lots of practice. I was used to playing music that almost played itself, just follow the formula. Dan became my drummer hero, he was the first person I took formal lessons from. He and Val played together since they were kids, and they worked together like they were the same person. They would simultaneously come up with parts, like they were of one mind. It was hard to keep up. It was the first time I was truly challenged.
Thirteen Dead Girls From the Yellow Skirted River
While I was developing as a drummer my brother was learning the bass. We started jamming together, and as we both got better, we kept trying to challenge each other. We'd go to shows on the weekends, take in all the great music happening all around us, and jam whenever we had the chance. We found a guitar player named Kennedy and as a trio, were able to actually develop a few finished songs. Although this was enthralling to actually have our own band (I had only played other people's music until then). Our music was disjointed, frenzied, in odd time signatures, and unlike any other music out there. Three people with three completely different sets of influences, with we stitched together like the body parts of our very own Frankenstein. Each song became a disparate collage of unlikely pieces slammed together without regard for how they fit. Like a child with one of those toys where he or she tries to fit the proper shapes into the right holes, only we gave the child a really big hammer. We became adept at jerking through transitions as if we were randomly splicing tape. We'd start with a 6/8 blues groove, then suddenly change to a 2/4 punk-metal, and just as suddenly go into a 9/8 funk extravaganza; kind of like being run over by several trains at once.
We became a creative powerhouse. New ideas, sounds, and songs flowed out of the three of us like the gush from a fire hydrant. When we wrote music it was like we were a three-headed six-armed sound monster. It seemed so easy, like it would never end. As the three of us practiced and improved, the urge to play out grew stronger. Our first singer was a friend named Jack Rabbit. He brought a vocal poetry to our Frankenstein, gave him a voice. Jack Rabbit's lyrics were like punk rock fairy tales on LSD:
Goats in heat
Sheep are meat
Halloween kids say, "trick or treat."
Bah bah gnu gnu, sheep are fucking you...
We needed a band name. Kennedy suggested "Drunk Women in High Heels." I think we played at a party under that name, but I don't clearly remember. Jack Rabbit liked "Dead Girls in Tight Skirts." We stuck with that. It had an irreverent sarcasm that was so lacking in the world. People really like to get offended so we thought we'd help them out.
Our first official gig as Dead Girls was at a place called "The Crypt" which was a large grey stone house on, I think, 46th and Walnut, not sure, but it stood by itself surrounded by empty parking lots. The people who lived in that house paid their rent by hosting punk shows in the basement. We opened for "Pussy Galore" and some other band from NYC. (I think one of those bands stole my high-hat cymbals.) Jack Rabbit was fantastic. We all were fantastic. People were thrashing around, I remember cheers and applause after songs. After a few other gigs Jack Rabbit became uncomfortable with the idea of playing in a working band with a regular practice schedule, gigs, and rigid commitments. He liked when things were more spontaneous. We tried to convince him to stay. I think he felt bad, but in the end he left. Tortured artists fly best solo.
In days of old
when knights were bold
there was a thing that was so bad,
it was the worst thing you'd ever seen...(Jack Rabbit Pollak, The Plague Song)
After Jack Rabbit left, we tried singing ourselves. Mostly we sounded like the backup singers for a poorly rehearsed muppet skit. After a long and disappointing search, we finally found a guy named Jay. Although he could sing, he was no Jack Rabbit. His style was more straightforward, less weirdness, less embellishment, less humor. His lyrics were like a teenage Dungeons & Dragons poem. Nonetheless we settled on Jay, and hoped we could tease out a little more creativity in time. I don't recall how long he played with us but we- played a bunch of gigs and recorded a demo on eight track reel to reel. It had a black and white xeroxed cover with a drawing by Kennedy on the front. Even though it was essentially a demo, we put it out for sale and it sold out.
I think we made enough selling those tapes to cover the recording costs. Eventually Jay started to flake out on rehearsals, then a gig. We heard from others that he developed a crack habit. We fired him.
Again we searched for another singer. We wanted another Jack Rabbit. What we found was Bernadette Rappold. She was the singer for a band called "Tons of Nuns", and also played with me in "Uh-Oh". She had a voice right out of a Broadway rock musical. She was solid, competent, and played guitar which added more fullness to the overall sound. Bernadette was nonplussed with the name "Dead Girls in Tight Skirts" she was after all a lesbian feminist, so we changed it to a phrase picked out of an obscure comic book where a samurai dude said in the heat of battle: "Oh no! Its the thirteen weirdos from the yellow river!" With Bernadette we played all over Philly, I think just about every club, basement, block party, you name it. We were playing at least a show a week for a while.
The weirdos at a block party. Note the footwear. This was probably 1989
We developed a strong following. When we played a club, it would be packed. We had actual fans. Two rehearsals a week and one to two gigs a week plus trying to record, all while Ben and I were holding down full-time jobs and Kennedy and Bernadette were in school began to take its toll. It was work.
We recorded "Woo-Woo" in 1990, where we had the time and freedom to make a-
good album. It took a full day just to get the drum sound right. After we finished recording we continued to play a lot of gigs but my inspiration began to wane. The creative drive sputtered and died. I remember trying to come up with new songs and falling flat. The fire hydrant ran out of water. I felt exhausted, drained, and was desperately trying to rekindle my part of that creative monster but it was gone. We stopped booking gigs, and tried to come up with new material but I lost interest.
All good things...
A year or so later a housing crash in Philly all but erased my construction day job. Then I was mugged, stabbed, and lavished with a fifteen thousand dollar bill from the prestigious Hospital of the University of Pennsylvania. Then my drum set was stolen. In debt, with little income, plagued by PTSD, and my creative well all dried up, I left Philly. My father had moved to Boulder, CO so I went there to try to patch myself together.
While I've played with many great bands, projects, recordings, and performances since I left, I've never had any musical experience that compared to those few years playing with the Weirdos/Dead Girls.
I could write an entire book about the Philly punk scene in the eighties. What's in this memoir covers a microscopic slice of a much larger story. It was a unique time, one of the most prolific underground music scenes from anytime, anywhere. If you google "Philly punk eighties" you'll get a long list of material devoted to the subject. I was fortunate enough to be right in the middle of it. Not just to be able to see and hear legendary bands playing at legendary venues, but to be an actual participant.
My brother and I recently started playing again. Its good. Maybe we'll start a band, who knows...

subset: commoncrawl


text:
This position is responsible for delivering revenue and profit goals for the Southern Region, which includes TX, OK, NM, AZ, UT and NV. The incumbent will actively manage all distributor sales, and take a leadership position with key accounts. Major duties include budget/sales planning, distributor selection and training, management of regional and local promotional activity, key account retention, and quarterly business reviews.
This position will manage manufacturers' representatives and staff in territory to achieve company's sales targets by focusing on new business opportunities and supporting existing accounts. The manager is expected to:
• Establish appropriate sales metrics and performance criteria for proper measurement of results and future trends;
• Recruit, hire, and train manufacturers' representatives and staff (where applicable) in assigned territory;
• Manage company policies and procedures as they pertain to the sales territory;
• Operate the assigned cost center within budget;
• Manage relationships with channel partners, contractors, resellers and distributors and end users to achieve maximum market penetration and to achieve agreed upon revenue and profit targets;
• Manage competitive pricing issues in a team fashion with other regional managers;
• Support customer requests for information and service as required;
• Create data, budgets and reports to support business, i.e.: Monthly report on sales/marketing activity; Quarterly report on industry trends, competition and activities;
• Represent the company at various activities (as required) such as trade shows, seminars, etc.
REQUIRED SKILLS Excellent communication, presentation, and relationship building skills, Technically savvy and effective in working with internal teams, clients and other industry related contacts
Computer proficiency in Microsoft Office and CRM systems such as
Goldmine or Salesforce.com
PREFERRED SKILLS Experience with strategic selling concepts
Strong preference given to someone with prior sales management


text:
We Luv these 6 Tips to Sell Your Home in the Winter Months.
It makes a lot of sense; when you selling a Denver, Parker, or Castle Rock CO home in the winter, make the home warm and inviting and get people to stay inside as long as possible. It can help a lot! The best thing you can do is make it look like you are expecting them. And if you have a gas fireplace leave the fire on...what's the harm? Helps with the WARM and INVITING part.


text:
Yes, you will get your own app in the app stores, after you have ordered it. The Blappsta Preview app, that you use while creating your app, is only for demonstration on how your app will look like.
Do I get my own app in the app stores?
Have more questions? Submit a request
Мейіржан Жылқыбай
Where is my app??? Why do not you answer the letters? Took my money and be silent. Already a month. I jo app
Please sign in to leave a comment.


text:
- Bachelor in General Studies
- Joint Degree
- Choosing a Major
The Individualized Major Program (IMP) has been discontinued as of January 2018 and no additional students will be able to declare an IMP major.
Welcome to the Individualized Major Program! We're very happy that your proposal was approved and you've joined this group. Because we don't have a departmental structure, it is even more important that we find ways to communicate, collaborate, and create a sense of community.
You should have received a t-shirt when you declared. If not, check with the IMP Advisor.


text:
- About Us
- Financial Services
- Membership
- Learning
- Career Center
- Advocacy
- Toolkit
|Career Center
|Search Openings Subscribe Find Postings
Searching for your next great employee?
Have an amazing job opportunity?
Look no further than NAM's Career Center!
NAM Members Post for FREE
Log in using your organization's master account profile. (If you are having issues logging in, please email Kelly or Thaddaeus). Click on "CAREER CENTER" in the main menu. At the top of the Career Center page, click on "SUBMIT AN OPENING". Follow the directions to post your opening. PLEASE ensure you post information in the 'HOW TO APPLY' section.
Not yet a NAM member?
You can purchase a career post. After the purchase, you will receive and email with instructions on how to post your job opening. (Please ensure you check your junk mail folder before contacting Kelly.)
Want to conduct a NATIONWIDE search?
If you are interested in doing a nationwide search, we have partnered with the National Council of Nonprofits to offer a 15% discount to NAM members on single or package postings. Their site is: https://careers.councilofnonprofits.org. Email Kelly or Thaddaeus for the promo code (NAM members only). Members may also use our state by state guide to nonprofit jobs here.
To view or inactivate a job posting
To view your active job postings, log in to your account. Click on 'Manage Profile', scroll down and click on 'Career Postings' and all of your job posts will appear. Email Kelly or Thaddaeus if you want a posting inactivated.
Job postings are intended for single open positions. Openings advertising a period of general hiring or multiple jobs will be deleted, and if a fee is paid it will not be returned.
Stay in touch with your professional network through NAM!
As a NAM member you will be able to join our online Google Groups:
HR/Finance Networking Forum
ED/CEO Networking Forum
Ask questions and share insights, best practices, templates and tools. We'll keep you up-to-date on all NAM networking events and learning opportunities in your professional community. Contact Kelly or Thaddaeus to be added to the appropriate forum.
6/19/2019 » 6/23/2019
Tour de Nebraska (Sidney|Kimball|Gering|Scottsbluff|Bridgeport)
Trauma & Recovery 101 (Omaha)
Grant Writing Palooza! 2019
Bullying in the Workplace (Omaha)


text:
George Costanza (Seinfeld episode "The Mango")
The other day while doing some errands I stopped by Tap Coffee in Fitzrovia. Not sure why but their shops are quite appealing to me, all the time I pass by they are inviting me in to indulge with coffee, maybe is the black facade with the huge number (No 114) and no mention of the name.
3 Locations around London with the same feel and offer, a relax place to share a coffee while catching up with friends.
I got a macchiato and my better half a Flat White. The coffee was a light body with mild bitterness. Modest citrus acidity, some chocolate and tobacco notes.
Beans: Tap Coffee
Espresso Machine: Nuova Simonelli
Score: 3/5
Decent cup of coffee, lovely cutlery, and inviting place
Tap Coffee
Coffee House
Approx Damage: £5
Area: Fitzrovia
Borough: Camden
Twitter: tapcoffee


text:
My post on Yoon Mi Rae probably gets the most hits on this blog (which is great, since it means more and more people are searching her out :)). I thought I'd post up a translation of the lyrics of 잊었니 (Did you forget) for anyone looking out for it. Its pretty much a tearful, sorrowful, break-up song. I enjoy her powerful and emotional singing and the music more than the lyrics themselves.
I didn't translate this (I wish my Korean was this good!); all credit goes to KBS World Radio. The host of the K-Pop Interactive show translates songs at the request of listeners, and these are available on the website. Explore this via http://rki.kbs.co.kr/english/radio/ and http://rki.kbs.co.kr/english/entertainment/enter_bbs_list.htm.
(KBS World has actually created a very impressive compilation of resources to reach out to the universal audience, and the radio shows are quite good, with some good hosting: many shows are broadcasted and in numerous languages, with great reception if listened to online (also broadcast in some states in the US). I've personally listened to some of the shows often and enjoyed them. Great for someone new to Korean music and culture :))
Thanks to DJ Sarah and the KBS World website for the translation and the romanization. So you can sing along when you're feeling dramatic and/or broken hearted!
Here's the song again. 잊었니, by 'T' (Yoon Mi Rae)
진실은 거짓이 되고 [Jinshireun geojishi dwego]
The truth has become lies
술은 눈물과 섞여 쓰라린 [sooreun noonmool-gwa sekkeo]
my tears mix with my drink sseurarin
내 마음 속을 태워 [nae ma-eum sogeul taewuo]
내려 가네 여전~히~ [naeryeo ganae yeojeonhi]
burning my bitter heart as it goes down, like always
니 품 안에 웃던 그날~ 그 날에 멈춰 [Ni poom ane ootdeon geu nal Geu nare meomchuo]
I remain in the days when I laughed in your embrace
미소 지어~ 보는~ 난 [miso jieo boneun nan]
I, who wore a smile on my face
내일이 되면 꿈~이라고~ [nae-ili dwemyeon ggoomirago]
will realize that it was a dream when tomorrow comes
오늘도~ 애써 잠들려 [oneuldo aesseo jamdeullyeo]
so today it's hard for me to fall asleep
난 눈~을 감~지만~ [Nan noon-eul gamjiman]
Though I close my eyes
눈물이 고인 내 가슴 속은 진실을 알려~
[noonmooli goin nae gaseum sogeun jinshireul allyeo]
as my tears well up, my heart tells me the truth 잊었니 우린 사랑했~잖~니 [Ijeonni woorin saranghaetjanni]
Did you forget? Didn't we love?
나만 사랑한단 우리의 약~속~을 잊었니~ [Naman saranghandan woori-eh yaksogeul Ijeonni]
Did you forget the promise that you would love only me?
우린 행복했~잖~니 [woorin haengbokhaetjanni]
Weren't we happy?
너 없이 난 천천히 [Neo eobshi nan cheoncheonhi]
죽어 가~는~데 [joogeo ganeunde]
Without you, I'm dying slowly
넌 어~딨니 [Neon eodinni]
Where are you? Ooh ooh ohh거짓은 진~실이 돼 [Geojiseun jinshili dwae]
Lies become the truth
술은 눈물과 섞여~ [sooreun noonmoolgwa seokkeo]
my tears mix with my drink
내 맘속에 아픔을 불 질러 [Nae mamsoge apeumeul bool jilleo]
pain is setting my heart on fire
네게 기대려 해~도~ [Nege gidaeryeo haedo]
Even if I try to lean on you
옆에 넌 있진 않지만~ [Yeope neon itjin anchiman]
Even if you're not by my side
내일이 되면 넌 날 깨~워주겠지 [Naeili dwemyeon neon nal ggaewuojoogetji]
When tomorrow comes you'll wake me up, won't you?
There's just no more you and me
오늘도~ 널 기다리며 [oneuldo neol gidarimyeo]
Even today, as I wait for you
눈~을 감~지만~ [nooneul gamjiman]
and close my eyes
눈물에 잠긴 내 가슴 속은 [noonmoole jamgin nae gaseum sogeun]
거짓을 알려~ [geojiseul allyeo]
flooded with tears, my heart tells me about the lies
벌써 딴 사랑을 [Beolsseo ddan sarangeul]
찾아 간거니 [chaja gan-geoni]
Have you already gone to search for another love?
넌 어떻게 나 없이 살아가 [Neon eoddeoke na oebshi saraga]
How can you live without me?
난 너 없이 살아지는데 [nan neo eobshi sarajinende]
When I'm fading away without you
내 영혼도 날 떠난대 [Nae younghondo nal ddeonandae]
Even my soul says it's leaving me
해는 너무 빨리[Haeneun neomoo bballi]
떠 오지만 [ddeo ojiman]
Though the sun rises so quickly,
너는 오지 않아 [neoneun oji ana]
you're not coming
돌아와 제발 [Dorawa jebal]
Come back, please


text:
Attackhead video diary from the Thrash Bash ll in Santa Ana
Posted: Sep 28, 2013 4:55 pm
We had a great time at the Thrash Bash ll at Malones in Santa Ana, CA. last night. The venue is pretty nice and it has 2 stages so the music never stopped. Saw some great opening acts and met a lot of new friends. Here is the video dairy from the event. Cheers [View Article]
Attackhead video diary from Tropics Lounge, Fullerton, CA
Posted: Sep 22, 2013 3:21 pm
Here is a quick video update from our last show ever at the Tropics Lounge before it gets demolished. Last time we performed here was in 2007. [View Article]
Interview on Metal Perv TV from London
Posted: Sep 10, 2013 10:18 pm
Here is a video interview we did with Metal Perv TV out of London. We had just played the Unicorn and were chilling in the van to get out of the rain. [View Article]
2 local shows added.
Posted: Sep 2, 2013 9:00 pm
Greetings to all our So Cal brothers and sisters. It has been a long time since we performed a local show but the wait is over. We were just added to the TLDS 4 day festival at the Tropics Lounge in Fullerton, CA. We first played at the Tropics in Aug of 2006 and were one of the first bands to do so. Since then the venue has become very popular with local and national acts. But now the venue is... [View Article]
Attackhead European tour diary #6 from Brighton, England
Posted: Aug 19, 2013 3:19 pm
Here is the final video of our European tour diary. This was filmed at the Green Door Store in Brighton, England. [View Article]
Attackhead European tour diary #5 from Leeuwarden, Netherlands
Posted: Aug 16, 2013 4:13 pm
Here is installment #5 live from Caffe Mukkes in Leeuwarden, Netherlands. [View Article]
Attackhead European tour diary #4 from Augsburg, Germany
Posted: Aug 14, 2013 1:50 pm
Here is installment #4 of the Attackhead European tour diary live from Augsburg, Germany. [View Article]
New European tour photos
Posted: Aug 11, 2013 6:50 pm
Greeting metal maniacs. We have uploaded a ton of new photos from our European tour to our Attackhead FB page. If you got some time head on over and take a look.http://www.facebook.com/attackhead Cheers [View Article]
Attackhead European tour diary #3 from Hamburg, Germany
Posted: Aug 11, 2013 6:47 pm
Here is installment #3 of the European tour diary live from the Hafenklang in Hamburg, Germany. [View Article]
Attackhead European tour diary #2
Posted: Aug 6, 2013 2:15 pm
Here is the second installment of our European tour diary. Cheers [View Article]


text:
Luke Bryan Earns Platinum With 'Kill the Lights'
Luke Bryan's newest album, Kill the Lights, has been certified platinum.
The news made an already-exciting weekend even better: Bryan learned of the album's certification as he was preparing for his tributes to Lionel Richie at the 2016 MusiCares Person of the Year Gala and the 2016 Grammy Awards.
Following its release in August, Kill the Lights debuted at No. 1 on both Billboard's Top Country Albums and all-genre Top 200 charts. It was Bryan's third consecutive No. 1 debut on the Top 200 and his sixth No. 1 album overall on the Top Country Albums chart. The record's No. 1 debut on the Top 200 also marked the highest debut for a country album on that chart since Bryan's own Crash My Party debuted at No. 1 in 2013.
Of the 13 songs on Kill the Lights, Bryan co-wrote six, including the title track. Other songwriters on the project, which was produced by Jeff and Jody Stevens, include Luke Laird, Ashley Gorley, Dallas Davidson, Rodney Clawson, Rhett Atkins, Shane McAnally and Hillary Lindsey.
On Thursday (Feb. 18), Bryan will begin his 2016 Kill the Lights Tour. The album is available for download on iTunes.
Luke Bryan's Goofiest (and Sexiest) Looks
Unforgettable Luke Bryan Moments


text:
The recent completion of new construction activity at The Residences at Palmer Square has produced outstanding opportunities for buyers at the charming and picturesque community in the heart of ...
Comprehensive credit & criminal screening.
Get access to bankruptcies, employment history, medical records, past addresses, evictions and more.
State-specific legal forms.
Lease agreements, rental applications, lease termination, eviction forms and much more.
Our paid membership plans now include free tenant screenings! Greater access to all of our features and the membership pays for itself!
© Copyright 2019 LandlordStation LLC


text:
Mr. President by Katy Evans
Release Date: October 31st Genre: Contemporary Romance
He's won the hearts of millions. But is he willing to lose his?
I met the president's son when we were both young. Matthew Hamilton was handsome, polished, and intelligent. I'd never met a guy like him.
He promised me that he'd never run for president. I promised that if he did, I'd be by his side.
Three terms later, an invitation to join Matthew Hamilton's campaign is the most exhilarating opportunity I've ever experienced. I'm determined to make a difference; he is determined to win.
Focused on his goal, Matt is steadfast, ruthless, and disarming. All eyes are on him and his popularity is surging. But soon, the next possible president of the United States is possessing me in more ways than one—and despite the risks, I'm helpless to resist.
We're stealing touches, stealing moments, and stealing away at night. But our chemical connection is quickly becoming dangerously combustive, putting not only my heart, but Matt's chance at the presidency on the line.
Winning will take everything. Walking away will be the hardest thing of all.
Add to Goodreads
Buy Links:
Amazon ➜ http://amzn.to/2dYH8wj iBooks ➜ http://apple.co/2dPYPQr Nook ➜ http://bit.ly/2dNp2hl Kobo ➜ http://bit.ly/2cQp2Rv
Katy Evans is a New York Times, USA Today, and Wall Street Journal bestselling author. Her debut REAL shot to the top of the bestselling lists in 2013 and since then 9 of her titles have been New York Times bestsellers. Her books have been translated into nearly a dozen languages across the world.
Email / Facebook / Twitter / Website
This was a genius release with the election right around the corner. I would totally vote for Matt Hamilton! I love how Katy writes all of these amazing characters that are vastly different, and manages to make me swoon every single time I pick up one of her books. I have never read any books featuring politics, so I was interested to see how this plot would play out since I don't really care for the subject. I was pleasantly surprised. The romance is split with the politics, so there is a nice blend of both. The forbidden aspect is sexy and adds a little suspense. I enjoyed both of the main characters and thought they had great chemistry together. The one thing I would change would be the age difference between the characters. (13 years- It didn't take away from my enjoyment of the story, but I thought the gap was on the large side.) But other than that, I loved everything about this book.
Mr. President is a well written romance novel with a unique plot and great characters. It was a quick and enjoyable read. I thought this was a standalone, so the ending was quite the shock. But I am happy to read more about these characters and see where the second book takes them. If you love contemporary romance or any of Katy Evans other books, definitely pick this one up! Katy never disappoints!


text:
The MTA and the city face dozens of lawsuits stemming from a May subway derailment in Queens that sent 10 people to the hospital and trapped about 1,000 people underground.
Since the F train went off the rails, 29 people have filed notices of claim.
They include Monifah Kidd, a Queens woman who plans to sue the city for $5 million because of her bad dreams about the derailment — as well as severe injuries she says she suffered.
Kidd filed a notice of claim with the MTA and city comptroller less than a week after the May 2 derailment.
The notice says she "sustained severe and protracted and permanent injuries to her neck, back, head and suffered psychological trauma, mental anguish, emotional distress and suffers from nightmares of the experience to date."
Another Queens rider, Helon Mioh, is suing for $10 million and says she's been left disabled.
Lawyer Frank Lomuscio, of Little Neck, also plans a lawsuit after suffering pain in his neck, back, legs and arms and filed a notice of claim on July 23.
But one straphanger who filed a claim has since decided not to sue.
Taiser Hussein had filed a notice of claim but told The Post she has since decided to drop it.


text:
St. James celebrated Lutheran Schools Week with all other Lutheran schools across our nation. Our purpose is to share the Gospel message to our children every day by our words and actions. Our mission is to equip our children to share the Gospel with others.
The Race to Jerusalem fundraiser ended on Friday of Lutheran schools week with a tailgate party and a Victory Lab Celebration. Thank-you to the Kindergarten class for starting us off in their race cars.


text:
Download detailed specifications for this productDownload PDF
Radix Plastic Bayonet RTD Sensors are designed for temperature measurement in plastic process machinery and for quick connecting applications. These RTD sensors may be constructed with fixed or adjustable sheath lengths as well as straight or 90° bent sheaths. The sensor is PTFE insulated with jacketed cables, we can also design them with SS over-braided cables. Each unit is supplied with one meter or multiples thereof of cable. The sensing element is spring-loaded in the bayonet cap of plated brass, with a SS304 cap also optionally available. Another option is a flex hose over the cable and threaded adaptors.
Please choose from our customization options and enter under comments while inquiring about this product`


text:
This listing is for a pair of a gorgeous large (6.8cm long) genuine Amethyst gemstone and Tibetan Silver Wing Earrings. Similar earrings with genuine Rose Quartz and Opalite gemstone available.
Note: Existing earring hooks (silver plated) could be exchanged for solid .925 Sterling Silver hooks for additional $5 (if you prefer .925 Sterling Silver hooks, please send a message).
Stone: Amethyst (Authentic)
Stone Size: 26mm x 10mm (2), 8mm beads (2)
Wing Material: Tibetan Silver
Wing Size: 20mm x 7mm
Width: 20mm (the widest part)
Length w/bail: 6.8cm
Weight: 12.5 Grams
The picture is of the actual item you will receive.
Amethyst Gemstone meaning and healing properties:
Ancient Egyptians used the amethyst guard against guilty and fearful feelings. It has been worn as protection from self-deception, as well as a protection against witchcraft. The amethyst has long been used to open the spiritual and psychic centers, making it one of the power stones. It is also used as a meditation aid when worn as a necklace. The amethyst symbolizes piety, humility, sincerity and spiritual wisdom. It is thought the amethyst is the perfect stone to symbolize The Age of Aquarius. Using an Amethyst as a meditation focus will increase the positive spiritual feelings. Amethyst helps overcome fears and cravings. It also helps relieve headaches.
If you intent to buy more items and use combined shipping (to combine more items in one parcel for just one shipping fee), please contact me after your first purchase.
1-3 DaysAuckland


text:
Sugar-Lime Sauce
Juice of 1 fresh lime (2 tablespoons)
1/2 cup brown sugar
Whipped Coconut Cream
1 cup heavy cream
2 tablespoons confectioners' sugar
2 tablespoons canned coconut cream (optional)
Pineapple Sticks
1 pineapple, peeled and quartered lengthwise (remove the prickly eyes with the point of a knife)
8 metal kabob skewers
To make the sauce: Put the lime juice and sugar in a small saucepan, bring to a boil, and stir until the sugar dissolves.
Cut the pineapple quarters into 2 to 3 wedges lengthwise. If preferred, slice off the core sections, then cut each wedge into slices about 1/2-inch thick. Thread long metal skewers through each wedge and brush with the lime mixture.
Light an outdoor grill or preheat a broiler. Grill or broil until the fruit is tinged dark brown. Serve drizzled with any leftover sauce, accompanied by the whipped coconut cream.
Serves 4


text:
When you visit London hospitals for treatment or to support relatives we understand that you need your own space and no additional stress. AllStay offer you the independent accommodation you need in locations close to some of the capital's biggest and most important hospitals.
Whether you're staying on your own or with family members, AllStay has accommodation to suit your requirements. Each modern, spacious apartment comes with a fully fitted and equipped kitchen, giving you control over the time and expense of meals, and free Wi-Fi internet allows you to keep in touch with the world. In addition, wheelchair users can be comfortably accommodated in our accessible apartments.
Why choose Serviced Apartments? We explain the benefits of our serviced apartments here: Find out more »
Call us on +44 (0) 203 465 9100 or
email us at email@example.com
Sign up to our newsletter to receive special offers and our latest news.


text:
HATTIESBURG, MS (WDAM) - The Pine Belt Quilters presented their 14th show at the Lake Terrace Convention Center October 5 – 7.
One of the unique features of the show is the Hattiesburg Visitor Center Challenge, in which participants must produce a quilt using the theme "Find Your True South".
"This is our largest show in the state. We put this show on every two years to show the public what is being done in the field of quilting," said Co-Chair Gloria Green. "Our goal is to maintain the interest of quilting, the interest in historic quilts and hopefully to groom a few new quilters."
Quilters from more than 65 cities in five states entered a total of 400 quilts to compete for special recognition in the judged show.
"I think the thing that always impresses me is the absolute, unbelievable, creativity of the human spirit. Everyone is different, everyone is special in its own way," said Green.
General information and details about the show are available HERE.


text:
Life Redefined: This Is Not Chicken Soup for the Thyroidectomized Soul
Cancer is a heavy burden to carry. The road you walk when you have cancer is hard and treacherous. Having cancer on your mind everyday will leave you weary and exhausted. In order to avoid an emotionally drained state of being, I think we need to focus on something a bit lighter every now and then. Rather than reading an I-want-to-be-motivational-but-actually-leave-you-drowning-in-a-pool-of-your-own-tears story from Chicken Soup for the Thyroidectomized Soul, I prefer to find humor in the midst of all the bad in my life. Today, we're going to try to find something to laugh about.
A few weeks ago, I was chatting with a friend and fellow cancer survivor. My friend and I have no problem discussing our experience with cancer with each other. After this particular conversation ended, however, I realized we spent nearly the entire time talking about the medicines and supplements we now take as a result of having our cancerous thyroids removed. It got me thinking—you know you have thyroid cancer when…
- You have friends with cancer and your conversations are based around the medicines you take
- You join a cancer-related organization and the membership gift is a pill organizer
- You're actually excited to have a new pill organizer
- You know what day of the week it is based on said pill organizer
- You have a favorite phlebotomist
- You know what a phlebotomist is
- You have a favorite tech in the hospital's nuclear medicine department
- You've actually been to the nuclear medicine department
- You can use Tg, nuke med, I-131, and whole body scan in a sentence with no problem. For example, "Yeah, I was in nuke med for my whole body scan after my tracer dose of I-131…the radiologist said I had some uptake and my Tg is elevated so my endo is ordering 150mCi of I-131 followed by a SPECT-CT."
- During your treatment, your pee is so radioactive that you have to flush the toilet three times whenever you go to the bathroom
- You meet your insurance deductible by February (and it's not because your deductible is really low)
- Friends/family start greeting you with, "How AAARRRRE you?" as though you're going to fall over dead any minute. And when you don't keel over, they don't know what to say or do, so they either stare at you awkwardly until you walk away or they start talking about the weather.
Aaaand, now it's your turn! I know some of these apply to other thyroid diseases, too, so I want to hear from all of you, lovely thymmunity, regardless of the type of disease that is waging war against your thyroid. Finish the sentence: you know you have thyroid cancer/disease when…
Tags: Health Community, Life Redefined, literary community, thyroid cancer patient, thyroid cancer support, Written by Joanna Isbill, you know you have thyroid cancer when, Young Adults with Thyroid Cancer
Follow Dear Thyroid on Twitter/@DearThyroid | See our Facebook Page | Become a Fan on Facebook | Join our Facebook Group
You Can Create a Dear Thyroid Profile and share with friends!
Reader Feedback
18 Responses to "Life Redefined: This Is Not Chicken Soup for the Thyroidectomized Soul"
You know you have thyroid cancer when…
You've augmented the Thyca.org LID recipe book with tips and new dishes from other people in the waiting room on scan day.
You know you have when….
You're oncologist, radiologist, and pharmacist are 1 – 2 – 3 on your cellphone's speed dial list….
You know you have medullary thyroid cancer when your daughter's friend is staring at the deep scar on your neck because the cancer metasized they had to cut you in two places and now everyone stares at your scar – thinks you are a former gang member.
When: 1) you've seen 3 drs in 2 days and discussed your constipation/fissures with each of them 2) a person you've just met says " wow gnarly scar on your throat" 3) your wake up time revolves around taking your pill early enough you can go back to bed for an hour while you wait to eat 4) you are on a first name basis with the hospital scheduling dept 🙂
When you explain that the scar on your neck if from defending the White House in the war of 1812.
You are happy to have a scar on your neck as an opening to the conversation about why you are SO weird…
My spouse, who works in an ENT clinic, adds: when you become an example for rotating medical students and when the doctors ask you to come speak to new Thyca patients!
Dear Joanna,
Yes to humor about your illness! Yes to your awesome life redefined series! …Boo to being able to say yes to most of those without having had cancer…haha.. 🙂
I'm not sure if you all remember me but I used to do this comic about my life with chronic illness, and well, I'm back to being sick so I'm back to writing it. I think you guys started out just a few months after I did and I'm so amazed and so impressed at how this site has grown! Well done Ladies (and Gents)!!!
Looking forward to keeping up with reading more in the future!
– Miss Waxie
Leave a Reply
Comments are moderated in an effort to control spam. If you have a previously approved Comment, this one should go right through. Thanks for your patience!


text:
Our vision is to provide Australia's leading ski racing and development programme, based on core values of PERFORMANCE, RESPECT and RESPONSIBILITY. We deliver this in a safe, fun and challenging environment, assisting each athlete to maximise their potential, and to develop the foundation skills for excellence that will extend beyond ski racing to all their personal endeavours.
TSRC is a not-for-profit sporting club run by a committee of past and current parents. The Club is supported by Kosciuszko Thredbo Limited (KT), which provides us with first class racing and training conditions.
Over the past 40 plus years our athletes have represented TSRC on Olympic, National and NSW State teams. They have competed in World Championships, World Cup races, Europa Cup races, World Junior Championships and international FIS and FIS Children's races including Topolino (Italy) and Whistler Cup (Canada), regularly achieving podium results.
Northern Hemisphere Safety Reminder
A safety message after recent northern hemisphere skiing tragedy.read more
SSA Children's Carnival Day 2 ResultsHi all Race results from today. You can now go to the TSRC website / Races tab to get all 2018 TSRC Race Results. SL Results Race 1 SL Results Race... read more
SSA Children's Carnival – GS ResultsResults from today's races: Results Race 1 Results Race... read more
Click to select a category:


text:
Misconceptions about Martial Arts
You need to be in shape to do Martial Arts
. NOT TRUE, we will get you into shape.
I am too old to start now
. NOT TRUE, you are never too old to start. Your training may vary slightly from that of other students.
I don't have enough time to train
. NOT TRUE Training can take as little 2 to 4 hours a week. It's up to you.
Karate is for people who want to learn to fight
. NOT TRUE, People get into Karate for many reasons. Those who are looking for a fight do not last long in Martial Arts.
Karate classes are just for men
. NOT TRUE, we have a large number of female students both kids and adults who train just as hard, if not harder than the men.


text:
Contemporary bedroom is the optimum design to you who wish for make a large and modern atmosphere. Some folks in the urban areas choose to modern design for their house, mainly their most private room like bedroom. We wish to present contemporary bedrooms to provide you an inspiration in decoration bathroom.
There are much ways for make contemporary bedrooms. For example, your can use the black and white adjustment for make your bedroom feels quiet but definitely modern. If you loathe that idea, you can add wooden interior which can make your feel warm and inviting. But you can design your bedrooms over creative.
Do you wish for discover over inspirations? Your can view at the gallery pictures of contemporary bedrooms below. We will indicate you much good interiors which can make your bedroom feels impeccable!


text:
Do you have a certain course that you'd like your learners to see front and center when they log in to the Learning Management System? With Knowledge Anywhere's LMS, featuring a course is simple. Follow the instructions below to learn how.
- From the Administrative side of the LMS, navigate to the "Content" tab in the main navigation header.
- Then, select the "Courses" tab.
- Once you're in the "Courses" tab you will see a library of courses within your LMS displayed. To feature a course, you'll need to select the "Edit" button next to the name of the course. Tip: If you have a large library of courses, you can use the filter table with the search bar to find the course you'd like to feature faster.
- After you select "Edit" next to the name of the course you'd like to feature, you will be redirected to a page where you can edit the course information. On this page, you'll want to click on the blank field next to the prompt "Featured On." A calendar will pop up where you can select the day that you'd like the course to be featured on. Once you've selected a day, select the "Save" button.
- Your featured course will appear as shown in the image below on the Learner side of the LMS.


text:
Death and Joblessness
Suicide Dogs the Long-Term Unemployed. What Can Be Done to Help Them?
By Annie Lowrey
He hit "publish" on the last Wednesday in July, in the middle of a long afternoon. "I also have become homeless and am on the verge of suicide. I slept out in the wood last night and didn't gett very much sleep. I hate to bring you people down with my problems but I thought you would like to know this. I don't know what else to say except I'm very sorry it turned out like this but I can take the strain of living like this very much longer." (All posts are reproduced as published.)
The post went up as part of a conversation about homelessness on Unemployed-Friends, a popular online forum for the unemployed to connect with one another. Most were discussing how to live in homeless shelters after eviction or foreclosure. But his post went further. "This is killing me physically and emotoinally. I am at the end of my rope and getting to the point of letting go. I have tried everything I know to get help. DHS won't help' Salvation Army won't help. 211 won't help. I have no idea as to where to go from here. If you don't hear from me by tomorrow I probably will be dead."
Thousands of users visit the web site daily, offering one another everything from advice about applying for unemployment insurance benefits to emotional support. It is one of dozens of such sites helping the nation's 14.6 million unemployed — particularly the long-term unemployed, the 6.6 million Americans who have been out of work for more than six months. "I am very tempted to walk in front of an oncoming semi right now. Sorry to go on ranting but I am getting to the point where I feel I have no choice. For those of you that want to know I am currently in Grand Rapids. I appreciate your words of encouragement but right now it doesn't seem to be enough to keep me going."
The post ended, "I will try to tough out another night. Goodbye for now."
The Rest…HERE


text:
Digital theft in the age of the internet is rampant and there is little that can be done to stop it. While the media format of digital music is very difficult to protect, digital graphics can contain a watermark, and with the right software to add the watermark to the photo, photos can safely be distributed in the proofing stages of the graphics process.
Image watermarking software protects your digital images and artwork by adding professional transparent watermarks that combine text, illustrations and graphics.
Adding a watermark to a photo is the best way to ensure that the photo is not used without permission, but watermarking photos can be a complicated process.
The reliable method of watermarking your photos is using software that has been specifically designed to do the job quickly and easily. The Batch Picture Protector has been designed from the ground up, to make it very simple to load a photo and then add a watermark to that photo. The software also allows the watermarked photo to be saved in any major image file format.
Simply load your photo into the program and then type the text to be used as a watermark or just add a logo as the watermark. The software will allow you to adjust the transparency, rotation and position of the watermark in just a few clicks. You can usefully save your created watermarks and load them again for another project.
It is just as easy to watermark multiple photos at once because you can load all of the photos you want to watermark and process them as one batch. Batch processing is a real time saver when adding a watermark to tens, hundreds or even more photos at once.
It might be important to protect your digital copyright and luckily this could be done efficiently with purpose built software. The Batch Picture Protector can guard your photos from unauthorized use with its advanced aesthetic watermarking capabilities and simplicity of use. That will give you the assurance to distribute photos with the peace of mind that the copyright of your work has been subtly protected.
Image watermarking software protects your digital images and artwork by adding professional transparent watermarks that combine text, illustrations and graphics.


text:
Fighting Words
Chris Murphy, the junior senator from Connecticut, has not given up on diplomacy, but he certainly looks ready for war. "Five years ago it was ridiculous to think Russia would invade Ukraine," says the first term senator in the echo of the Cannon Rotunda. "Today it may be ridiculous to think that they'll march on a NATO ally in five or ten years." Senator Murphy finds himself in good company wishing to protect NATO against the scourge of Russia: Dean Acheson and the Dulles brothers, to name a few. CNN's Jake Tapper chimes in echoing George F. Kennan, and suggests that the US "contain" Russia.
Murphy, a vivacious, energetic cold warrior at the young age of 40, intends to thwart "Putin's ambitions to re-establish the old soviet empire." "Putin," the Senator triumphantly declared, has "become an international pariah."
But, at risk of reprising an argument already made in this publication, Senator Murphy's fears just do not make strategic sense.
Let's first put aside Murphy's qualm regarding a Russian attack against a NATO ally. It is not in Russia's interest—nor will it ever be in Russia's interest—to invade a NATO member in a way similar to its recent attacks on non-NATO states (Georgia in 2008, and, of course, Ukraine today). As much as the United States might not care to go to war to protect the sovereignty of a NATO member, take Latvia, for example, the Senate-ratified North Atlantic Treaty would compel such action. Clause five of the treaty, first invoked by the United States after 9/11/01, equates an attack on Latvia to an attack on France, the UK, or the United States, and compels a response by all NATO members. Russia conquering Latvia, or any other Baltic state, is essentially declaring war against five of the world's ten most powerful militaries, at the same time. Because of this, the "ridiculous" notion Murphy mentions will likely still be ridiculous in five or ten years. His slippery slope concern is, therefore, limited when it comes to NATO states (which, due to the West's Cold War strategies, already effectively boxes in present day Russia).
Completely aside from the strategic conundrum of the NATO tripwire, Senator Murphy's assumption that Crimea represents the first conquest of the New Russian Empire puts US policy in an extremely difficult position. Acting on the 'Empire' assumption, and following the 'line in the sand' drawn by Sen. Murphy, commits the United States to expanding its engagement in Europe in order to counteract Russia with the same type of containment that led the United States into Vietnam.
But why not be cautious assume the worst about Russia's intentions? Because the 'worst' that Senator Murphy is worried about in Washington is almost as bad for Moscow. First, a serious, sustained attempt at building an Empire to the west would trigger harsh economic consequences. Though the most commonly told economic story about the current crisis has been Russia's control of petroleum and natural gas flowing to Europe, Russia's economy would suffer greatly. Russia's export economy makes up one-sixth of its GDP, and six of its seven largest customers are in Europe. Russia would not just lose customers if Europe cut economic ties; they would lose important industrial resources and capital goods like heavy machinery, plastics, and finished metal products.
Further, even accounting for the 'pivot to Asia,' America's vital strategic interests still include protecting the European industrial core from a potential hegemon. When Russia challenged this interest in the Cold War, the US proved much stronger. Today, the US is not just much stronger than Russia: it is astronomically stronger. Putin knows this, and knows it is not in his country's interest to challenge the existing order in Europe.
Russia's aims in the Ukraine crisis have little to do with expanding its power or dominion. Russia would be satisfied with a return to status quo ante rerum, where Ukraine was not wholly in the EU's camp or Russia's. That Putin is seeking to protect own security interests from NATO/EU expansion right on his doorstep is overall a much more plausible explanation than the theory of neo-imperialism. Assuming the worst, as Senator Murphy has clearly done, is not America's best course of action.
The 'Murphy/Tapper Cold War Pep Rally' is not the only misrepresentation of the situation in Ukraine in the US media, but it is uniquely scary, because it stars the Chairman of the Senate's Subcommittee on Europe, who is also a member of the Senate Foreign Relations Committee. Vladimir Putin likely doesn't watch Jake Tapper, but he certainly pays attention to US Senators because they have the tools—sanction authority, treaty power, and influence over foreign affairs—to turn outdated, jingoistic Cold-War rhetoric into a reality.